WO2010012077A1 - Compositions, methods and kits for reprogramming somatic cells - Google Patents

Compositions, methods and kits for reprogramming somatic cells Download PDF

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Publication number
WO2010012077A1
WO2010012077A1 PCT/CA2009/001026 CA2009001026W WO2010012077A1 WO 2010012077 A1 WO2010012077 A1 WO 2010012077A1 CA 2009001026 W CA2009001026 W CA 2009001026W WO 2010012077 A1 WO2010012077 A1 WO 2010012077A1
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Prior art keywords
cell
cells
reprogramming
transposon
reprogrammed
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PCT/CA2009/001026
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French (fr)
Inventor
Andras Nagy
Keisuke Kaji
Knut Woltjen
Iacovos Michael
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Mount Sinai Hospital
University Court Of The University Of Edinburgh
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Priority claimed from GB0813770A external-priority patent/GB0813770D0/en
Priority claimed from GB0815496A external-priority patent/GB0815496D0/en
Priority claimed from GB0900845A external-priority patent/GB0900845D0/en
Application filed by Mount Sinai Hospital, University Court Of The University Of Edinburgh filed Critical Mount Sinai Hospital
Publication of WO2010012077A1 publication Critical patent/WO2010012077A1/en

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    • G—PHYSICS
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    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/10—Vectors comprising a special translation-regulating system regulates levels of translation
    • C12N2840/102—Vectors comprising a special translation-regulating system regulates levels of translation inhibiting translation
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    • C12N2840/00—Vectors comprising a special translation-regulating system
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/60—Vectors comprising a special translation-regulating system from viruses

Definitions

  • the invention relates to compositions, methods and kits for reprogramming somatic cells.
  • the invention relates to a method of reprogramming somatic cells including somatic stem cells to produce pluripotent cells or cells of increased pluripotency, the cells produced by the methods, as well as reagents, including vectors and kits suitable for use in the methods.
  • iPS induced pluripotent stem
  • viral vectors silenced in iPS cells can be re-activated when the cells differentiate [16, 22].
  • the present invention is based on the development of non-viral based systems for reprogramming somatic cells.
  • the invention provides non-viral based vector systems which are able to express the proteins necessary for reprogramming somatic cells.
  • the present invention is directed to the reprogramming of somatic cells including somatic stem cells and therefore does not employ eggs, embryos or embryonic stem cells.
  • reprogrammed cells of the present invention display pluripotency and growth ability and may be similar in some respect to embryonic stem cells.
  • the systems and methods of the invention represent significant improvements over known methods.
  • the traceless removal of reprogramming factors is an improvement of particular significance.
  • Systems and methods of the invention enable high-fidelity post-reprogramming transgene removal once exogenous factor expression becomes dispensable.
  • the controlled reduction of reprogramming factors from established iPS cell lines is an invaluable tool.
  • the systems and methods of the invention also provide technical simplification of reprogramming methodology. In particular, there is no need for specialized biohazard containment facilities or the production of high-titer, limited-lifetime viral stocks.
  • the methods employ autonomously replicating nucleic acid constructs, such as plasmid/transposon DNA preparations and/or vectors which may be introduced into cells directly using commercial transfection products and well known techniques in the art such as electroporation.
  • the methods also allow the option of xeno-free production of human iPS cells contrary to current viral production protocols, which use xenobiotic conditions.
  • the methods of the invention permit somatic cell reprogramming to be achieved with single-copy, removable, multi-transgene expression.
  • a method of reprogramming a somatic cell comprising: a) providing a non-viral based vector system which is capable of expressing reprogramming factors necessary to reprogram said somatic cell; b) transfecting said vector into said somatic cell; and c) expressing said reprogramming factors, such that reprogramming of said somatic cell can take place.
  • a method of reprogramming a differentiated somatic cell or unipotent or multipotent somatic stem cell, which generates only limited lineages of cells, in order to provide a pluripotent reprogrammed cell comprising the steps of: a) providing a non-viral based vector system which is capable of expressing at least two proteins necessary for reprogramming (i.e. reprogramming factors) of said somatic cell or somatic stem cell to occur; b) transfecting said vector into said somatic cell or somatic stem cell; and c) allowing/causing expression of said at least two genes encoding the proteins, such that reprogramming of said somatic cell or somatic stem cell can take place.
  • the non-viral based vector system is capable of expressing at least two, three or four reprogramming factors.
  • the non-viral based vector system comprises a single vector capable of expressing said reprogramming factors, such as three or four reprogramming factors.
  • the non-viral based vector system comprises a single vector comprising reprogramming sequences encoding three or four reprogramming factors.
  • two or more genes encoding the reprogramming factors are expressed as a single polycistronic messenger RNA which is subsequently expressed into a polypeptide comprising a polypeptide sequence encoding the proteins corresponding to the two or more genes.
  • the protein sequences of the two or more genes are preferably separated by self cleaving/processing sequences known in the art.
  • the nucleic acid between each of the two or more genes in a vector encodes a self cleaving peptide.
  • Preferred self cleaving peptides are 2A peptides from Aphthoviruses, a typical example of which is foot and mouth virus, or 2A like sequences from other viruses in particular Picornaviridae like the Equine rhinitis A virus as well as the insect Thosea asigna virus, and Porcine teschovirus-1 (Donnelly et. al. J Gen Virol (2001) 82, 1027-41.)
  • the same or different self-cleaving sequence may be used, with different sequences being preferred.
  • the methods of the invention may further comprise isolating the reprogrammed cells, and optionally producing cell lines comprising the cells.
  • Such cells/cell lines are commonly known in the art as iPS cells/cell lines.
  • a method of the invention for reprogramming a somatic cell can utilize a non-viral based vector system comprising one or more or at least one transposon region(s) each region comprising a transposon and one or more reprogramming sequences encoding or expressing reprogramming factors.
  • a method for reprogramming a somatic cell utilizing at least one transposon region, each region comprising a transposon with 5' and 3' inverted repeat sequences for insertion/excision of the transposon sequence and one or more reprogramming sequences inserted within the transposon, encoding or expressing reprogramming factors(s).
  • the present invention relates to a method for generating a reprogrammed cell or reprogramming a somatic cell comprising integrating into a somatic cell (e.g. fibroblast) one or more transposon region(s) thereby generating a reprogrammed cell wherein said transposon region(s) comprises a transposon and one or more reprogramming sequences.
  • a somatic cell e.g. fibroblast
  • the transposon region(s) comprise a piggyBac transposable element.
  • the transposon region(s) comprise a DNA transposon, in particular Sleeping Beauty transposon.
  • a method for producing or generating reprogrammed cells or reprogramming somatic cells comprising:
  • transfecting somatic cells with a non-viral based vector system comprising one or more transposon region(s) each comprising a transposon and one or more reprogramming sequences;
  • a method for producing or generating reprogrammed cells comprising:
  • Nanog and Lin28 gene families and a transposase expression plasmid Nanog and Lin28 gene families and a transposase expression plasmid
  • a method for producing or generating reprogrammed cells comprising:
  • Nanog and Lin28 gene families (b) culturing the transfected somatic cells and isolating reprogrammed cells, in particular iPS cells.
  • the non-viral based vector system comprises two, three or four, preferably three or four separate transposon regions, each transposon region comprising a transposon and one or more reprogramming sequences, preferably one or two, most preferably one reprogramming sequence.
  • the somatic cells are transfected with three transposon regions each comprising a transposon and a reprogramming sequence.
  • the somatic cells are transfected with three transposon regions each comprising a transposon and a reprogramming sequence of each of an Oct gene family, a KIf gene family, and a Sox gene family.
  • somatic cells are transfected with three transposon regions each comprising a transposon and an Oct 3/4, Klf4 or Sox2 gene.
  • the somatic cells are transfected with four transposon regions each comprising a transposon and a reprogramming sequence.
  • somatic cells are transfected with four transposon regions each comprising a transposon and a reprogramming sequence of each of an Oct gene family, a KIf gene family, a Sox gene family and a Myc gene family.
  • somatic cells are transfected with four transposon regions each comprising a transposon and an Oct 3/4, c-Myc, Klf4 or Sox2 gene.
  • the non-viral based vector system comprises a single transposon region comprising a transposon and two or more reprogramming sequences, preferably three or four reprogramming sequences.
  • somatic cells may be transfected with a single transposon region comprising a transposon and at least two, three or four reprogramming sequences.
  • the somatic cells are transfected with a single transposon region comprising a transposon and three reprogramming sequences of each of an Oct gene family, a KIf gene family, and a Sox gene family.
  • the somatic cells are transfected with a single transposon region comprising a transposon and Oct 3/4, Klf4 and Sox2.
  • the somatic cells are transfected with a single transposon region comprising a transposon and four reprogramming sequences of each of an Oct gene family, a KIf gene family, a Myc gene family and a Sox gene family.
  • the somatic cells are transfected with a single transposon region comprising a transposon and c-Myc, Oct 3/4, Klf4 and Sox2.
  • a vector or a transposon region may comprise a promoter, in particular an inducible promoter, a polyadenylation signal and/or excision or cleaving sequences.
  • the transposon region comprises a promoter, in particular an inducible promoter.
  • the vector or transposon region comprises a tet ⁇ 2 tetracycline/doxycycline (dox) inducible promoter and the transfected somatic cells are cultured in the presence of dox.
  • the transposon region(s) comprise excision or cleaving sequences and the transposon region(s) are excised in the reprogrammed iPS cells.
  • the non-viral based vector system is integrated into the genome of the somatic cell for expression of the reprogramming factors, or it is not integrated and the reprogramming factors are episomally expressed.
  • the non-viral based vector system is integrated and comprises means for excising the reprogramming sequences from the genome of the reprogrammed cell.
  • the invention also provides pluripotent stem cells induced by reprogramming somatic cells where the reprogramming comprises contacting the somatic cells with a non- viral based vector system of the invention, in particular a non-viral based vector system which is capable of expressing at least two reprogramming factors, preferably three reprogramming factors, more preferably four reprogramming factors.
  • the invention provides reprogrammed somatic cells obtained by a method of the invention or somatic cells derived by inducing differentiation of somatic cells reprogrammed using a non-viral based vector system of the invention.
  • a method of the invention may further comprise differentiating a reprogrammed cell to a desired cell type.
  • a method of the invention may further comprise culturing the reprogrammed cells with at least one agent that induces differentiation i.e. a differentiation agent.
  • a differentiation factor which directs differentiation may be introduced into the somatic cells with the non-viral based vector system.
  • the invention thus also provides cells differentiated from reprogrammed cells of the invention.
  • the differentiated cell may be a cardiac cell, neural cell, hepatic cell, haemopoietic cell, lymphoid cell, bone cell, epithelial cell, kidney cell, pancreatic cell, endothelial cell, or muscle cell type.
  • the present invention also provides a method for expanding, preferably selectively expanding, the reprogrammed cells or cells derived or differentiated therefrom.
  • the method comprises culturing the cells under proliferation conditions; and isolating increased numbers of the cells.
  • Increased numbers of the cells refers to an increase in the number of cells by at least about 2-fold relative to the number of cells that are present in a parallel control culture of cells that are not subjected to the same proliferation conditions.
  • the invention also relates to an expanded cell preparation obtained by this method.
  • the term “expanding” or “expansion” contemplates the proliferation of the cells.
  • the invention also provides reprogrammed cells or cells derived therefrom generated using a method of the invention, and compositions comprising same.
  • the invention provides a reprogrammed cell comprising a non-viral based vector system, in particular a non-viral based system described herein, which is capable of expressing reprogramming factors necessary to reprogram said somatic cell.
  • a reprogrammed cell of the invention may have one or more of the reprogramming sequences excised from the reprogrammed cell, such that one or more of the other reprogramming sequences remains in the reprogrammed cell.
  • the genome of a reprogrammed cell of the invention may comprise excision sequences or portions of excision sequences following excision of the non-viral based vector system.
  • the genome of the reprogrammed cell comprises LoxP sites.
  • the invention further contemplates reprogrammed cells or cells derived or differentiated therefrom and compositions comprising same, in combination with a substrate or matrix, preferably a substrate or matrix adapted for transplantation into a patient.
  • the substrate may be an engineered biomaterial or porous tissue culture insert.
  • the invention also contemplates a cell line comprising reprogrammed cells or cells derived or differentiated therefrom produced by a method of the invention.
  • the invention also contemplates reprogrammed cells or cells derived or differentiated therefrom and compositions comprising same, in combination with a substrate or matrix, preferably a substrate or matrix adapted for transplantation into a patient.
  • the substrate may be an engineered biomaterial or porous tissue culture insert.
  • the invention also provides reprogrammed cells or cells derived or differentiated therefrom that have the ability to migrate and localize to specific regions in a patient where they differentiate into somatic cells typical of the region and they integrate into the tissue in a characteristic tissue pattern.
  • the invention provides a pharmaceutical composition comprising an effective amount of a reprogrammed cell, or a cell differentiated or derived therefrom.
  • the invention provides a pharmaceutical composition comprising cells of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • a pharmaceutical composition may include a targeting agent to target cells to particular tissues or organs.
  • a non-viral based vector system capable of expressing at least two proteins for use in reprogramming somatic cells including somatic stem cells (i.e. reprogramming factors), the vector system comprising at least two genes encoding said proteins or reprogramming factors necessary for reprogramming said somatic cells including somatic stem cells; and preferably a single promoter capable of directing expression of said genes.
  • a non-viral based vector system of the invention comprises a single vector which is capable of expressing said proteins or reprogramming factors, such as 3 or 4 or more proteins or reprogramming factors necessary for reprogramming to occur.
  • the invention contemplates a non-viral based vector system comprising a plurality of transposon regions each comprising a transposon and a reprogramming sequence and its use in the methods of the invention.
  • a non-viral based vector system is provided comprising one or more transposon region(s) comprising a transposon and one or more reprogramming sequences.
  • the non-viral based vector system comprises two, three or four transposon regions each comprising a transposon and one or more reprogramming sequences, preferably one reprogramming sequence.
  • the one or more reprogramming sequence is selected from the group consisting of members of the Myc, Oct, KIf, Sox, Nanog and Lin28 gene families.
  • the transposon region(s) comprise a piggyBac transposable element.
  • the transposon region(s) comprise a DNA transposon, in particular Sleeping Beauty transposon.
  • the vector system comprises a single transposon region comprising a transposon and at least two, three or four reprogramming sequences.
  • the single transposon region comprises a transposon and two or more, preferably three or four, reprogramming sequences of each of an Oct family gene, a KIf family gene, and a Sox family gene.
  • the transposon region comprises a transposon and Oct 3/4, Klf4 and Sox2.
  • the single transposon region comprises a transposon and c-Myc, Oct 3/4, Klf4 and Sox2.
  • a non-viral based vector system may further comprise a promoter, in particular an inducible promoter, means to enable excision of sequences (i.e. excision or cleaving sequences), an IRES site, a polyadenylation signal, an origin of replication and/or an antibiotic selection marker gene.
  • a promoter in particular an inducible promoter, means to enable excision of sequences (i.e. excision or cleaving sequences), an IRES site, a polyadenylation signal, an origin of replication and/or an antibiotic selection marker gene.
  • the non-viral based vector system comprises the vectors identified in Figures 4, 10, 12, 14 or 20 and the Examples herein.
  • Cells and compositions of the invention may be used in both cell therapies and gene therapies aimed at alleviating disorders and diseases.
  • the invention contemplates a method of treating a subject suffering from a condition where an increase or replacement of a particular cell type is desirable comprising transferring to a patient an effective amount of cells of the invention, in particular, a composition comprising reprogrammed cells of the invention.
  • the reprogrammed cells and compositions obtained using the systems and methods of the invention may be administered to a subject for the repair or regeneration of a tissue or organ.
  • the method further comprises increasing the function of the tissue or organ.
  • the invention also relates to the use of cells and compositions of the invention for the repair or regeneration of a tissue or an organ in a subject or to increase the function of a tissue or organ in a subject.
  • the invention relates to the use of cells and compositions of the invention in the manufacture of a medicament for the repair or regeneration of a tissue or an organ in a subject or to increase the function of a tissue or organ in a subject.
  • the invention provides a method for repairing or regenerating tissue in a subject comprising obtaining reprogrammed cells of a previous aspect of the invention and administering the cells to a subject suffering from a condition where an increase or replacement of a particular cell type is desirable.
  • the subject has a myocardial infarction, congestive heart failure, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, cirrhosis, Parkinson's disease, Alzheimer's disease, diabetes, cancer, arthritis, or wound healing, or similar diseases.
  • a subject has damage to a tissue or organ and a method of the invention involves administering a dose of reprogrammed cells or cells differentiated or derived therefrom sufficient to increase a biological function of the tissue or organ or increase the number of cells in the tissue or organ.
  • the subject has a condition, disorder or disease and a method of the invention involves administering a dose of reprogrammed cells or cells differentiated or derived therefrom to ameliorate or stabilize the condition, disorder or disease.
  • the method increases the biological activity of the tissue or organ by at least about 5%, 10%, 15%, 20%, 25%, 50%, 60%, 75%, or more compared to a corresponding untreated tissue or organ.
  • the reprogrammed cells or cells differentiated or derived therefrom are administered directly to a subject at a site where an increase in cell number is desired, in particular a site of tissue damage or injury, or disease.
  • the invention provides a method for obtaining reprogrammed cells for autologous transplantation from a patient's own cells comprising (a) obtaining a sample comprising somatic cells from the patient; (b) transfecting the somatic cells with a non-viral based vector system in accordance with the invention which is capable of expressing reprogramming factors necessary to reprogram said somatic cell;(c) expressing said reprogramming factors, such that reprogramming of said somatic cells occurs.
  • the invention also provides a tissue or organ comprising reprogrammed cells or cells differentiated or derived therefrom. Further the invention provides the use of a reprogrammed cell of the invention for tissue repair or regeneration. The invention also relates to a method of using the cells and cell compositions described herein in rational drug design.
  • Cells and compositions of the invention may be used to screen for potential therapeutics that modulate development or activity of such cells or cells differentiated therefrom, or in toxicology testing or studies.
  • the invention relates to the use of reprogrammed or differentiated cells prepared by a method of the invention for drug screening and/or toxicity testing.
  • the cells and cell compositions of the invention may be used as immunogens that are administered to a heterologous recipient.
  • the cells and compositions of the invention may be used to prepare model systems of disease.
  • the cells and cell compositions of the invention can also be used to produce growth factors, hormones, etc.
  • the invention also relates to a method for conducting a regenerative medicine business. Still further the invention relates to a method for conducting a stem cell business involving identifying agents that affect the proliferation, differentiation, function, or survival of cells of the invention.
  • An identified agent(s) can be formulated as a pharmaceutical composition, and manufactured, marketed, and distributed for sale.
  • the invention also contemplates a method of treating a patient comprising administering an effective amount of an agent identified in accordance with a method of the invention to a patient with a disorder or disease.
  • the invention also contemplates a method for conducting a drug discovery business comprising identifying factors or agents that influence the proliferation, differentiation, function, or survival of cells of the invention, and licensing the rights for further development.
  • the invention further contemplates a method of providing drug development wherein cells described herein are used as a source of biological components of cells in which one or more of these biological components are the targets of the drugs that are being developed.
  • the invention provides a method for identifying an agent that can replace one or more reprogramming factors excised from reprogrammed cells.
  • the invention also relates to the use of a reprogrammed cell of the invention for identifying an agent to replace one or more reprogramming factors excised from the reprogrammed cell.
  • An agent may include but is not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members of protein or peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids, polysaccharides, oligosaccharides, monosaccharides, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), nuclei acids (e.g. siRNAs, cDNAs), and small organic or inorganic molecules.
  • the agent may be an endogenous physiological compound or it may be a natural or synthetic compound.
  • the invention provides a method for identifying a small molecule mimic to replace one or more reprogramming factors excised from reprogrammed cells.
  • the invention also relates to the use of a reprogrammed cell of the invention for identifying a small molecule mimic to replace one or more reprogramming factors excised from the reprogrammed cell.
  • the invention provides a kit for use in reprogramming somatic cells or somatic stem cells, the kit comprising: a) one or more non-viral based vector systems according to the present invention; and b) reagents for facilitating transfection of said one or more vectors into a somatic cell including a somatic stem cell of choice.
  • the invention also provides a kit for carrying out a method of the invention comprising a transposon system for reprogramming cells.
  • the kit comprises a transposon region or components thereof in a package.
  • the invention provides a kit for tissue repair or regeneration comprising a reprogrammed cell or a cell differentiated or derived therefrom, and instructions for use of the cell in tissue repair or regeneration.
  • the invention is also directed to a kit for transplantation of reprogrammed cells or cells derived or differentiated therefrom comprising a flask with medium and cells or a cell composition of the invention.
  • the invention relates to a kit for rational drug design comprising reprogrammed cells and cells derived or differentiated therefrom obtained by a process of the invention.
  • the kit comprises reprogrammed cells and instructions for their use in drug screening or toxicity assays.
  • the kit comprises reprogrammed cells and instructions for their use in an absorption assay.
  • the kit comprises reprogrammed cells with one or more reprogramming factors excised from the cells and instructions for their use in identifying mimics to replace reprogramming factors excised from the reprogrammed cells.
  • Figure 1 Generation of iPS cells using a non-viral multiprotein expression vector
  • a Schematic of reprogramming cassette.
  • the four reprogramming factors are translated from a single mRNA coding of c-Myc, KIf 4, Oct4 and Sox2 linked with three different 2A sequences, F2A, T2A and E2A [SEQ ID NOs. 153-155].
  • Different 2A sequences were chosen to avoid homologus recombination in the vector, however it might not be necessary.
  • Additional peptide sequences to N-terminal and C-terminal protein of the 2A peptides are shown.
  • Full maps of the vectors with the reprogramming cassette are show in Figure 4.
  • b Schematic of reprogramming cassette.
  • the four reprogramming factors are translated from a single mRNA coding of c-Myc, KIf 4, Oct4 and Sox2 linked with three different 2A sequences, F2A, T2A and E2A [SEQ ID NO
  • FIG. 1 Efficient reactivation of pluripotent markers and fewer vector integration sites
  • a Quantitative PCR for total and endogenous c-Myc, Klf4, Oct4 and Sox2 expression. Data is shown as relative expression to an ES cell line, E14Tg2a (E 14). Note that higher relative expression of total c-Myc (2-8 fold compared to ES cells) is consistent with the multiprotein expression system for the reprogramming factors and lower expression level of c-Myc in ES cells compare to other three genes (data not shown), b. Immunoblot analysis of c-Myc, Klf4, Oct4 and Sox2. Asterisks indicate exogenous Klf4.
  • c Quantitative PCR for pluripotent markers, d.
  • a single integration site of imO7 (white circle in e), and a single integration site with tandem integration (same orientation) of imOl and imO3 (black circles in e) was identified by inverse PCR (data not shown) and validated by genomic PCR.
  • Asterisk band from wild-type allele. Arrowhead; integration site-specific band. Detail of the integration site is shown in Figure 9. Note different band size of tandem repeats is caused by vector degradation accompanied by random integration.
  • FIG. 3 Reprogramming cassette excision and pluripotency of the non-viral iPS cells, a. b.
  • a FGF receptor inhibitor, PD 173074 inhibits differentiation caused by reprogramming cassette excision. Many undifferentiated colonies at day 5 differentiate in the absence of PD 173074 (-PD) by day 9 (a). Percentage of remaining reprogramming cassette-free undifferentiated colonies in the absence (-PD) and presence (+PD) of PD 173074 is shown in b. Numbers of monitored reprogramming cassette-excised colonies are indicated in parentheses. Experiments were performed in three cell lines, imO2, imO7 and TNGimO5. c.
  • Quantitative PCR for pluripotency markers e. Undifferentiated cells in imO7 teratoma (upper left) and various tissues in imO7c8 teratoma (the other three panels), f. imO3Ec5 derived 10.5 dpc chimeric embryo (left) and non-chimeric embryo (right), g. imO7c8 derived chimeric mice. All blastocyst injection experiments are summarized in Table 3. Figure 4.
  • Maps of MKOS vectors. c-Myc, KIf 4, Oct4 and Sox2 coding regions linked with three different 2 A peptide sequences, F2A, T2A and E2A are located under CAG promoter.
  • the reprogramming cassette is followed by either ires EGFP (pCAGMKOSiE) or ires mOrange (pCAG2LMKOSimO).
  • the reprogramming cassette and ires mOrange are flanked by loxP sites in pCAG2LMKOSimO.
  • pCAGMKOSiE and pCAG2LMKOSimO are linearized with Fspl or Pvul before transfection, respectively.
  • FIG. 5 Robust expression of reprogramming factors using a multiprotein expression vector
  • a Immunoblot analysis of c-Myc, Klf4, Oct4 and Sox2 in HEK293 cells 48 hrs post transfection with pCAGMKOSiE (293+MKOS). Nuclear extract of ES cells (ES) and HEK293 cells without nucleofection (293) are used to identify exogenous protein expression. An anti- ⁇ tubulin antibody is used as a loading control
  • b Immunofluorescence of HEK293 cells for Oct4 and Sox2 24 hrs post transfection of pCAGMKOSiE. ES cells and HEK293 cells without nucleofection is used as positive and negative control, respectively.
  • Figure 6 Estimation of stable transfection efficiency in MEF using nucleofection.
  • a vector that derives EGFP expression via the CAG promoter was introduced into MEFs and the percentage of EGFP positive cells were monitored for 20 days after nucleofection without selection. Data represent two independent experiments. Averages of EGFP positive population were 86% and 3% of total cells at day 2 and day 20, respectively. We estimate stable integration occurred in 3.6% (3% of 86%) of transiently transfected cells.
  • FIG. 7 Schematic presentation of vector integration sites. Integration sites of imOl, imO3 and imO7 were found in chromosome 5 (a), chromosome 1 (b) and chromosome 17 (c), respectively. A red box in chromosome diagrams is enlarged below with indication of the vector integration site. Primers used for validation of the integration sites in Figure 2f are shown as black arrows. Diagrams and gene abbreviations were referred from Ensemble genomic databases.
  • FIG. 9 Efficient differentiation of reprogramming cassette-free iPS cell lines in vitro, a. Embryoid body (EB) differentiation.
  • imO2Ec3, imO3c8 and imO7c8 EBs show similar down-regulation of pluripotent markers (Oct4, Nanog, Rexl), and up-regulation of endoderm (Gata ⁇ ), ectoderm (Fgf5), mesoderm ⁇ Brachyury, Flkl) markers, while their parental cell lines, imO2El, imO3 and imO7, differentiate less efficiently, b.
  • EB Embryoid body
  • PB-iPS cell lines carry multiple copies of mFx transgenes.
  • PB-derived iPS cell lines bear multiple random transposon insertions, as revealed by Southern analysis.
  • Each clone tested was derived from treatment with a cocktail containing lOOng of each PB-TET-mFx transposon.
  • Total genomic DNA from MEFs and derived PB-iPS cell lines was digested with Bar ⁇ l, and screened with a probe detecting the entire neo coding region of ⁇ geo.
  • PB-iPS lines were found to contain a minimum of ⁇ 10 PB-TET transposon copies. Note that no fragmented transgene constructs (banding below the predicted lower limit of ⁇ 1.5kb) were detected.
  • the red arrow indicates the band predicted from the neomycin resistance gene detected in contaminating multi-drug resistant feeder cells (MEF) used for PB-iPS maintenance (band not detected in rtTA- MEF).
  • FIG. 11 Reprogramming factors delivered by PB-TAB are dox-dependent and permit contribution of induced cells to embryonic development, a) Contribution of the induced cell lines to chimaera formation supports the notion that PB-mediated factor delivery results in pluripotent reprogramming. Chimaeras with the strongest contribution of GFP-positive cells are shown beside their corresponding littermates which displayed no GFP signal (WT). Chimaeras from IB were dissected at 12.5dpc, while those from 6C were dissected at 15.5dpc. Scale bars shown are 2mm (IB) and 5mm (6C).
  • iPS-derived fibroblasts from IB and 6C chimaeric embryos respond to dox treatment (24 hours) by reactivating ⁇ geo expression.
  • dox treatment 24 hours
  • few cells stain positive for lacZ, implicating that at a low frequency some transgenes have residual expression.
  • Scale bars are 50OuM.
  • FIG. 12 PB vectors and colony- forming efficiencies using transposition of inducible mouse factors
  • (a) Schematic of the Gateway-compatible PB-CAG transposon vector used to deliver constitutively expressed mouse factors (mFx) by PB transposition. 5'/3'TR: PB 5' and 3' terminal repeats; CAG: pCAGGS promoter; Bl, B2: lambda attBl and attB2 sites; pA: rabbit ⁇ -globin polyadenylation signal
  • tetO dox-inducible promoter
  • IRES internal ribosomal entry signal
  • ⁇ geo lacZ-neo fusion protein
  • pA bovine growth hormone polyadenylation signal
  • Induction cocktails were prepared containing lOOng of each of the four transposons and lOOng transposase plasmid. The dox concentration was varied in the media as indicated. Colonies were counted on the indicated days post-dox treatment. Total colony number per 10cm 2 is indicated on the Y-axis. Transfections were performed and analyzed in triplicate.
  • FIG. 13 Activation of pluripotency markers and characterization of transgene inducibility in PB-TET clonal cell lines,
  • Stable dox-independent PB-TET induced cell lines activate alkaline phosphatase (AP), SSEAl and Nanog, indicative of reprogramming. Representative images from a single cell line (IB) are shown for each marker analysis. Scale bars are lOO ⁇ m.
  • RT-PCR analysis of four stable dox-independent PB-TET-mFx reprogrammed clones reveals additional pluripotency marker gene expression.
  • Rl ES cells and parental rtTA-MEFs serve as positive and negative controls, respectively.
  • FIG. 14 Seamless factor removal from iPS cells using transposase-stimulated PB excision, (a) Schematic of the MKOS (mouse reprogramming factors linked with 2A peptide sequences) containing PB-TET transposon. (b) Genomic integration site of the individual transposons in scBl and scC5 lines. The capital letters represent the flanking genomic sequences while the lower case letters are 5' and 3' transposon sequences. Both integrations occurred in the plus-strand orientation. [SEQ ID NOs 146-149] (c) Doxycycline inducibility of single-copy transposon iPS cell lines; scBl and scC5.
  • Lane 3 ChI l- and Chl ⁇ -specific PCR across the TTAA tetranucleotide site of PB transposon integration. Note that scBl and scC5 are hemizygous for the transposon insertion and the PCR amplifies from the wild type allele. Lanes 4 and 5: Specific PCR for the 5' and 3' junction of transposon insertions, respectively. Lane 6: 3-primer simultaneous PCR amplification from the wild type allele and the transposon-genome junction, (f) RT-PCR analysis of the single transposon induced iPS cell lines and their factor-removed derivatives reveals maintenance of hallmark pluripotency gene expression. Rl ES cells and parental rtTA-MEFs serve as positive and negative controls, respectively. Amplification of the GAPDH housekeeping gene was used as an internal loading control.
  • FIG. 15 Cell lines reprogrammed by PB-mediated factor transposition are pluripotent.
  • FIG. 17 Colony-forming efficiencies using PB transposition of constitutively expressed mouse factors in mouse embryonic fibroblasts, (a) Cocktails were prepared containing 10, 100, or 400ng each of the four transposons, while the transposase plasmid amount was kept constant at lOOng. MEFs were transfected 24hrs after seeding at 1.25x105 cells/well of a 6-well dish (10cm2). Distinct changes in morphology were noted in the fibroblasts within 3 days post-transfection, with noticeable foci forming over d4-6, and the first ES cell-like colonies arising as early as d6. Colonies were counted on the indicated days post-transfection.
  • Diminution of the overall colony number using dox-inducible versus constitutive-expression PB vectors for reprogramming may be the result of fundamental differences between the tetO2 and CAG promoters (such as sensitivity to genomic position effects) or core PB vector design differences (such as transcript length or the effect of IRES function). Transfections were performed and analyzed in triplicate. Error bars represent the standard error. Note that the scale is logarithmic. The panel on the right shows representative alkaline phosphatase staining of the resulting colonies on a 10cm 2 surface 20 days post-transfection.
  • Colonies with abnormal morphology were not able to generate pure ES cell- like cultures upon initial cloning and passage, and were not considered during numeration as described above.
  • the underlying cause of clonal diversity is unknown, but may be directly related to over/underexpression of certain factors reflecting position effects or insertion copy number. Differences may also represent various stages in reprogramming, however no obvious progression through morphological classes was noted.
  • FIG. 18 Reprogramming induced in human embryonic fibroblasts with PB- CAG transposons expressing mouse factors, (a) Typical cell morphology changes and colony formation observed over two weeks in the transfected fibroblast culture and following expansion of a representative clone (#39). (b) Three established PB-CAG lines were selected to demonstrate the activation of endogenous pluripotency marker genes using human-specific (with the exception of DNMT3b) RT-PCR primers. Lines #10 and #39 are alkaline phosphatase (AP) positive while #40 is AP negative.
  • AP alkaline phosphatase
  • CAl and CA2 human ES cell lines 1 served as positive controls, while parental human fibroblasts and mouse fibroblasts served as negative controls.
  • the AP negative cell line #40 appeared to activate only endogenous hTert. Line #10 activated most endogenous genes except for hTert. Line #39 was most similar to the human ES cell controls, (c) Immunohistochemistry revealed the expression of Nanog, SSEA-4 and TRA- 160 in the AP positive line #10.
  • Figure 20 PB transposon copy number and mobilization in reprogrammed cells, (a) Diagnostic Southern analysis of seven clonal PB-TET-mFx induced lines using Bar ⁇ l digestion and a neo probe to determine the number of transposon integrations. The neo probe detects all PB-TET transgene insertions, regardless of the mFx transgene delivered at the insertion site. The estimated copy number for each line is indicated below the image. Note that no fragmented transgene constructs (banding below the predicted lower limit of ⁇ 1.5kb) were detected.
  • Asterisk indicates the neo band resulting from inactivated G418-resistant mouse embryonic fibroblast feeder DNA contamination (this band was not detected in G418-sensitive parental rtT A-MEFs).
  • FIG 22 Differentiation of PB-induced iPS cells into complex tissues,
  • (a) Whole mount immunohistochemistry of the genital ridge of a chimaeric embryo obtained by aggregating IB iPS cells with C57BL/6 eight cell stage embryos. The Vasa and GFP double positive cells are iPS cell-derived germ cells. Scale bars are 40 ⁇ m.
  • (b) PB-TET clones were capable of differentiating in teratoma formation assays, generating tissues derived from all three embryonic germ layers. Sections from clone IB are shown as representatives. Scale bars are 50 ⁇ m.
  • Cells staining positive only for DAPI are fibroblast feeders. Scale bars are 50 ⁇ m.
  • FIG. 25 Immunohistochemistry to detect in vitro differentiated human iPS cell- derivatives positive for markers representing each of the three embryonic germ layers.
  • A- SMA alpha smooth muscle actin
  • vWF Von Willebrand factor
  • GFAP glial fibrillary acidic protein
  • AFP alpha-fetoprotein. Scale bars are 50 ⁇ m.
  • a reprogrammed cell includes reference to one or more reprogrammed cells and equivalents thereof, and so forth.
  • reprogramming refers to changing the differentiation ability of a somatic cell including a somatic stem cell (i.e. making the cell pluripotent or increasing the degree of pluripotency - this is often now termed "induced pluripotency"), or being able to change the fate of the cell, for example changing a skin cell to a pancreatic cell. This may be achieved, for example, by expressing multiple genes which are specific for a particular cell type, such as pancreatic specific genes, in a cell of another cell type.
  • reprogrammed cell refers to a somatic cell that has undergone reprogramming or de-differentiation in accordance with a method of the invention.
  • a reprogrammed cell may be subsequently induced to re-differentiate.
  • a reprogrammed cell may express one or more cell specific markers, morphology and/or biological function that are not characteristic of the cell prior to reprogramming, de-differentiation or re- differentiation.
  • the reprogrammed cell expresses an embryonic stem cell marker not expressed in the somatic cell, in particular Nanog, Daxl, Eras, Fbxo, FoxD3, Rexl and/or Zfp296.
  • differentiation refers to the development process of lineage commitment.
  • a “lineage” refers to a pathway of cellular development in which progenitor cells undergo progressive physiological changes to become a particular cell type with a characteristic function such as neural, muscle, endothelial or pancreatic cells. Differentiation can be determined by assaying for the presence of, or increases in one or more cell specific markers compared to their expression in corresponding undifferentiated control cells.
  • somatic cell refers to a cell that generally is obtained from a tissue of a subject generally at a post-natal stage of development (e.g. infant, child, adult).
  • a somatic cell may be a differentiated somatic cell or a unipotent or multipotent somatic stem or progenitor cells.
  • the somatic cells are embryonic fibroblast cells, more particularly human fibroblast cells.
  • non-viral based vector system refers to a system comprising non-viral sequence(s) that are capable of expressing proteins necessary for reprogramming somatic cells.
  • the system may include promoters, excision or cleaving sequences, an IRES site, a polyadenylation signal, an origin of replication and/or an antibiotic selection marker gene.
  • the system may include promoters, excision sequences etc. of viral origin but viral sequences are generally excised from the reprogrammed cell or they are selected so that they cannot be re-activated when the cells differentiate.
  • reprogramming factor refers to a factor which can be used to induce reprogramming of somatic cells in particular induce reprogramming of differentiated cells to establish a pluripotent stem cell, more particularly an inducible pluripotent stem cell having similar pluripotency and growing ability to those of an embryonic stem cell.
  • a reprogramming factor may be produced from a single copy or multiple copies of reprogramming sequences or genes or in the form of a fusion protein.
  • a "fusion protein” comprises all or part (preferably biologically active) of a reprogramming sequence operably linked to a heterologous polypeptide (i.e., a polypeptide other than the reprogramming sequence).
  • the term "operably linked" is intended to indicate that reprogramming sequence and the heterologous polypeptide are fused in-frame to each other.
  • the heterologous polypeptide can be fused to the N-terminus or C-terminus of the reprogramming sequence.
  • heterologous polypeptides that can be incorporated in a fusion protein include green fluorescence protein (GFP) or glutathione-S-transferase (GST). Fusion proteins can be produced by standard recombinant DNA techniques.
  • a "reprogramming sequence(s)" refers to a sequence or gene encoding a reprogramming factor.
  • the term refers to a sequence encoding a reprogramming factor that can induce reprogramming of differentiated cells to establish a pluripotent stem cell, in particular an inducible pluripotent stem cell, having similar pluripotency and growing ability to those of an embryonic stem cell.
  • a reprogramming sequence may be introduced into a vector system and in particular a transposon region using methods known to a skilled artisan (see, for example, the methods described in the Examples herein).
  • Oct family gene examples include, for example, Oct4, OctlA, Oct6, and the like.
  • the use of Oct4 is preferred.
  • Oct4 is a transcription factor belonging to the POU family, and is reported as a marker of undifferentiated cells (K. Okamoto et al., Cell, 60, pp461-72, 1990).
  • Oct4 is also reported to participate in the maintenance of pluripotency (J. Nicholas et al., Cell, 95, pp379-91, 1998).
  • KIf family gene examples include KIf 1, Klf2, Klf4, Klf5 and the like. The use of Kif4 is preferred.
  • Klf4 (Kruppel like factor -4) is reported as a tumor repressing factor (A.M.
  • c-Myc is a transcription control factor involved in differentiation and proliferation of cells (S. Adhikary, M. Eilers, Nat. Rev. MoI. Cell Biol. 6, pp.635-45, 2005), and is also reported to be involved in the maintenance of pluripotency (P. Cartwright et al., Development, 132, pp.885-96, 2005).
  • Sox family gene examples include, for example, Soxl, Sox3, Sox7, Soxl5, Soxl7 and Soxl8, and a preferred example includes Sox2.
  • Sox2 expressed in an early development process, is a gene encoding a transcription factor (A.A. Avilion et al., Genes Dev., 17, pp.126-40, 2003). Nanog and Lin28 have been proposed as other inducing factors which may be used (Yu J, et al. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells, Science 2007 Nov 23;318(5854)).
  • NCBI accession numbers of representative genes of families that can be employed in the present invention are listed in the following Table: Accession Numbers of Representative Reprogramming Sequences/Genes
  • a reprogramming factor may be replaced with a cytokine or the vector system may include a cytokine.
  • the gene product may be replaced with a cytokine.
  • cytokines that may be used in the present invention include without limitation basic fibroblast growth factor, stem cell factor or the like.
  • telomere shortening sequences or genes may be replaced by small molecules such as BIX as shown by Y. Shi, et. al. (Y. Shi, et. al. Cell Stem Cell, 2, pp. 525-528, 2008).
  • the present invention is not however limited to use of the aforementioned mouse or human genes; other mammalian homologues to these genes are known in the databases and can easily be used, as will be appreciated by the skilled reader. For example, if one wished to reprogram a rat somatic cell or somatic cell progenitor, it may be preferred to use a rat homologue of the aforementioned genes/proteins.
  • mutant genes including substitutions, insertions, and/or deletions of several (for example, 1 to 10, preferably 1 to 6, more preferably 1 to 4, still more preferably 1 to 3, and most preferably 1 or 2) amino acids and having similar function to that of the wild-type gene/protein can also be used.
  • a gene product of c-Myc a stable type product (T58A) may be used as well as the wild-type product. All this is well within the understanding of the skilled reader and does not depart from the scope of the present invention.
  • preferred combinations of reprogramming factors comprise one or more gene products of each of the following families: Oct family, KIf family and Sox family.
  • preferred combinations of reprogramming factors can include at least a gene product of an Oct family member and a Sox family member, optionally with a KIf and/or Myc family member.
  • preferred combinations of reprogramming factors comprise one or more gene products of each of an Oct gene family, a KIf gene family and a Myc gene family.
  • preferred combinations of reprogramming factors can include one or more gene products of each of an Oct gene family member, a Sox gene family, a KIf gene family and a Myc gene family.
  • the reprogramming factors include products of the following genes: Oct 3/4, Klf4, and Sox2.
  • the reprogramming factors are one or more Yamanaka factors.
  • the reprogramming factors include products of the following genes: c-Myc, Oct 3/4, Klf4, and Sox2.
  • transposon refers to a short piece of nucleic acid bounded by inverted repeat sequences. Active transposons encode enzymes that facilitate the insertion of the nucleic acid into DNA sequences.
  • a vector system with a transposon may comprise regulatory elements including without limitation promoters, enhancers, or polyadenylation signals.
  • a transposon may also comprise a sequence encoding a transposase. Any suitable transposon can be used in the present invention including a DNA transposon or retrotransposon sequence or a sequence substantially similar to that of any known retrotransposon or DNA transposon.
  • the transposon is a DNA transposon including without limitation those substantially similar to the TcI family of DNA transposons (Plasterk, R.
  • DNA transposons comprise inverted or direct repeat sequences flanking the sequence to be integrated, sequences encoding a transposase which catalyzes the excision of the transposon from its original location and promotes its reintegration elsewhere, and a promoter sequence operably linked to the transposase encoding sequence.
  • transposons include those with sequences substantially similar to naturally occurring transposons such as Mariner (Gueiros-Filho and Beverly, 1997, Science 276(5319): 1716- 9) as well as those substantially similar to natural sequences such as Sleeping Beauty (Ivies, Z. et al., 1997, Cell. 91(4):501-10).
  • the transposon is a piggyBac (PB) transposable element from the cabbage looper moth, Trichoplusia ni [1; Cary et al., Virology, Volume 161, 8-17, 1989].
  • the transposon is a PB transposable element with a CAG promoter (CMV enhancer and ⁇ -actin promoter), optionally terminated with a ⁇ -globin polyadenylation sequence.
  • the transposon is a PB transposable element with an inducible promoter, for example a tetC ⁇ tetracycline/doxycycline inducible promoter.
  • the transposon is a PB-CAG or PB-TET described in the examples herein.
  • a transposon region may be in combination with a helper plasmid which expresses a transposase enzyme.
  • expression of the transposase enzyme is provided in trans from a helper plasmid.
  • a transposon region comprises one or more reprogramming sequences, i.e. a sequence encoding a reprogramming factor which is integrated into the cell along with the transposon during the process of transposon integration.
  • the reprogramming sequence can also be operably linked to a promoter, polyadenylation signal, and other sequences in order to facilitate its expression in the host cell.
  • "Means to enable excision of the reprogramming sequences" refers to sequences that may be included in a non-viral based vector system of the invention which are capable of excising the reprogramming sequences from a reprogrammed cell. Suitable means to enable excision of the reprogramming sequences include site-specific recombination systems such as the cre/lox P system.
  • the transposon includes excision or cleaving sequences which facilitate the excision of the transposon from a reprogrammed cell.
  • cleaving sequences include sequences encoding the self cleaving peptides including 2A peptides from Aphthoviruses , a typical example of which is foot and mouth virus, or 2A like sequences from other viruses in particular Picornaviridae like the Equine rhinitis A virus as well as the insect Thosea asigna virus, and Porcine teschovirus- 1 (Donnelly et. al.
  • a non-viral based vector system comprises a transposon region and an excision or cleaving sequence comprising one or more transcription factor open reading frames and/or self-cleaving sequences such as 2A peptide sequences.
  • an excision sequence comprises a MKOS sequence (c-Myc, Klf4, Oct4 and Sox2 ORFs linked with 2A peptide sequences).
  • subject refers to a vertebrate, in particular a mammal.
  • the term refers to a human.
  • the term also includes domestic animals bred for food, sport, or as pets, including horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo animals.
  • the methods herein for use on subjects contemplate prophylactic as well as curative use. Typical subjects for treatment include persons susceptible to, suffering from or that have suffered a condition, disorder, disease and/or injury.
  • Compositions, Methods and Kits The invention features compositions, methods and kits that are useful for reprogramming somatic cells without the disadvantages of viral transfection systems.
  • Somatic cells may be obtained from a number of sources such as cell lines, biopsies or autopsies using conventional methods.
  • the somatic cells are allogenic cells or autologous cells isolated by biopsy from a subject.
  • Cells may also be obtained from other donors of the same species using techniques known to those skilled in the art.
  • a tissue or organ can be mechanically disaggregated (e.g. using grinders, blenders, sieves, homogenizers, pressure cells or sonicators), or treated with digestive enzymes (e.g.
  • Cell suspensions can be fractionated into subpopulations using standard techniques such as positive selection or negative selection. Selection techniques may utilize for example fluorescence-activated cell sorting, electrophoresis, cell agglutination, freeze-thaw procedures, differential adherence properties of cells, filtration, conventional and zonal centrifugation, unit gravity separation, and countercurrent distribution. [See for example, Freshney, Culture of Animal Cells, A Manual of Basic Techniques, 2d Ed, A.R. Liss, Inc., New York, Chapters 11 and 12, 1987.]
  • a non-viral based vector system that can be used in the present invention to reprogram somatic cells may comprise an integrating vector.
  • the vector includes means to enable excision of a major portion (e.g. at least the genes themselves) of the vector from a host cell's genome, following cell reprogramming.
  • Such means may be a site-specific recombination system such as the cre/lox P system.
  • the multiple protein expression vector system described herein may be applied to episomal vector systems such as transposon based systems and Epstein-Barr virus based episomal vector, pCEP4 (Invitrogen), which can avoid integration of the vector into genome.
  • the non-viral vector of the present invention is a plasmid vector which may or may not integrate into a host's genome. If an integrating vector is used, the vector may comprise sequences specifically designed to facilitate integration and may for example comprise sequences designed to cause integration at a specific locus or loci.
  • the expression of the genes encoding the reprogramming factors is generally under the control of a promoter, in particular a single promoter.
  • the promoter may be a ubiquitous promoter rather than inducible promoter, but an inducible promoter may be used in certain aspects of the invention.
  • a particularly preferred promoter is the CAG promoter (Niwa, H., Yamamarua, K. & Miyazaki, K. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193-9 (1991)).
  • the CAG promoter is a composite promoter that combines the human cytomegalovirus immediate-early promoter and a modified chicken beta-actin promoter and first intron.
  • vectors of the present invention may include other features that are not essential, but nevertheless may be desirable/advantageous.
  • other preferred features vectors of the present invention may include one or more of the following an IRES (internal ribosome entry site), antibiotic resistance marker gene, polyadenylation signal sequence, origin of replication site and one or more site-specific recombination sites.
  • IRES internal ribosome entry site
  • antibiotic resistance marker gene antibiotic resistance marker gene
  • polyadenylation signal sequence origin of replication site
  • origin of replication site origin of replication site and one or more site-specific recombination sites.
  • An IRES site and/or polyadenylation signal sequence may facilitate expression and/or processing of two or more reprogramming sequences or genes.
  • An origin of replication site and/or antibiotic selection marker gene may facilitate production and/or maintenance of the vector in an in vitro culture prior to its use in transfecting somatic or somatic cell precursors.
  • Site-specific recombination sites can assist with removal of the vector sequence, especially two or more reprogramming genes, once reprogramming has occurred.
  • a variety of site-specific recombination systems are well known to those skilled in the art, including Cre/Lox, Dre/rox, Att/ ⁇ integrase, frt/Flp, gamma delta resolvase, Tn3 resolvase and 0C231 integrase (see Gover et al, 2005, to which the skilled reader is directed).
  • the vector Prior to transfection into the somatic cell or somatic stem cell, the vector may be linearised by, for example, cleavage using a restriction endonuclease.
  • Preferred vectors in accordance with the present invention are identified in Figures 4, 10, 12, 14 and 20.
  • Transfection of the non-viral vector may be carried out by any suitable means including electroporation, liposome mediated transfection, dendrimers, calcium phosphate mediated transfection and the like, all well known to those skilled in the art (see for example Nature Methods 2, 875 - 883 (205)).
  • a particularly preferred method is nucleofection developed by Amaxa which is now part of Lonza Group, (http ://www. amaxa. com/products/technology/) (Efficient transfection method for primary cells. Hamm A, Krott N, Breibach I, Blindt R and Bosserhoff AK.
  • a transposon region or multiple transposon regions may be produced and introduced into a somatic cell using methods known to those skilled in the art.
  • one or more reprogramming factors may be placed via cloning (e.g. Gateway cloning) into a transposon (e.g. PB transposon plasmid) and transposon regions containing one or more of the factors may be delivered by standard transfection techniques into somatic cells. If the transposon region does not contain a transposase sequence the cells are transfected in conjunction with a transposase vector or plasmid.
  • the somatic cells are grown under suitable conditions, for example, embryonic stem cell media.
  • Reprogrammed cells generated using the methods of the invention may be identified using methods known to a skilled artisan including methods based on cell morphology or expression of cell markers, gene products, antigens, nucleic acids (DNA, cDNA, RNA, antisense RNA, microRNA) and portions thereof.
  • iPS cells may be identified based on morphology and/or expression of ES cell markers such as SSEAl, ES cell pluripotency markers (e.g.
  • a reprogrammed cell can also be identified by down-regulation of markers characteristic of the somatic cells from which the reprogrammed cell is induced.
  • Thyl a fibroblast cell marker
  • a reprogrammed cell produced by a method of the invention may be cultured with at least one agent that induces differentiation i.e. a differentiation agent.
  • the agent induces differentiation of reprogrammed cells to cardiomyocytes and is for example one or more of LIF, BMP-2, retinoic acid, trans-retinoic acid, dexamethasone, insulin and indomethacin.
  • the agent induces differentiation of reprogrammed cells into endothelial cells and is for example fibronectin and/or fetal bovine serum (e.g. 10% fetal bovine serum).
  • the agent induces differentiation of reprogrammed cells into neuronal cells and is for example all-trans-retinoic acid.
  • the agent induces differentiation of reprogrammed cells into adipocytes and is for example retinoic acid, dexamethasone, insulin, and indomethacin.
  • the invention thus provides cells differentiated or derived from reprogrammed cells of the invention. Differentiated cells can be used to prepare a cDNA library relatively uncontaminated with cDNA preferentially expressed in cells from other lineages, and they can be used to prepare antibodies that are specific for particular markers of non-hematopoietic cells.
  • non-viral transfection with a single multiprotein expression vector comprising the coding sequences of c-Myc, KIf 4, Oct4 and Sox2 linked with intervening 2A peptides, in which peptide bond formation is impaired [HA]
  • a single multiprotein expression vector comprising the coding sequences of c-Myc, KIf 4, Oct4 and Sox2 linked with intervening 2A peptides, in which peptide bond formation is impaired [HA]
  • This one vector system allows the generation of iPS cells by a single vector integration and subsequently complete elimination of the exogenous reprogramming factors by Cre-mediated excision.
  • These non-viral iPS cells show robust expression of pluripotent markers and genuine pluripotency was confirmed by in vitro differentiation assays and formation of adult chimeric mice.
  • This non-viral single vector system minimizes genome modification and eliminates the unpredictable reactivation of reprogramming factors, providing iPS cells more applicable to regenerative medicine, reliable drug screening and establishment of trustworthy disease models
  • murine and human embryonic fibroblasts were reprogrammed using tetracycline inducible reprogramming factors delivered by transposition.
  • the stable iPS cells generated with this transposon vector approach expressed hallmark pluripotency markers and performed in a series of rigorous differentiation assays. Additionally, the traceless removal of the reprogramming factors joined with excision sequences and delivered by a single transposon was demonstrated.
  • Cells described herein, in particular reprogrammed cells can be modified by introducing mutations into genes in the cells (or the cells from which they are obtained) or by introducing transgenes into the cells. Insertion or deletion mutations may be introduced in a cell using standard techniques.
  • a transgene may be introduced into cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated trans fection, lipofection, electroporation, or microinjection. Suitable methods for transforming and transfecting cells can be found in Sambrook et al. (Sambrook, Fritsch, & Maniatis, DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed.
  • a transgene may be introduced into cells using an appropriate expression vector including but not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses). Transfection is easily and efficiently obtained using standard methods including culturing the cells on a monolayer of virus-producing cells (Van der Putten, 1985, Proc Natl Acad Sci U S A.;82:6148-52; and Stewart et al. 1987, EMBO J. 6:383-388).
  • a selection marker gene may be integrated into cells described herein.
  • a gene which encodes a protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or a fluorescent protein marker may be integrated into the cells.
  • fluorescent protein markers are the Green Fluorescent Protein (GFP) from the jellyfish A. victoria, or a variant thereof that retains its fluorescent properties when expressed in vertebrate cells. (For example, the EGFP commercially available from Clontech Palo Alto, CA).
  • Another aspect of the present invention relates to genetically engineering the cells described herein in such a manner that they or cells derived or differentiated therefrom produce, in vitro or in vivo, polypeptides, hormones and proteins not normally produced in the cells in biologically significant amounts, or produced in small amounts but in situations in which regulatory expression would lead to a therapeutic benefit.
  • the cells could be engineered with a gene that expresses a molecule that specifically inhibits bone resorption, but does not otherwise interfere with osteoclasts binding to bone, or the cells could be engineered with a gene that expresses insulin at levels compatible with normal injected doses.
  • the cells could be modified such that a protein normally expressed will be expressed at much lower levels. These products would then be secreted into the surrounding media or purified from the cells.
  • the cells formed in this way can serve as continuous short term or long term production systems of the expressed substance.
  • reprogrammed cells can be modified with genetic material of interest.
  • the modified cells can be cultured in vitro under suitable conditions so that they are able to express the product of the gene expression or secrete the expression product.
  • These modified cells can be administered to a target tissue where the expressed product will have a beneficial effect.
  • reprogrammed cells may be engineered to express a gene of interest whose expression promotes survival, proliferation, differentiation, maintenance of a cellular phenotype, or otherwise enhances the engraftment of the cell.
  • Expression of a gene of interest in reprogrammed cells of the invention may also promote repair or regeneration of a tissue or organ having a deficiency in cell number or excess cell death.
  • proteins that may be expressed in reprogrammed cells include without limitation, growth factors such as BMP, FGF, VEGF, TGF ⁇ , PD-ECGF, PDGF, TNF, HGF, IGF, erythropoietin, CSF, M-CSF, and fragments or variants thereof.
  • Reprogrammed cells may also comprise a component of the extracellular matrix, such as collagen and elastin; proteins having specialized functions such as fibrillin, fibronectin, and laminin, and proteoglycans such as hyaluronan, chonodroitin sulphate, dermatan sulphate, heparin sulphate, heparin, keratin sulphate and aggrecan.
  • the reprogrammed cells and cells differentiated or derived therefrom can be used in a variety of methods (e.g. transplantation) and they have numerous uses in the field of medicine.
  • Transplantation or grafting can include the steps of producing reprogrammed cells or cells differentiated or derived therefrom and transferring cells in the preparation into a patient, in particular a mammal. Transplantation can involve transferring the cells into a patient by injection of a cell suspension into the patient, surgical implantation of a cell mass into a tissue or organ of the patient, or perfusion of a tissue or organ with a cell suspension.
  • the route of transferring the cells may be determined by the requirement for the cells to reside in a particular tissue or organ and by the ability of the cells to find and be retained by the desired target tissue or organ. Where the transplanted cells are to reside in a particular location, they can be surgically placed into a tissue or organ or simply injected into the bloodstream if the cells have the capability to migrate to the desired target organ.
  • the invention may be used for autografting (cells from an individual are used in the same individual), allografting cells (cells from one individual are used in another individual) and xenografting (transplantation from one species to another).
  • the cells described herein may be used in autologous or allogenic transplantation procedures to improve a cell deficit or to repair tissue.
  • the newly created reprogrammed cells and cells differentiated or derived therefrom can be used in both cell therapies and gene therapies aimed at alleviating disorders and diseases.
  • the invention obviates the need for human tissue to be used in various medical and research applications.
  • the cell therapy approach involves the use of transplantation of the newly created cells as a treatment for injuries and diseases.
  • the steps in this application include: (a) allowing reprogrammed cells or cells derived or differentiated therefrom to form functional connections either before or after a step involving transplantation of the cells.
  • the gene therapy approach also involves reprogrammed cells or cells derived or differentiated therefrom, however, the newly created cells are transfected with an appropriate vector containing a cDNA for a desired protein, followed by a step where the modified cells are transplanted.
  • reprogrammed cells, or cells or tissues differentiated from the cells can be transplanted in, or grafted to, a patient in need.
  • the cells can be used to replace particular cells in a patient in a cell therapy approach which will be useful in the treatment of tissue injury and diseases.
  • These cells can be also used as vehicles for the delivery of specific gene products to a patient.
  • One example of how these newly created cells or cell differentiated therefrom can be used in a gene therapy method is in treating the effects of Parkinson's disease.
  • tyrosine hydrolase a key enzyme in dopamine synthesis
  • tyrosine hydrolase may be delivered to a patient via the transplantation of reprogrammed cells that can differentiate into neuronal cells, or transplantation of neuronal cells differentiated from the reprogrammed cells, which have been transfected with a vector suitable for the expression of tyrosine hydrolase.
  • the invention provides methods of using reprogrammed cells of aspects of the invention to repair or regenerate diseased or damaged tissues and organs.
  • the reprogrammed cells are used to increase the number of cells in a tissue or organ having a deficiency in cell number or an excess in cell death.
  • Methods of the invention may also stabilize a damaged tissue or organ in a subject. Methods of the invention may be carried out in vitro, in vivo or ex vivo and they may include prophylactic treatment.
  • the invention contemplates methods of treating a disease and/or disorder or symptoms thereof characterized by a deficiency in cell number or excess cell death comprising administering a therapeutically effective amount of reprogrammed cells or pharmaceutical compositions of aspects of the invention to a subject in need thereof, in particular to a mammal such as a human.
  • the invention provides a method of treating a subject suffering from or susceptible to a disease characterized by a deficiency in cell number or excess cell death or symptom or disorder thereof.
  • the disease, disorder or condition is a heart attack, heart failure, stroke, a neurodegenerative disease such as Parkinson's disease or Alzheimer's disease, diabetes, an inflammatory disease such as arthritis, or cancer.
  • the invention provides a method for stem cell therapy comprising: (a) isolating and collecting somatic cells from a patient; (b) inducing reprogramming of the somatic cells by contacting or transfecting the cells with a vector system of the invention; (c) inducing differentiation of the reprogrammed cells; and (d) transplanting the differentiated cells into the patient or another patient.
  • the treatment methods of the invention may be administered to subjects (e.g. humans) suffering from, susceptible to, or at risk for a disease characterized by a deficiency in cells or increase in cell death, disorder or symptom thereof.
  • a subject at risk may be identified by any objective or subjective determination of a subject or health care professional using a diagnostic method or test including without limitation genetic tests, biological markers, family history and the like.
  • Therapeutic efficacy may be determined by measuring, for example, the biological function of the treated tissue or organ (see for example, the Textbook of Medical Physiology, Tenth Edition, Guyton et al, W.B. Saunders Co., 2000).
  • a method of the invention may increase the biological function of a tissue or organ by at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, or 200%.
  • Therapeutic efficacy can also be measured by assaying the increase in cell number in the treated tissue or organ compared to a control that did not receive treatment.
  • Cell number may be increased by at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, or 200% relative to a control.
  • Cell proliferation can be assayed using methods known to a skilled artisan (see for example, Bonifacino et al, Current Protocols in Cell Biology Loose-leaf, John Wiley and Sons, Inc., San Francisco, Calif).
  • compositions comprising reprogrammed cells of the invention or cells derived or differentiated therefrom and a pharmaceutically acceptable carrier, excipient or vehicle.
  • the compositions include, albeit not exclusively, the reprogrammed cells in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • a pharmaceutical composition may be provided as a sterile liquid preparation (e.g. isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions).
  • compositions herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective amount of the reprogrammed cells are combined in a mixture with a pharmaceutically acceptable vehicle.
  • Suitable vehicles are described, for example, in the standard texts Remington: The Science and Practice of Pharmacy (21 st Edition. 2005, University of the Sciences in Philadelphia (Editor), Mack Publishing Company), and in The United States Pharmacopeia: The National Formulary (USP 24 NF 19) published in 1999.
  • Reprogrammed cells of the invention may be administered directly or indirectly to a tissue or organ, and preferably engraft within the tissue or organ.
  • the cells of the invention may be provided to a site where an increase in the number of cells is desired, for example, due to disease, damage, injury or excess cell death.
  • Reprogrammed cells can also be indirectly administered to a tissue or organ by administration into the circulatory system. Cells may be delivered to a portion of the circulatory system that supplies the target tissue or organ. Administration may be autologous or heterologous. Cells obtained from one subject may be administered to the same subject or a different compatible subject. Agents including expansion and/or differentiation agents may be provided prior to, during or after administration of reprogrammed cells to increase, maintain, or enhance production or differentiation of the cells in vivo.
  • Cells can be administered to subjects using a variety of means apparent to those of skill in the art. Suitable methods include injection of the cells into a target site in a subject. Cells may be inserted into a delivery device to facilitate injection or implantation into the subjects. Examples of delivery devices include tubes, e.g., catheters, for injecting cells and fluids into the body of a subject. Cells can be prepared for delivery in a variety of different forms. For example, the cells may be suspended in a solution or gel, or mixed with a pharmaceutically acceptable carrier, excipient, or diluent in which the cells remain viable. Pharmaceutically acceptable carriers, excipients, and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media.
  • the use of such carriers and diluents is well known in the art.
  • the solution is generally sterile, and will often be isotonic.
  • a solution of cells is preferably selected that is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • Modes of administration of cells include without limitation systemic intracardiac, intracoronary, intravenous, intradermal, or intra-arterial injection and injection directly into the tissue or organ at the intended site of activity, or in proximity to the site of activity.
  • a cell preparation can be administered by any convenient route, for example by infusion or bolus injection and can be administered together with other biologically active agents. Administration in some aspects is preferably systemic.
  • reprogrammed cells and compositions of the invention are administered by catheter administration, systemic injection, localized injection, intravenous injection, intramuscular, intracardiac injection or parenteral administration.
  • a pharmaceutical composition is formulated in a unit dosage injectable form (solution, suspension, or emulsion).
  • Kits may include instructions for the treatment regime or assay, reagents, equipment (test tubes, needles, syringes, etc.) and standards for calibrating or conducting the treatment or assay.
  • the instructions in a kit may be directed to suitable operation parameters in the form of a label or a separate insert.
  • a kit may additionally comprise standard or control information to compare the results of a test sample.
  • Kits for transplantation of reprogrammed cells or cells derived or differentiated therefrom may also be supplied comprising a flask with medium and the cells or compositions of the invention.
  • Kits are also provided for carrying out methods of the invention to generate reprogrammed cells of the invention.
  • Such kits may comprise non-viral based vector systems of the invention, and optionally somatic cells (e.g. control mouse, canine or human fibroblasts), transfection reagents for the vector system, and/or characterization reagents such as antibodies to the pluripotent markers and the like.
  • a kit may also contain culture media products to carry out the reprogramming methods of the invention.
  • the kit comprises one or more transposon region and transposon expression plasmids.
  • the kit comprises a transposon region or components thereof in a package.
  • Kits are also contemplated for rational drug design comprising reprogrammed cells and cells derived or differentiated therefrom obtained by a process of the invention. Such kits may comprise reprogrammed cells and instructions for their use in toxicity assays or instructions for their use in an absorption assay.
  • Kits of the invention preferably comprise the vectors identified in Figures 4, 10, 12, 14 and 20 and the Examples.
  • the invention also contemplates methods for identifying modulators which enhance or inhibit reprogramming of somatic cells. Screening methods may involve identification of an agent that modulates the generation of reprogrammed cells, or increases proliferation or survival of reprogrammed cells, or progenitors or cells derived therefrom. These methods may involve contacting somatic cells with a non-viral based vector of the invention and a test agent and quantitating the number of reprogrammed cells produced, and comparing to an untreated control. An increase in the number of reprogrammed cells relative to a control may be indicative of a desired activity. These methods may be used to evaluate a physiological function or toxicity of a test compound.
  • iPS cells from which one or more of the reprogramming factors are removed by transient PB transduction can be generated. Such cells can be used for screening of small molecules/siRNA libraries, for example, in order to identify molecules which could replace the one or more reprogramming factors. Differentiated cells generated from the one or more reprogramming factor(s) removed from the iPS cells, may be cultured and then exposed to the small molecules/siRNA together with doxycycline, to induce the remaining factor(s). Efficiency of reprogramming can be measured by alkaline phosphate staining, immunofluorescense, or morphological criteria. The following non-limiting examples are illustrative of the present invention:
  • the fragments containing c-Myc, Oct4 and Sox2 coding region were amplified with the following conditions, 95°C for 3 min xl cycle, 95°C for 20 sec - 55°C for 20 sec - 72°C for 1 min x 5 cycles, 95°C for 20 sec - 68°C for 20 sec - 72°C for 1 min x 25 cycles, 72°C for 5 min xl cycle.
  • the fragment containing Klf4 coding region was amplified with 95°C for 3 min xl cycle, 95°C for 20 sec - 46°C for 20 sec - 72°C for 1 min x 5 cycles, 95°C for 20 sec - 70 0 C for 20 sec - 72°C for 1 min x 30 cycles, 72°C for 5 min xl cycle, c- Myc-F2A-Klf4-T2A, Oct4-E2A-Sox2 fragments are amplified by PCR using their flanking primers used in the first PCR after annealing and extension take advantage of complimentary 2A peptide regions of the first PCR products.
  • c-Myc-F2A-Klf4-T2A, Oct4- E2A-Sox2 fragments were amplified using EcoRI Koz 5'-Myc and Klf4-3' GSG T2A Xho primers, Xho 5'-Oct4 and Sox2-3' EcoRI Xho primers, respectively, under a PCR condition, 95°C 20 sec - 70 0 C 20 sec - 72°C 90 sec x30 cycles, 72°C for 5 min xl cycle.
  • pCAGMKOSiE is constructed by inserting the MKOS fragment into EcoRI site of pCIG2, which is generated by replacing CMV promoter of pIRES2-EGFP (BD biosciences Clontech) with CAG promoter, ires fragment was amplified by NotlRES, mOrangeIRES primers using pIRES2-EGFP as a template (95°C for 3 min x 1 cycle, 95°C 20 sec - 50 0 C for 20 sec - 72°C for 30sec x5 cycles, 95°C 20 sec - 65°C for 20 sec - 72°C for 30sec xlO cycles, 72°C for 5 min xl cycle).
  • mOrange fragment was amplified by IRESmOrange, XbaBamloxPmOrange primers using pmOrange (clontech) as a template using the same PCR cycles as the ires fragment, ires mOrange loxP fragment (imOloxP) was amplified using these fragments and the flanking primers at 95°C for 3 min x 1 cycle, 95°C 20 sec - 70 0 C for 20 sec - 72°C for 30sec x30 cycles without primers, and then subsequently 95°C 20 sec - 72°C for 45 sec xlO cycles, 72°C for 5 min xl cycle with NotlRES and Xba Bam loxP mOrange primers, and subcloned into pTOPO-bluntll.
  • MKOS was inserted into EcoRI site of pcDNA3 (Invitrogen), and then one loxP site made by annealing KpnBamloxPBamX F/R oligos, and imOloxP was inserted before c-Myc and after Sox2, respectively (pCMV2LMKOSimO).
  • pCAG2LMKOSimO is constructed by transferring a BamHI-BamHI fragment containing MKOSimO flanked by 21oxP sites from pCMV2LMKOSimO into pCAGDNA3 vector, which is made by replacing its CMV promoter with CAG promoter.
  • MEFs from 13.5-14.5 dpc 129 mice embryos were generated following a standard method.
  • MEFs from C57BL/6 strain TNG mice [19A] (TNG MEFs) were kindly provided by I. Chambers.
  • MEFs and HEK293 cells were cultured in GMEM containing 10% FCS, penicillin/streptomycin, L-glutamine, ⁇ -mercaptoethnol and nonessential amino acids.
  • Leukemia inhibiting factor (LIF) 1000 U/ml was added for iPS cell derivation and maintenance.
  • ES cell-like colonies derived from 129 MEFs were picked 4 weeks after nucleofection, trypsinised with 0.25% Trypsin, 0.1% EDTA in PBS, and seeded on irradiated MEF or directly on gelatin in 4 well plates as the first passage.
  • a wild type ES cell line, E14Tg2a, and established iPS cells were passaged on gelatin-coated dishes every 2-3 days.
  • Cell lines, imO2Ec3, imO3Ec5 and imO7Ec3 were generated by excising the reprogramming cassette from imO2El, imO3El and imO7El which has random integration of EGFP cassette under CAG promoter.
  • Embryoid body formation and neural differentiation was performed used for RT-PCR and immunofluorescence as described before [19, 22A]. Chimeras were produced by microinjection into C57BL/6 blastocysts Immunoblotting
  • MEF Nucleofector Kit 2 (Amaxa) and program T-20 was used for Nucleofection following manufacturer's instruction.
  • 2xlO 6 MEFs were transfected with 2-10 ⁇ g of linearized DNA, and 10 5 cells were seeded in 6 well plates on either irradiated MEFs or gelatin in the presence of LIF. Forty-eight hours later living cells were harvested from one of gelatin wells, and after counting cell number mOrange positive population was measured by flow cytometry (CyAn ADP, Dako). mOrange positive cell numbers per well were estimated using the results in each experiment as shown in Table 1. The rest of the wells were kept for 4 weeks changing medium every 2-3 days.
  • Nanog-GFP positive colonies from TNG MEFs were picked and seeded on irradiated MEFs after trypsinization. Colonies from 129 MEFs were fixed with 4% PFA and stained with anti-Nanog antibody (Abeam) followed by Alexa Fluor 488 conjugated anti-rabbit IgG antibody (Molecular Probes).
  • Genomic DNA extracted from imOl, imO3, and imO7 was digested with AIuI, Spel or Taql. After self-ligation for 18 hours at 16°C, the DNA was used as PCR templates for primers in Table 5. Amplified fragments were purified, cloned into pCRII- TOPO vector (Invitrogen) and sequenced. Integration site of imOl and imO7 was identified with invAluF/invAluR2 primers in AIuI digested genome and Spel digested genome, respectively. Integration site of imO3 was identified with invAluF/invAluRl primers in AIuI digested genome. Inv3TaqFl/inv3TaqR primers identified concatemer in Taql digested imOl and imO3 genome. The integration site and concatemer were verified using specific primers listed in Table 5. Reprogramming cassette excision
  • PD 173074 Cells were cultured in the presence and absence of 100 ng/ml PD 173074 (Sigma) for 24 hours before Cre transfection with Lipofectamine2000 (Invitrogen). Forty-eight hours after Cre transfection cells were harvested and seeded at 10 3 cells per 10 cm dishes with gelatin in the presence or absence of PD 173074. Undifferentiated mOrange negative colonies were marked 5 days later and morphology of the colonies was monitored to day 9. Colonies were picked between day 12-14, propagated in 96 well plates, and reprogramming cassette excision in each clone was verified by genomic PCR using CAG F, ires mOrange and BGH R primers ( Figure 8b, c, Primer sequences are in Table 5).
  • IVS internal ribosomal entry site
  • F2A foot-and-mouth disease virus
  • the F2A sequence is proposed to modify the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNA(Gly) ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows a ribosomal 'skip' from one codon to the next without the formation of a peptide bond [17A].
  • Nanog-EGFP positive colonies from TNG MEFs and Nanog positive colonies from 129 MEFs at day 28 is summarized in Table 1. Robust numbers of Nanog-GFP/Nanog positive colonies appeared in the absence of selection drug (Figure Ie). G418 selection during the culture dramatically reduced number of colonies (data not shown). This may have occurred because the neo-resistance gene was under the control of the SV40 promoter, which was not suitable for iPS cell derivation in this system. Five of nine Nanog-GFP positive colonies from TNG MEFs, and eight of twelve colonies picked based on ES like-morphology from 129 MEFs in independent experiments successfully propagated as stable cell lines. All cell lines grow without feeders.
  • iPS cells with this non-viral single vector system has many advantages. Firstly, making iPS with only one integration site extensively reduces risks of unexpected effects by multiple viral vector integrations, which is easily more than 20 in iPS cells derived from MEFs [8A]. Secondly, the reprogramming factors can be completely removed, and which is independent from an unstable silencing mechanism or another expression regulatory system like a doxycycline-inducible system. Complete elimination of unpredictable reactivation of exogenous reprogramming factors is important not only for regenerative medicine but also for drug screening since some of small molecules could affect epigenetic genome modification [26A].
  • this single vector system has high potential to improve the reprogramming technology further, such as targeting of the reprogramming cassette into well-characterized loci [27A], combining with episomal vector systems [28A] or transposon systems.
  • Example 2 Transgenic expression of key genes involved in maintaining embryonic stem (ES) cell self-renewal has proven sufficient to reprogram somatic cells to a pluripotent state.
  • the resulting induced pluripotent stem (iPS) cells resemble ES cells in their properties and have potential to differentiate into a spectrum of adult cells types.
  • iPS induced pluripotent stem
  • a possibility of generating a broad range of cell types from individual patients has become reality, promising a powerful tool for generating new disease models and eliminating concerns associated with the risk of immune rejection in future cell therapies of degenerative diseases.
  • the current method to achieve somatic cell reprogramming requires viral delivery of the defined factors.
  • PB-CAG transposon plasmid Figure 12a
  • CAG promoter CMV enhancer + chicken ⁇ -actin promoter
  • H rabbit ⁇ -globin polyadenylation signal
  • Circular PB-CAG-mFx plasmid DNA 'cocktails' containing each of the four factors were delivered in conjunction with a PB transposase expression plasmid [5] using Fugene HD transfection of 15.5dpc-derived mouse embryonic fibroblasts (MEFs) as described below in Methods. From the time of transfection onwards, MEFs were maintained in ES cell culture conditions.
  • the tet ⁇ 2 tetracycline/doxycycline inducible promoter 14] was used to regulate factor expression in PB-TET ( Figure 12b).
  • the mFx coding regions in PB-TET are linked to an IRES- ⁇ geo-pA cassette, which may be used as a reporter to assess the approximate factor expression level in both the induced (doxycycline (dox) plus) and non-induced (dox minus) states.
  • the inducible expression unit in PB-TET is placed in the 3'->5' TR orientation to minimize any effects of the 5 'TR promoter/enhancer activity [4] on the basal level expression from tet ⁇ 2, as determined prior to using tet ⁇ 2 test constructs (Table 7).
  • the reverse tetracycline transactivator protein (rtTA) was provided by parental MEFs (rtTA-MEFs) established from 15.5dpc Rosa26 rtTA-IRES-GFP knock-in [15] transgenic embryos. Based on results employing PB-CAG-mFx, lOOng of each PB-TET-mFx vector was used in the transfection cocktail.
  • PB-TET-mFx transposition provided ample, easily isolated colonies of high morphological quality and growth stability for subsequent applications. Endogenous pluripotency gene activation To establish stable cell lines, 48 PB-TET-mFx induced colonies were picked between dlO and dl2 from fibroblast induction fields, and passaged routinely (-1 :6 every 48hrs) on inactivated wildtype MEF feeder layers (Figure 12d). Clones were maintained in 1.5ug/mL dox during establishment, until found to be dox independent in duplicate wells. Dox independence occurred as early as dl2 for 3 lines (4D, 4E, 6C), but was most often achieved between dl6-18. All clones isolated which could be expanded became dox- independent by d22-24.
  • Genomic Southern analysis was used to determine the copy number in selected dox-independent PB-induced cell lines ( Figure 10).
  • the neo probe used detects all PB- TET transgene insertions, regardless of the mFx transgene delivered at the insertion site ( Figure 10).
  • the average estimated PB transposon copy number was 10, although co- migrating bands which may obscure the exact total for each cell line could not be ruled out using this analysis.
  • Considering the necessity of 4 independent transgene insertions (3 if one assumes c-Myc is dispensable [22]), it is likely that most reprogrammed cell lines carry 2 or more copies of each mFx reprogramming factor.
  • An interesting exception is line 3D which contained 6 transposon insertions.
  • PB-TET-reprogrammed clones cultured in the presence and absence of doxycycline were screened for lacZ expression (Figure 13d) to determine general transgene activity under either condition.
  • LacZ expression is an indicator of all or any of the transgenes being activated, while only RT-PCR can distinguish which of the four factors remains active in the absence of dox ( Figure 13c).
  • Four clones tested by RT-PCR revealed that transgene expression levels are near that of their corresponding endogenous transcripts in the presence of dox. Interestingly, there was variable low level of expression of some transgenes in the uninduced state (dox-).
  • Oct3/4 transgene expression could be detected in IB and to a lesser extent in 3B, and c-Myc was maintained at minimal levels in both IB and 6C ( Figure 13c, lane 4).
  • IB and 3D Sox2 expression could be detected, while Klf4 appeared silent in all lines except for IB.
  • RT-PCR detection of mFx transcripts reflects the expression level for the population as a whole, and does not reflect the possibility of mosaic expression (as seen in Figure 13d), where there is only a small population of cells maintaining factor expression, while in others, the expression remains silent.
  • PB reprogrammed cells are iPS cells From the panel of dox-independent, PB -reprogrammed cell lines, IB and 6C were chosen to test pluripotency in aggregation chimaeras with wildtype 8-cell stage mouse embryos. These lines showed differential responses to dox withdrawal, where 6C showed minimal expression of the PB-mFx transgenes (Figure 13c) and minor lacZ staining ( Figure 13d), and line IB displayed residual expression of all four transgenes by RT-PCR ( Figure 13c) and strongest lacZ staining of all cell lines tested ( Figure 13d).
  • Embryos resulting from aggregations were dissected on 12.5dpc (IB) and 15.5dpc (6C), and scored for GFP fluorescence indicating contribution of reprogrammed Rosa26-rtTA-IRES-GFP parental MEFs (Table 8). Chimaeras were obtained with high-level contribution from both lines ( Figure Ha). Clone 6C produced a single strong chimaera as determined by GFP fluorescence. For clone IB, three out of seven embryos demonstrated high-level chimaeric contribution despite the residual mFx factor expression detected ( Figure 13 c) suggesting a threshold level of tolerance which permits differentiation of these cells in the embryonic environment.
  • PB transposition may achieve the induction efficiency required for high-throughput peptide or chemical screens.
  • Regulable factor expression by PB-TET transposition permits further study of post-reprogramming processes.
  • the Rosa26 knocked in rtTA in MEF provided convenience in these experiments, but it was not essential. Reprogramming with co-transfection of PB-TET-mFx and PB-CAG-rtTA expresser transposons has also been initiated (not shown).
  • PB-transposition allows technical simplification of reprogramming methodology. Without a viral vector intermediate, there is no need for specialized biohazard containment facilities or the production of high-titer, limited-lifetime [25], viral stocks.
  • PB transposition makes use of simple plasmid DNA preparation, and vectors may be introduced into cells directly using commercial transfection products. Thus, accessible somatic cell types are limited by the effective range of the transfection reagent employed, rather than decreased susceptibility of certain cells to viral infection [26]. These practical simplifications will make reprogramming technology more accessible.
  • PB holds the potential for high- fidelity post-reprogramming transgene removal, once exogenous factor expression becomes dispensable. This approach is more thorough than FIp- or Cre-mediated deletion [27], since the PB terminal repeat elements themselves are removed during the excision process and no footprint remains in 95% of insertion sites [2, 5-8].
  • PB transposase expression results in transposon mobilization in 1% of cells, with reinsertion occurring at a rate of 40%. Therefore, transposon loss may be expected to occur in more than 0.5% of cells, a rate which is amenable to negative selection schemes and would allow cells to be seamlessly cured of genomic modifications.
  • the removal process could be further improved by first minimizing the initial number of transposon insertions into target somatic cells.
  • PB-mediated delivery allows the option of xeno-free production of human iPS cells contrary to current viral production protocols which use xenobiotic conditions.
  • factor consolidation should allow somatic cell reprogramming to be achieved with single-copy, removable, multi-transgene expression, opening the door to potential clinical applications of iPS cells and their derivatives.
  • Fibroblasts (rtT A-MEFs) for PB transposition were isolated from 15.5dpc embryos resulting from heterozygous breeding of Rosa26 knock-in rtTA-IRES-GFP mice [15]. Passage three MEFs were seeded in ES media at 1.25xl0 5 cells/10cm 2 and transfected with PB-mFx transposon vectors and PB transposase expression plasmid using Fugene HD (Roche) 24hrs later. For PB-TET-mFx transfections, expression was induced with 1.5 ⁇ g/mL dox after 24hrs. After 48hrs transfection, cells were fed with fresh ES media daily without passage.
  • iPS clones and ES cell controls were maintained on inactivated feeders, or gelatin for DNA and RNA isolations. lacZ staining was performed on 0.2% glutaraldehyde fixed cells overnight. AP staining was carried out according to the manufacturer's specifications (Vector Labs). Immunostaining used cells grown and fixed on chambered slides (Nunc) and primary antibodies directed to SSEAl (MC-480, Developmental Hybridoma Bank) or Nanog (AB5731, Chemicon). RT-PCR used cDNA prepared with the QuantiTect Reverse Transcription Kit (Qiagen). Southern blotting was carried out using standard methods and DIG-labeled probes for immunodetection (Roche).
  • PB-iPS cell lines were grown on feeders in the absence of dox for at least three passages prior to aggregation with 8 cell stage ICR host embryos [29]. The resulting embryos were dissected on the indicated days, scored for GFP, and dissociated to establish fibroblasts and confirm chimaerism via lacZ staining. Full Methods
  • PB-CAG Plasmid vector construction.
  • PB-CAG was constructed by exchanging the 573 'TR- flanked neo cassette of PB-PGK-neo-bpA [5] digested with MeI (polished with T4 DNA polymerase) and HmdIII with a CBA-Rf A-r ⁇ gpA cassette released by OHI and HindIII digestion from a constitutive gene expression plasmid.
  • the PGK-neo cassette was replaced with a blunt Notl/HindUI fragment containing tetCh via digestion with MeI and HindIII (both polished), resulting in PB-tetO 2 .
  • the four reprogramming factors were shuttled from retroviral backbones (Addgene) into the PB-CAG and PB-TET transposon vectors using their flanking attBl/2 Gateway sites and pDONR221 (Invitrogen) as an intermediate. Sequence confirmation at the intermediate stage and restriction analysis of the final PB-mFx constructs was used to ensure correct recombination events.
  • PB transfection and cell culture PB transfection and cell culture. Defrosted MEFs were allowed to reach 90% confluency (36-48hrs at 37°C, 5% CO 2 ). Cells (1.25xl0 5 /10cm 2 ) were seeded in ES cell medium (DMEM, 15% FBS, penicillin-streptomycin, glutamax, ⁇ -mercaptoethanol, sodium- pyruvate, non-essential amino acids, 2 x leukemia inhibitory factor) to a gelatinized (0.1%) 6-well dish.
  • DMEM 15% FBS, penicillin-streptomycin, glutamax, ⁇ -mercaptoethanol, sodium- pyruvate, non-essential amino acids, 2 x leukemia inhibitory factor
  • dox for PB-TET -mFx transfections, each well was supplemented at 24hrs with dox at the appropriate concentration (d ⁇ ). 48hrs post-transfection, the media was replaced with fresh ES media. Cells were fed daily at the appropriate dox concentrations until analyzed.
  • Colonies were picked in 96-well format on mitomycin-c arrested feeder layers over dlO-12. Routine cultivation (1 :6 passage with trypsin every 2 days) of clones and control Rl ES cells was performed with maintenance in ES media on a feeder layer, while preparation for DNA or RNA required preplating on an untreated surface to deplete feeders (30-40min) and growth on gelatin (0.1%). For PB-TET induced clones, dox treatment was maintained until dl6-24, or removed earlier if replica dox- free cultures appeared stable for at least 48hrs (one passage). / ⁇ cZ/AP staining and immunofluorescence.
  • lacZ staining was performed on cells fixed in situ with 0.2% glutaraldehyde, and stained overnight ( ⁇ 16hrs) in lacZ staining solution: 2OmM MgCl 2 , 5mM K 4 Fe(CN) 6 , 5mM K 4 Fe(CN) 6 and lmg/mL X-gal in PBS.
  • neo probe was isolated by PCR amplification of the neomycin resistance gene coding region from plasmid, gel purified, and labeled with DIG High Prime DNA Labeling and Detection Kit II (Roche). Transposon insertions were detected with the neo DIG-labeled probe ( ⁇ 25ng/mL hybridization solution), processing the membrane and performing DIG immunodetection according to the manufacturer's instructions. RT-PCR.
  • iPS cells Diploid aggregations and chimaera production.
  • dox- independent iPS cells were preplated and passaged at low density on gelatinized dishes.
  • ES cell clumps of -8-12 cells were collected by gentle trypsinization and suspended in ES cell medium.
  • Diploid ICR host embryos were collected ⁇ 20hrs prior to aggregation and maintained overnight in KSOM medium to delay development.
  • Aggregation with iPS cells (lines IB and 6C, 120 embryos each) was performed using standard techniques [29]. The resulting embryos were transferred after 24hrs in groups of 20 to recipient pseudopregnant mothers.
  • Example 3 Example 3
  • iPS induced pluripotent stem
  • PB piggyBac
  • Stable iPS cells generated with this PB transposon vector approach express hallmark pluripotency markers and perform in a series of rigorous differentiation assays. Additionally, by taking advantage of the natural propensity of PB for seamless excision [6], the traceless removal of reprogramming factors joined with 2A sequences (see Example 1) and delivered by a single transposon is demonstrated. By the same mechanism individual factors can also be removed in a combinatorial manner. Such controlled reduction of reprogramming factors from established iPS cell lines is an invaluable tool.
  • PB transposon/transposase system is an effective transgene delivery system, which requires only the inverted terminal repeats (5 'TR and 3'TR) flanking a transgene and transient expression of the transposase enzyme [I].
  • PB transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines [2, 3], including mouse embryonic stem (ES) cells [4,5].
  • PB-CAG transposon plasmid Figure 12a
  • Human (HEF) and mouse (MEF) embryonic fibroblasts were transfected with circular PB-CAG -mFx plasmid DNA 'cocktails' containing each of the four factors in conjunction with a PB transposase expression plasmid [5]. From the time of transfection onwards, fibroblasts were maintained in relevant ES cell culture conditions without passage before picking.
  • PB-CAG transposition was effectively harnessed to deliver factors and initiate early reprogramming, PB transposons are not purposefully subjected to the same silencing process that diminish retroviral (and less extensively lentiviral) expression [33]. It was therefore necessary to achieve temporal expression control and transgene silencing to demonstrate the reprogrammed cells' capacity for autonomous maintenance and subsequent differentiation.
  • the tetCh tetracycline/doxycycline (dox) inducible promoter [14] was used to regulate factor expression in PB-TET ( Figure 12b).
  • PB-TET The mFx coding regions in PB-TET are linked to an IRES- ⁇ geo-pA cassette, which may be used as a reporter to approximate factor expression in both the induced (dox +) and non-induced (dox -) states.
  • the inducible expression unit in PB-TET is placed in the 3'->5' TR orientation to minimize any effects of the 5 'TR promoter/enhancer activity [4] on the basal level expression from tet ⁇ 2 , as determined prior to using tet ⁇ 2 test constructs (Table 7).
  • rtTA reverse tetracycline transactivator
  • Equal efficiency of colony formation was achieved by lOOng-mixtures of PB- TET-mFx and IOng-mixtures of PB-CAG-mFx (compare the graph in Figure 12c with that of Figure 17a), possibly due to more consistent expression levels from the CAG promoter.
  • PB-TET-reprogrammed clones were screened for lacZ expression to determine general transgene activity in the presence and absence of doxycycline ( Figure 13d).
  • RT- PCR which allows further distinction of individual transgene activity, revealed variable trace expression levels of some transgenes in the uninduced (dox -) state ( Figure 13 c).
  • Oct4 transgene expression could be detected in IB and to a lesser extent in 3B, and c-Myc was maintained minimally in both IB and 6C.
  • IB and 3D Sox2 expression could be detected, while Klf4 appeared silent in all lines except for IB.
  • RT-PCR detection of mFx transcripts reflects average expression levels for each cell population, and does not expose mosaic expression patterns (as revealed in Figure 13d).
  • Genomic Southern analysis was used to determine transgene copy number in selected dox-independent mouse PB-TET lines ( Figure 20a). The average estimated PB transposon copy number was 9, although co-migrating bands which may obscure the exact total could not be ruled out.
  • Line 3D contains only five transposon insertions. As all four PB-TET-mFx transgenes are represented in 3D ( Figure 13c), three of the four factors are sufficient in single copy for reprogramming.
  • Transposons located at the ROSA26 locus in mouse ES cells are mobilized by additional transient transposase expression at a rate of 0.7% [5]. Furthermore, for -60% of these mobilization events reinsertion does not occur, resulting in loss of the transposable element.
  • the MKOS sequence (c-Myc, Klf4, Oct4 and Sox2 ORFs linked with 2 A peptide sequences (see Example 1) was inserted into the PB-TET transposon ( Figure 14a).
  • induction of rtTA-MEFs with PB-TET- MKOS in the presence of dox yielded colonies after 12-14 days, at which point 48 iPS cell-like colonies were picked, expanded in the absence of dox, and subjected to diagnostic Southern analysis (not shown) to determine the absolute number of transposon insertions.
  • the analysis revealed two single-copy cell lines, designated as scBl and scC5.
  • flanking genomic sequences of the single-copy transposon insertions were determined by splinkerette PCR [5]. PB inserted into the first intron of the Myold gene (Chrl 1) in csBl, and into an intragenic region of Chrl 6 in scC5 ( Figure 14b); insertion events were also confirmed by locus-specific genomic PCR ( Figure 14e). These lines showed typical ES cell morphology, and indirect measurement through the lacZ reporter revealed no expression of the MKOS reprogramming factors in the absence and high inducibility in the presence of dox ( Figure 14c).
  • Pluripotency of PB-reprogrammed cell lines was demonstrated by their contribution to chimaera development following aggregation with 8- cell stage ICR embryos ( Figure 15a,b).
  • the resulting embryos were dissected at 10.5dpc (IB and 3D), 12.5dpc (IB), 14.5 (scBl and scC5) and 15.5dpc (6C), and scored for GFP fluorescence indicating contribution of reprogrammed ROSA26-rtTA-IRES-GFP parental MEFs (Table 10). Chimaeras with high-level contribution were obtained ( Figure 15a).
  • Pluripotency achieved by PB reprogramming was stringently confirmed by the production of completely iPS cell-derived 13.5dpc embryos via tetraploid embryo complementation assay [34], including germ cell formation detected by Vasa expression in the genital ridge ( Figure 15c). Germ cell formation from iPS cells was also detected in genital ridges of standard diploid chimaeric embryos ( Figure 22a). Teratomas containing derivatives from all three embryonic germ layers are additional proof that these cells are pluripotent and able to differentiate to complex tissues (Figure 22b).
  • PB-CAG-rtTA transposon was successfully employed as a "fifth factor" in the reprogramming of human embryonic fibroblasts.
  • Doxycycline tightly regulates the expression of the reprogramming factors; expression in the uninduced state was not detectable by lacZ staining for the factor-linked IRES- ⁇ geo reporter ( Figure 23a) and only trace amounts of transcription was detected in colonies by semi-quantitative RT-PCR ( Figure 23b).
  • PB-iPS cell derived fibroblasts displayed a much more rapid initial response to reprogramming factor induction than that previously reported for the dox inducible lentivirus system [37], which may reflect the high level of instability of lentivirus-inserted transgene expression.
  • single cell sorting of 2°F/6C cells into 96 well plates revealed that 56 of 192 single cells (29%) were capable of forming colonies. Thirty-nine out of fourty-four (89%) established single-cell clones evaluated for Nanog expression were found positive on day 13 of induction.
  • PB transposition permits technical simplification and improved accessibility of the reprogramming methodology by making use of effortless plasmid DNA preparation and commercial transfection products for delivery. This eliminates the need for specialized biohazard containment facilities or the production of high-titer, limited-lifetime viral stocks [25].
  • the range of somatic cell types that could be used for reprogramming are not limited by the decreased susceptibility to viral infection [25].
  • PB-mediated delivery will allow the option of xeno-free production of human iPS cells contrary to current viral production protocols that use xenobiotic conditions.
  • PB-CAG and PB-TET expression vectors were generated using standard cloning procedures. Reprogramming factors were shuttled into PB plasmid vectors from retroviral sources using Gateway cloning (Invitrogen). MEFs were isolated from 15.5dpc ROSA26 knock-in rtTA-IRES-GFP [15] embryos. HEFs were derived from 12 week abortuses. Fibroblasts were seeded in ES media at 1.25XIO 5 CeIIsZlOCm 2 (MEFs) or 6.25XIO 4 CeIIsZlOCm 2 (HEFs) and transfected with PB-mFx transposon vectors and PB transposase expression plasmid 24hrs later.
  • MEFs were isolated from 15.5dpc ROSA26 knock-in rtTA-IRES-GFP [15] embryos. HEFs were derived from 12 week abortuses. Fibroblasts were seeded in ES media at 1.
  • PB- TET-mFx transfections expression was induced with 1.5 ⁇ gZmL dox the following day. 48hrs after transfection, cells were fed with fresh ES media daily without passage. The resulting colonies were picked on dlO-12. iPS clones were maintained on inactivated feeders, or gelatin coated dishes. lacZ staining was performed overnight on 0.2% glutaraldehyde fixed cells. AP staining was done according to the manufacturer's specifications (Vector Labs). RT-PCR used cDNA prepared with the QuantiTect Reverse Transcription Kit (Qiagen). Southern blotting was carried out using standard methods and DIG-labeled probes for immunodetection (Roche).
  • Immunostaining was performed on cells grown and fixed on chambered slides (Nunc) or 4-well dishes. Flow cytometry used live cells stained with SSEAl. Mouse PB-TET clones were grown in the absence of dox for at least three passages prior to aggregation with diploid or tetraploid ICR or diploid C57BLZ6 host embryos [29]. The prenatal embryos were dissected, scored for GFP and then further processed to perform IH or to derive secondary MEFs. Embryos prepared for lacZ staining and sectioning were treated with dox in utero 20hrs before dissection. Teratoma formation using PB-TET clones was performed as previously described [9]. Differentiation of human iPS was spontaneous on Matrigel following plating of EBs formed in AggreWell dishes. Full Methods Plasmid vector construction.
  • PB-CAG was constructed by exchanging the 5' /3' TR- flanked PGK-neo cassette of PB-PGK-neo-bpA [5] digested with MeI (polished with T4 DNA polymerase) and Hin ⁇ lll with a CBA-RfA-r ⁇ gpA cassette released by OHI and HindIII digestion from a constitutive gene expression plasmid (unpublished data).
  • the PGK-neo cassette was replaced with a blunt Notl/HindUI fragment containing tetO 2 (provided by S. Mohammadi) via digestion with Nhel and HmdIII (both polished), resulting in PB-tet ⁇ 2 .
  • the four reprogramming factors were Gateway cloned from retroviral backbones (Addgene) into the PB-CAG and PB-TET transposon vectors using pDO ⁇ R221 (Invitrogen) as an intermediate.
  • the MKOS element from pCAG2LMK0Sim0 was cloned into pENTR2B using EcoRl prior to Gateway shuttling.
  • the Tet transactivator was amplified from pTet-On Advanced (Clontech) using attBl/2 primers (Table 9). Fibroblast isolation. 15.5dpc ROSA26-rtTA-IRES-GFP embryos (from Gt(ROSA)26Sor tml [
  • rt ⁇ A,EGFP Na g y ⁇ a nS g 6n J 0 crosses
  • MEF medium DMEM, 10% FBS, penicillin-streptomycin, glutamax
  • Primary rtTA-MEFs were passaged once for expansion and frozen (p2).
  • ⁇ EFs were isolated from 12 week-old abortion material, with maintenance and expansion in ⁇ EF media (DMEM, 15% human serum, IOng/mL bFGF, penicillin- streptomycin, glutamax, ⁇ -mercaptoethanol, sodium-pyruvate, non-essential amino acids).
  • DMEM 15% human serum, IOng/mL bFGF, penicillin- streptomycin, glutamax, ⁇ -mercaptoethanol, sodium-pyruvate, non-essential amino acids).
  • MEFs were grown to 90% confluency (36-48hrs at 37°C, 5% CO 2 ) and seeded in mouse ES cell medium (DMEM, 15% FBS, penicillin-streptomycin, glutamax, ⁇ - mercaptoethanol, sodium-pyruvate, non-essential amino acids, LIF) to a gelatinized (0.1%) 6-well dish (p3; 1.25x10 cells/lOcm ).
  • Fugene ⁇ D (Roche) was used to transfect cells with IOng, lOOng, or 400ng of each mFx transposon (25ng, 50ng, or lOOng for PB-TET-MKOS) plus lOOng of pCyL43 PB transposase plasmid [5] (normalized to 2 ⁇ g total DNA with empty pBluescriptKS+) at a Fugene:DNA ratio of 8uL:2 ⁇ g (day zero for PB-CAG inductions).
  • media was supplemented at 24hrs with dox (d ⁇ ). 48hrs post-transfection, the media was replaced with fresh ES media.
  • dox treatment was typically maintained until dl6-24.
  • iPS cells for DNA or RNA preparation were grown on gelatin. Established iPS cells were passaged 1:6 every 48 hours.
  • HEFs Transfection of HEFs was performed similarly, except fibroblasts were initially seeded in DMEM supplemented with 15% human serum, IOng/mL bFGF, penicillin- streptomycin, glutamax, non-essential amino acids at a density of 6.25x10 4 cells/l 0cm 2 , and grown in HEScGRO (Millipore) 48 hours after transfection.
  • Doxycycline (1.5 ⁇ g/ml) was added 24h post transfection to the medium. From four wells of a six- well plate 17 colonies were picked between day 14 and 29, five of them were AP positive and four of those grew doxycycline independently to a permanent cell line. Doxycycline was withdrawn a week after picking.
  • Human iPS cells were maintained on inactivated MEF feeders in human ES medium (KO-DMEM, 20% serum replacement, IOng/mL bFGF, penicillin- streptomycin, glutamax, non-essential amino acids). Southern blotting.
  • neo probe was isolated by PCR amplification of the neomycin resistance gene from plasmid, gel purified, and labeled with DIG High Prime DNA Labeling and Detection Kit II (Roche). Transposon insertions were detected with the neo DIG-labeled probe ( ⁇ 25ng probe/mL hybridization solution) after processing the membrane and performing DIG immunodetection according to the manufacturer's instructions. Splinkerette, Genomic, and RT-PCR.
  • PCR products TA-cloned and sequenced bidirectionally with Ml 3 forward and reverse primers. PB insertion loci were determined using BLAST. Genomic PCR on factor-removed PB-iPS lines was performed using primer sets described in Table 9. Approximately lOOng of genomic template DNA was amplified using Qiagen Taq (Qiagen) with the inclusion of Q-Solution. Due to highly repetitive sequence, chromosome 16 amplification required the use of nested PCR. Three-primer PCR amplification used PB-3F in conjunction with the chromosome-specific primer set. Standard PCR conditions were: 95°C for 30 sec, 55°C for 30sec, 72°C for 45sec; x35 cycles. Reaction products were resolved using standard gel electrophoresis.
  • Stable PB-TET clones were treated transiently with pCyL43 transposase plasmid (2 ⁇ g DNA: 8uL FugeneHD). Non-transfected cells were eliminated by treatment with puromycin (l ⁇ g/mL) for 3 days. Viable cells were counted and plated at clonal density ⁇ SOO-lOOOcells/ ⁇ Ocm 2 ) on inactivated feeders. Clones were picked after 5 days for expansion, lacZ staining and DNA preparation. lacZ/AP staining.
  • lacZ staining was performed on cells or embryos fixed in situ with 0.25% glutaraldehyde, and stained overnight ( ⁇ 16hrs) in lacZ staining solution: 2OmM MgCl 2 , 5mM K 3 Fe(CN)6, 5mM K 4 Fe(CN)O and lmg/mL X-gal in PBS. Embryos were rinsed in wash buffer (2mM MgCl 2 , 0.01% Sodium deoxycholate, and 0.02% Nonidet-P40 in PBS) prior to whole-mount lacZ staining. Embryos were embedded in paraffin, sectioned using Microm Ergostar HM200 microtome, and counterstained with neutral red.
  • Staining for alkaline phosphatase activity was performed on cells without fixation using the Vector Red Kit (Vector Labs), according to the manufacturer's specifications. Immunostaining and flow cytometry. Immunostaining used cells grown on an inactivated feeder layer plated on gelatin- coated Lab-Tek borosilicate glass slides (Nunc) or 4-well dishes containing gelatinized or Matrigel-treated glass coverslip inserts. Cells were washed with PBS, fixed in 4% PFA/PBS for lOmin at 25°C, and permeabilized with 0.3% Triton X-IOO in PBS for 10 min at 25°C.
  • Genital ridges were pre-fixed with 4% paraformaldehyde in PBS for 1 hour at 25°C, then embedded and cryosectioned at 30 ⁇ m thickness. Sections were washed, blocked (5% goat serum for lhr), and incubated overnight at 4°C with rabbit anti- primordial germ cell marker (DDX4/MVH, abl3840, abeam, 1 :400). Sections were washed in PBS and secondary Ab (cy3 IgG, 1:500) was added for 2hrs at 25°C. All immunofluorescence was visualized and obtained using a Zeiss LSM 510 confocal microscope equipped with UV, argon and helium-neon lasers (Zeiss).
  • Dox-induced fibroblast pools from PB-iPS chimaeric mice for FACS analysis were diluted to -15% GFP positive 2°F/1B and /6C representation with wildtype ICR MEFs prior to seeding.
  • Cells were harvested on the indicated day, washed once in PBS containing 5% FBS, and incubated with anti-SSEAl antibody (1 :200) for 30min on ice.
  • Cells were washed twice in PBS:5% FBS, and incubated with Alexa647 conjugated secondary antibody for 30min on ice.
  • Cells were washed twice and resuspended in PBS:5% FBS for analysis on a FACS-Calibur.
  • Single cell plating from PB-iPS chimeric MEF pools was performed using a FACS-Aria, gating on the GFP positive cell fraction. In vitro differentiation assays.
  • PB-iPS lines Human PB-iPS lines were dissociated and used to generate embryoid bodies (EBs) by aggregation in AggreWell 400 plates (StemCell Technologies) in 15% FBS DMEM with all additives except LIF and bFGF, in the absence of dox. After 14 days growth, EBs were collected and permitted to attach on Matrigel coated cover slips or 4 chamber slides in the same medium. After 10 days outgrowth the resulting cultures were analyzed by immunohistochemistry.
  • EBs embryoid bodies
  • PB-iPS cell clumps of ⁇ 8-12 cells were collected by gentle trypsinization and suspended in mouse ES cell medium.
  • ICR or C57BL/6 embryos were collected at 2.5dpc, aggregated with PB-iPS cell clumps, cultured overnight and transferred into pseudopregnant recipients using standard techniques [29].
  • Embryos were dissected in cold F 12 media plus HEPES and examined for GFP on the indicated day, or left to term to verify iPS contribution by coat color.
  • lacZ detection in embryos pregnant dams were treated with dox (1.5 ⁇ g/mL dox; 5% sucrose in water) 20hrs prior to dissection.
  • tetraploid embryo complementation the blastomeres of two-cell stage embryos (1.5dpc) from superovulated ICR females were electro fused by using a CF- 150B Pulse Generator (BLS). The fused embryos were cultured overnight at 37°C in 5% CO2 in KSOM medium (Specialty Media). Two tetraploid embryos at the four-cell stage were aggregated with a clump of ⁇ 8- 15 ES cells. The following day, embryos were transferred into pseudopregnant recipient ICR females [29]. Embryos were dissected at 13.5dpc and contribution of GFP-positive cells (iPSC-derived) was visualized by using Leica MZ 16 FA stereomicroscope.
  • BLS CF- 150B Pulse Generator
  • Reprogramming efficiency was estimated using number of Nanog-GFP/Nanog positive colonies, mOrange positive cell number at day 2 in Table 1, and stable transfection efficiency (3.6% of transient transfected cells as estimated in Figure 5). -; no data.
  • Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues.
  • Maherali, N. et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1, 55-70 (2007).

Abstract

The invention relates to non-viral based vector systems, methods, reprogrammed cells and kits for reprogramming somatic cells. Reprogrammed cells are generated by integrating into somatic cells one or more non-viral based vector systems capable of expressing reprogramming factors necessary to reprogram somatic cells.

Description

TITLE; Compositions, Methods and Kits for Reprogramming Somatic Cells FIELD OF THE INVENTION
The invention relates to compositions, methods and kits for reprogramming somatic cells. In aspects, the invention relates to a method of reprogramming somatic cells including somatic stem cells to produce pluripotent cells or cells of increased pluripotency, the cells produced by the methods, as well as reagents, including vectors and kits suitable for use in the methods. BACKGROUND OF THE INVENTION
Reprogramming of somatic cells to pluripotent cells has great benefits for medicine. Recently many groups have achieved direct reprogramming of somatic cells by forced expression of defined factors using multiple viral vectors [2A, 5A, 6A, 9, 18, 26,
30]. However, the induced pluripotent stem (iPS) cells contain a number of viral vector integrations [8A, 9] and therefore a risk of possessing unexpected genomic abnormalities.
Moreover, viral vectors silenced in iPS cells can be re-activated when the cells differentiate [16, 22].
It would therefore be desirable to develop processes which would enable reprogramming to be carried out, without the use of viral vectors. SUMMARY OF THE INVENTION
The present invention is based on the development of non-viral based systems for reprogramming somatic cells. In particular the invention provides non-viral based vector systems which are able to express the proteins necessary for reprogramming somatic cells.
It is to be understood that the present invention is directed to the reprogramming of somatic cells including somatic stem cells and therefore does not employ eggs, embryos or embryonic stem cells. However, reprogrammed cells of the present invention display pluripotency and growth ability and may be similar in some respect to embryonic stem cells.
The systems and methods of the invention, and particularly the transposon-based systems of the invention, represent significant improvements over known methods. The traceless removal of reprogramming factors is an improvement of particular significance. Systems and methods of the invention enable high-fidelity post-reprogramming transgene removal once exogenous factor expression becomes dispensable. The controlled reduction of reprogramming factors from established iPS cell lines is an invaluable tool. The systems and methods of the invention also provide technical simplification of reprogramming methodology. In particular, there is no need for specialized biohazard containment facilities or the production of high-titer, limited-lifetime viral stocks. The methods employ autonomously replicating nucleic acid constructs, such as plasmid/transposon DNA preparations and/or vectors which may be introduced into cells directly using commercial transfection products and well known techniques in the art such as electroporation. The methods also allow the option of xeno-free production of human iPS cells contrary to current viral production protocols, which use xenobiotic conditions.
In addition, the methods of the invention permit somatic cell reprogramming to be achieved with single-copy, removable, multi-transgene expression.
In an aspect there is provided a method of reprogramming a somatic cell comprising: a) providing a non-viral based vector system which is capable of expressing reprogramming factors necessary to reprogram said somatic cell; b) transfecting said vector into said somatic cell; and c) expressing said reprogramming factors, such that reprogramming of said somatic cell can take place.
In a particular aspect, there is provided a method of reprogramming a differentiated somatic cell or unipotent or multipotent somatic stem cell, which generates only limited lineages of cells, in order to provide a pluripotent reprogrammed cell, the method comprising the steps of: a) providing a non-viral based vector system which is capable of expressing at least two proteins necessary for reprogramming (i.e. reprogramming factors) of said somatic cell or somatic stem cell to occur; b) transfecting said vector into said somatic cell or somatic stem cell; and c) allowing/causing expression of said at least two genes encoding the proteins, such that reprogramming of said somatic cell or somatic stem cell can take place.
In an aspect of the invention, the non-viral based vector system is capable of expressing at least two, three or four reprogramming factors. In a particular aspect, the non-viral based vector system comprises a single vector capable of expressing said reprogramming factors, such as three or four reprogramming factors. In a particular aspect, the non-viral based vector system comprises a single vector comprising reprogramming sequences encoding three or four reprogramming factors.
In aspects of the invention, two or more genes encoding the reprogramming factors are expressed as a single polycistronic messenger RNA which is subsequently expressed into a polypeptide comprising a polypeptide sequence encoding the proteins corresponding to the two or more genes. The protein sequences of the two or more genes are preferably separated by self cleaving/processing sequences known in the art. Thus, preferably the nucleic acid between each of the two or more genes in a vector encodes a self cleaving peptide. Preferred self cleaving peptides are 2A peptides from Aphthoviruses, a typical example of which is foot and mouth virus, or 2A like sequences from other viruses in particular Picornaviridae like the Equine rhinitis A virus as well as the insect Thosea asigna virus, and Porcine teschovirus-1 (Donnelly et. al. J Gen Virol (2001) 82, 1027-41.) When two or more self-cleaving sequences are used to separate 3 or more genes suitably for reprogramming, the same or different self-cleaving sequence may be used, with different sequences being preferred.
The methods of the invention may further comprise isolating the reprogrammed cells, and optionally producing cell lines comprising the cells. Such cells/cell lines are commonly known in the art as iPS cells/cell lines.
In an aspect of the invention, the potential of transposition systems was harnessed to deliver reprogramming factors to somatic cells. Thus, a method of the invention for reprogramming a somatic cell can utilize a non-viral based vector system comprising one or more or at least one transposon region(s) each region comprising a transposon and one or more reprogramming sequences encoding or expressing reprogramming factors.
In an aspect of the invention, a method is provided for reprogramming a somatic cell utilizing at least one transposon region, each region comprising a transposon with 5' and 3' inverted repeat sequences for insertion/excision of the transposon sequence and one or more reprogramming sequences inserted within the transposon, encoding or expressing reprogramming factors(s).
In an aspect, the present invention relates to a method for generating a reprogrammed cell or reprogramming a somatic cell comprising integrating into a somatic cell (e.g. fibroblast) one or more transposon region(s) thereby generating a reprogrammed cell wherein said transposon region(s) comprises a transposon and one or more reprogramming sequences. In an aspect, the transposon region(s) comprise a piggyBac transposable element. In an aspect, the transposon region(s) comprise a DNA transposon, in particular Sleeping Beauty transposon.
In an aspect of the invention, a method is provided for producing or generating reprogrammed cells or reprogramming somatic cells comprising:
(a) transfecting somatic cells with a non-viral based vector system comprising one or more transposon region(s) each comprising a transposon and one or more reprogramming sequences; and
(b) culturing the transfected somatic cells and isolating reprogrammed cells, in particular iPS cells.
In an embodiment of the invention, a method is provided for producing or generating reprogrammed cells comprising:
(a) transfecting somatic cells with one or more transposon region(s) each comprising a transposon and one or more reprogramming sequences selected from the group consisting of members of the Myc, Oct, KIf, Sox,
Nanog and Lin28 gene families and a transposase expression plasmid; and
(b) culturing the transfected somatic cells and isolating reprogrammed cells, in particular iPS cells.
In an embodiment of the invention, a method is provided for producing or generating reprogrammed cells comprising:
(a) transfecting a somatic cell with multiple transposon region(s) each comprising a transposon and one or more reprogramming sequences selected from the group consisting of members of the Myc, Oct, KIf, Sox,
Nanog and Lin28 gene families; and (b) culturing the transfected somatic cells and isolating reprogrammed cells, in particular iPS cells.
In an aspect, the non-viral based vector system comprises two, three or four, preferably three or four separate transposon regions, each transposon region comprising a transposon and one or more reprogramming sequences, preferably one or two, most preferably one reprogramming sequence.
In an aspect, the somatic cells are transfected with three transposon regions each comprising a transposon and a reprogramming sequence. In an aspect, the somatic cells are transfected with three transposon regions each comprising a transposon and a reprogramming sequence of each of an Oct gene family, a KIf gene family, and a Sox gene family. In embodiments, somatic cells are transfected with three transposon regions each comprising a transposon and an Oct 3/4, Klf4 or Sox2 gene. In an aspect, the somatic cells are transfected with four transposon regions each comprising a transposon and a reprogramming sequence. In an aspect, the somatic cells are transfected with four transposon regions each comprising a transposon and a reprogramming sequence of each of an Oct gene family, a KIf gene family, a Sox gene family and a Myc gene family. In embodiments, somatic cells are transfected with four transposon regions each comprising a transposon and an Oct 3/4, c-Myc, Klf4 or Sox2 gene.
In an aspect, the non-viral based vector system comprises a single transposon region comprising a transposon and two or more reprogramming sequences, preferably three or four reprogramming sequences. Thus, somatic cells may be transfected with a single transposon region comprising a transposon and at least two, three or four reprogramming sequences.
In an aspect, the somatic cells are transfected with a single transposon region comprising a transposon and three reprogramming sequences of each of an Oct gene family, a KIf gene family, and a Sox gene family. In an embodiment, the somatic cells are transfected with a single transposon region comprising a transposon and Oct 3/4, Klf4 and Sox2. In an aspect, the somatic cells are transfected with a single transposon region comprising a transposon and four reprogramming sequences of each of an Oct gene family, a KIf gene family, a Myc gene family and a Sox gene family. In an aspect, the somatic cells are transfected with a single transposon region comprising a transposon and c-Myc, Oct 3/4, Klf4 and Sox2.
A vector or a transposon region may comprise a promoter, in particular an inducible promoter, a polyadenylation signal and/or excision or cleaving sequences.
In an aspect, the transposon region comprises a promoter, in particular an inducible promoter. In an embodiment, the vector or transposon region comprises a tetθ2 tetracycline/doxycycline (dox) inducible promoter and the transfected somatic cells are cultured in the presence of dox. In an aspect, the transposon region(s) comprise excision or cleaving sequences and the transposon region(s) are excised in the reprogrammed iPS cells.
In embodiments of the invention, the non-viral based vector system is integrated into the genome of the somatic cell for expression of the reprogramming factors, or it is not integrated and the reprogramming factors are episomally expressed. In a particular embodiment, the non-viral based vector system is integrated and comprises means for excising the reprogramming sequences from the genome of the reprogrammed cell.
The invention also provides pluripotent stem cells induced by reprogramming somatic cells where the reprogramming comprises contacting the somatic cells with a non- viral based vector system of the invention, in particular a non-viral based vector system which is capable of expressing at least two reprogramming factors, preferably three reprogramming factors, more preferably four reprogramming factors. In an aspect, the invention provides reprogrammed somatic cells obtained by a method of the invention or somatic cells derived by inducing differentiation of somatic cells reprogrammed using a non-viral based vector system of the invention.
A method of the invention may further comprise differentiating a reprogrammed cell to a desired cell type. In an embodiment, a method of the invention may further comprise culturing the reprogrammed cells with at least one agent that induces differentiation i.e. a differentiation agent. In another embodiment, a differentiation factor which directs differentiation may be introduced into the somatic cells with the non-viral based vector system. The invention thus also provides cells differentiated from reprogrammed cells of the invention. The differentiated cell may be a cardiac cell, neural cell, hepatic cell, haemopoietic cell, lymphoid cell, bone cell, epithelial cell, kidney cell, pancreatic cell, endothelial cell, or muscle cell type. The present invention also provides a method for expanding, preferably selectively expanding, the reprogrammed cells or cells derived or differentiated therefrom. The method comprises culturing the cells under proliferation conditions; and isolating increased numbers of the cells. "Increased numbers of the cells", refers to an increase in the number of cells by at least about 2-fold relative to the number of cells that are present in a parallel control culture of cells that are not subjected to the same proliferation conditions. The invention also relates to an expanded cell preparation obtained by this method. The term "expanding" or "expansion" contemplates the proliferation of the cells. The invention also provides reprogrammed cells or cells derived therefrom generated using a method of the invention, and compositions comprising same.
In an aspect, the invention provides a reprogrammed cell comprising a non-viral based vector system, in particular a non-viral based system described herein, which is capable of expressing reprogramming factors necessary to reprogram said somatic cell. A reprogrammed cell of the invention may have one or more of the reprogramming sequences excised from the reprogrammed cell, such that one or more of the other reprogramming sequences remains in the reprogrammed cell. The genome of a reprogrammed cell of the invention may comprise excision sequences or portions of excision sequences following excision of the non-viral based vector system. In an embodiment, the genome of the reprogrammed cell comprises LoxP sites.
The invention further contemplates reprogrammed cells or cells derived or differentiated therefrom and compositions comprising same, in combination with a substrate or matrix, preferably a substrate or matrix adapted for transplantation into a patient. The substrate may be an engineered biomaterial or porous tissue culture insert.
The invention also contemplates a cell line comprising reprogrammed cells or cells derived or differentiated therefrom produced by a method of the invention.
The invention also contemplates reprogrammed cells or cells derived or differentiated therefrom and compositions comprising same, in combination with a substrate or matrix, preferably a substrate or matrix adapted for transplantation into a patient. The substrate may be an engineered biomaterial or porous tissue culture insert.
The invention also provides reprogrammed cells or cells derived or differentiated therefrom that have the ability to migrate and localize to specific regions in a patient where they differentiate into somatic cells typical of the region and they integrate into the tissue in a characteristic tissue pattern.
In another aspect, the invention provides a pharmaceutical composition comprising an effective amount of a reprogrammed cell, or a cell differentiated or derived therefrom. In an embodiment, the invention provides a pharmaceutical composition comprising cells of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent. A pharmaceutical composition may include a targeting agent to target cells to particular tissues or organs. In a further aspect there is provided a non-viral based vector system capable of expressing at least two proteins for use in reprogramming somatic cells including somatic stem cells (i.e. reprogramming factors), the vector system comprising at least two genes encoding said proteins or reprogramming factors necessary for reprogramming said somatic cells including somatic stem cells; and preferably a single promoter capable of directing expression of said genes.
In aspects, a non-viral based vector system of the invention comprises a single vector which is capable of expressing said proteins or reprogramming factors, such as 3 or 4 or more proteins or reprogramming factors necessary for reprogramming to occur. The invention contemplates a non-viral based vector system comprising a plurality of transposon regions each comprising a transposon and a reprogramming sequence and its use in the methods of the invention. In an aspect of the invention, a non-viral based vector system is provided comprising one or more transposon region(s) comprising a transposon and one or more reprogramming sequences. In a particular aspect, the non-viral based vector system comprises two, three or four transposon regions each comprising a transposon and one or more reprogramming sequences, preferably one reprogramming sequence. In an embodiment, the one or more reprogramming sequence is selected from the group consisting of members of the Myc, Oct, KIf, Sox, Nanog and Lin28 gene families. In an embodiment, the transposon region(s) comprise a piggyBac transposable element. In another embodiment, the transposon region(s) comprise a DNA transposon, in particular Sleeping Beauty transposon.
In an aspect of the invention, the vector system comprises a single transposon region comprising a transposon and at least two, three or four reprogramming sequences. In an embodiment, the single transposon region comprises a transposon and two or more, preferably three or four, reprogramming sequences of each of an Oct family gene, a KIf family gene, and a Sox family gene. In another embodiment, the transposon region comprises a transposon and Oct 3/4, Klf4 and Sox2. In a further embodiment, the single transposon region comprises a transposon and c-Myc, Oct 3/4, Klf4 and Sox2.
A non-viral based vector system may further comprise a promoter, in particular an inducible promoter, means to enable excision of sequences (i.e. excision or cleaving sequences), an IRES site, a polyadenylation signal, an origin of replication and/or an antibiotic selection marker gene. In particular embodiments of the invention, the non-viral based vector system comprises the vectors identified in Figures 4, 10, 12, 14 or 20 and the Examples herein.
Cells and compositions of the invention may be used in both cell therapies and gene therapies aimed at alleviating disorders and diseases. The invention contemplates a method of treating a subject suffering from a condition where an increase or replacement of a particular cell type is desirable comprising transferring to a patient an effective amount of cells of the invention, in particular, a composition comprising reprogrammed cells of the invention.
The reprogrammed cells and compositions obtained using the systems and methods of the invention may be administered to a subject for the repair or regeneration of a tissue or organ. In another aspect, the method further comprises increasing the function of the tissue or organ.
The invention also relates to the use of cells and compositions of the invention for the repair or regeneration of a tissue or an organ in a subject or to increase the function of a tissue or organ in a subject. In addition, the invention relates to the use of cells and compositions of the invention in the manufacture of a medicament for the repair or regeneration of a tissue or an organ in a subject or to increase the function of a tissue or organ in a subject.
The invention provides a method for repairing or regenerating tissue in a subject comprising obtaining reprogrammed cells of a previous aspect of the invention and administering the cells to a subject suffering from a condition where an increase or replacement of a particular cell type is desirable.
In aspects of the invention, the subject has a myocardial infarction, congestive heart failure, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, cirrhosis, Parkinson's disease, Alzheimer's disease, diabetes, cancer, arthritis, or wound healing, or similar diseases.
In an aspect, a subject has damage to a tissue or organ and a method of the invention involves administering a dose of reprogrammed cells or cells differentiated or derived therefrom sufficient to increase a biological function of the tissue or organ or increase the number of cells in the tissue or organ. In another aspect, the subject has a condition, disorder or disease and a method of the invention involves administering a dose of reprogrammed cells or cells differentiated or derived therefrom to ameliorate or stabilize the condition, disorder or disease. In an aspect of methods of the invention, the method increases the biological activity of the tissue or organ by at least about 5%, 10%, 15%, 20%, 25%, 50%, 60%, 75%, or more compared to a corresponding untreated tissue or organ. In another aspect, the reprogrammed cells or cells differentiated or derived therefrom are administered directly to a subject at a site where an increase in cell number is desired, in particular a site of tissue damage or injury, or disease.
The invention provides a method for obtaining reprogrammed cells for autologous transplantation from a patient's own cells comprising (a) obtaining a sample comprising somatic cells from the patient; (b) transfecting the somatic cells with a non-viral based vector system in accordance with the invention which is capable of expressing reprogramming factors necessary to reprogram said somatic cell;(c) expressing said reprogramming factors, such that reprogramming of said somatic cells occurs.
The invention also provides a tissue or organ comprising reprogrammed cells or cells differentiated or derived therefrom. Further the invention provides the use of a reprogrammed cell of the invention for tissue repair or regeneration. The invention also relates to a method of using the cells and cell compositions described herein in rational drug design.
Cells and compositions of the invention may be used to screen for potential therapeutics that modulate development or activity of such cells or cells differentiated therefrom, or in toxicology testing or studies. In an aspect, the invention relates to the use of reprogrammed or differentiated cells prepared by a method of the invention for drug screening and/or toxicity testing.
The cells and cell compositions of the invention may be used as immunogens that are administered to a heterologous recipient.
The cells and compositions of the invention may be used to prepare model systems of disease. The cells and cell compositions of the invention can also be used to produce growth factors, hormones, etc.
The invention also relates to a method for conducting a regenerative medicine business. Still further the invention relates to a method for conducting a stem cell business involving identifying agents that affect the proliferation, differentiation, function, or survival of cells of the invention. An identified agent(s) can be formulated as a pharmaceutical composition, and manufactured, marketed, and distributed for sale. The invention also contemplates a method of treating a patient comprising administering an effective amount of an agent identified in accordance with a method of the invention to a patient with a disorder or disease. The invention also contemplates a method for conducting a drug discovery business comprising identifying factors or agents that influence the proliferation, differentiation, function, or survival of cells of the invention, and licensing the rights for further development.
The invention further contemplates a method of providing drug development wherein cells described herein are used as a source of biological components of cells in which one or more of these biological components are the targets of the drugs that are being developed.
The invention provides a method for identifying an agent that can replace one or more reprogramming factors excised from reprogrammed cells. The invention also relates to the use of a reprogrammed cell of the invention for identifying an agent to replace one or more reprogramming factors excised from the reprogrammed cell.
An agent may include but is not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members of protein or peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids, polysaccharides, oligosaccharides, monosaccharides, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), nuclei acids (e.g. siRNAs, cDNAs), and small organic or inorganic molecules. The agent may be an endogenous physiological compound or it may be a natural or synthetic compound. In an embodiment, the invention provides a method for identifying a small molecule mimic to replace one or more reprogramming factors excised from reprogrammed cells. The invention also relates to the use of a reprogrammed cell of the invention for identifying a small molecule mimic to replace one or more reprogramming factors excised from the reprogrammed cell. The invention provides a kit for use in reprogramming somatic cells or somatic stem cells, the kit comprising: a) one or more non-viral based vector systems according to the present invention; and b) reagents for facilitating transfection of said one or more vectors into a somatic cell including a somatic stem cell of choice. In an aspect, the invention also provides a kit for carrying out a method of the invention comprising a transposon system for reprogramming cells. In an aspect, the kit comprises a transposon region or components thereof in a package.
In another aspect, the invention provides a kit for tissue repair or regeneration comprising a reprogrammed cell or a cell differentiated or derived therefrom, and instructions for use of the cell in tissue repair or regeneration. The invention is also directed to a kit for transplantation of reprogrammed cells or cells derived or differentiated therefrom comprising a flask with medium and cells or a cell composition of the invention.
In an aspect, the invention relates to a kit for rational drug design comprising reprogrammed cells and cells derived or differentiated therefrom obtained by a process of the invention. In an embodiment, the kit comprises reprogrammed cells and instructions for their use in drug screening or toxicity assays. In another embodiment, the kit comprises reprogrammed cells and instructions for their use in an absorption assay. In another embodiment, the kit comprises reprogrammed cells with one or more reprogramming factors excised from the cells and instructions for their use in identifying mimics to replace reprogramming factors excised from the reprogrammed cells.
Although this should not be construed as limiting, this specification will exemplify methods of reprogramming somatic cells including somatic stem cells, so that they become embryonic or embryonic like cells displaying pluripotency and an ability to be maintained in an undifferentiated state. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in which: Figure 1. Generation of iPS cells using a non-viral multiprotein expression vector, a. Schematic of reprogramming cassette. The four reprogramming factors are translated from a single mRNA coding of c-Myc, KIf 4, Oct4 and Sox2 linked with three different 2A sequences, F2A, T2A and E2A [SEQ ID NOs. 153-155]. Different 2A sequences were chosen to avoid homologus recombination in the vector, however it might not be necessary. Additional peptide sequences to N-terminal and C-terminal protein of the 2A peptides are shown. Full maps of the vectors with the reprogramming cassette are show in Figure 4. b. Appearance of ES cell like-colonies 5-6 days after pCAGMKOSiE nucleofection. Merged images of phase-contrast and EGFP images of two independent colonies are shown with higher magnification of a white square (right panels), c. ALP positive colonies at day 9 after nucleofection. Some, but not all (right bottom), colonies are ALP positive (red), d. An established iPS cell line cultured on feeders (top) or gelatin (bottom). EGFP expression (green) indicates expression of the reprogramming cassette, e. Reactivation of Nanog gene at 4 weeks after transfection with pCAG2LMKOSimO. Nanog-EGFP/Nanog expression became active (green) in some mOrange expressing colonies (red). TNG MEFs (top row) or non-genetically modified 129 MEFs (bottom two rows) were seeded on irradiated MEFs (γMEFs) or gelatin after nucleofection.
Figure 2. Efficient reactivation of pluripotent markers and fewer vector integration sites a. Quantitative PCR for total and endogenous c-Myc, Klf4, Oct4 and Sox2 expression. Data is shown as relative expression to an ES cell line, E14Tg2a (E 14). Note that higher relative expression of total c-Myc (2-8 fold compared to ES cells) is consistent with the multiprotein expression system for the reprogramming factors and lower expression level of c-Myc in ES cells compare to other three genes (data not shown), b. Immunoblot analysis of c-Myc, Klf4, Oct4 and Sox2. Asterisks indicate exogenous Klf4. c. Quantitative PCR for pluripotent markers, d. Schematic diagram of restriction enzyme sites, AfIII and Kpnl in pCAG2LMKOSimO and two probes, CAG probe and Orange probe, used for Southern blotting (bars). Black arrows (invAluR2, inv3TaqFl) indicate position of primers used to detect tandem repeat integration in f e. Southern blotting analysis for AfIII (left two panels) and KpnII (right two panels) digested genome of imO 1 - 8 and TNGimol-5, using CAG probe (the first and third panels) and Orange probe (the second and forth panels), f Validation of the integration site and tandem repeat integration. A single integration site of imO7 (white circle in e), and a single integration site with tandem integration (same orientation) of imOl and imO3 (black circles in e) was identified by inverse PCR (data not shown) and validated by genomic PCR. Asterisk; band from wild-type allele. Arrowhead; integration site-specific band. Detail of the integration site is shown in Figure 9. Note different band size of tandem repeats is caused by vector degradation accompanied by random integration.
Figure 3. Reprogramming cassette excision and pluripotency of the non-viral iPS cells, a. b. A FGF receptor inhibitor, PD 173074, inhibits differentiation caused by reprogramming cassette excision. Many undifferentiated colonies at day 5 differentiate in the absence of PD 173074 (-PD) by day 9 (a). Percentage of remaining reprogramming cassette-free undifferentiated colonies in the absence (-PD) and presence (+PD) of PD 173074 is shown in b. Numbers of monitored reprogramming cassette-excised colonies are indicated in parentheses. Experiments were performed in three cell lines, imO2, imO7 and TNGimO5. c. Quantitative PCR for total and endogenous c-Myc, KIf 4, Oct4 and Sox2 expression before (imO2El, imO3, imO7) and after (imO2Ec3, imO3c8, imO7c8) reprogramming cassette excision, d. Quantitative PCR for pluripotency markers, e. Undifferentiated cells in imO7 teratoma (upper left) and various tissues in imO7c8 teratoma (the other three panels), f. imO3Ec5 derived 10.5 dpc chimeric embryo (left) and non-chimeric embryo (right), g. imO7c8 derived chimeric mice. All blastocyst injection experiments are summarized in Table 3. Figure 4. Maps of MKOS vectors. c-Myc, KIf 4, Oct4 and Sox2 coding regions linked with three different 2 A peptide sequences, F2A, T2A and E2A (reprogramming cassette) are located under CAG promoter. The reprogramming cassette is followed by either ires EGFP (pCAGMKOSiE) or ires mOrange (pCAG2LMKOSimO). The reprogramming cassette and ires mOrange are flanked by loxP sites in pCAG2LMKOSimO. pCAGMKOSiE and pCAG2LMKOSimO are linearized with Fspl or Pvul before transfection, respectively.
Figure 5. Robust expression of reprogramming factors using a multiprotein expression vector, a. Immunoblot analysis of c-Myc, Klf4, Oct4 and Sox2 in HEK293 cells 48 hrs post transfection with pCAGMKOSiE (293+MKOS). Nuclear extract of ES cells (ES) and HEK293 cells without nucleofection (293) are used to identify exogenous protein expression. An anti-α tubulin antibody is used as a loading control, b. Immunofluorescence of HEK293 cells for Oct4 and Sox2 24 hrs post transfection of pCAGMKOSiE. ES cells and HEK293 cells without nucleofection is used as positive and negative control, respectively.
Figure 6. Estimation of stable transfection efficiency in MEF using nucleofection. A vector that derives EGFP expression via the CAG promoter was introduced into MEFs and the percentage of EGFP positive cells were monitored for 20 days after nucleofection without selection. Data represent two independent experiments. Averages of EGFP positive population were 86% and 3% of total cells at day 2 and day 20, respectively. We estimate stable integration occurred in 3.6% (3% of 86%) of transiently transfected cells.
Figure 7. Schematic presentation of vector integration sites. Integration sites of imOl, imO3 and imO7 were found in chromosome 5 (a), chromosome 1 (b) and chromosome 17 (c), respectively. A red box in chromosome diagrams is enlarged below with indication of the vector integration site. Primers used for validation of the integration sites in Figure 2f are shown as black arrows. Diagrams and gene abbreviations were referred from Ensemble genomic databases. Figure 8. Differentiation induced by Cre-mediated reprogramming cassette excision, a. Flat differentiated colonies with no mOrange expression and tight three- dimensional colonies with mOrange expression appeared seven days after Cre transfection. b. Diagram of the vector before and after Cre-mediated reprogramming cassette excision. Arrows indicate primers used to validate Cre-mediated excision, c. Examples of genomic PCR before and after reprogramming cassette excision. Data represents one of the clones (imO2c and imO7c) generated from cell lines, imO2 and imO7 by Cre transfection.
Figure 9. Efficient differentiation of reprogramming cassette-free iPS cell lines in vitro, a. Embryoid body (EB) differentiation. imO2Ec3, imO3c8 and imO7c8 EBs show similar down-regulation of pluripotent markers (Oct4, Nanog, Rexl), and up-regulation of endoderm (Gataό), ectoderm (Fgf5), mesoderm {Brachyury, Flkl) markers, while their parental cell lines, imO2El, imO3 and imO7, differentiate less efficiently, b. In vitro neural differentiation. Reprogramming cassette excised iPS cells, imO3c8, differentiate into β-tubulin positive neuron (green in left panels) as efficient as ES cells. Reprogramming cassette intact iPS cells, imO3, hardly generate neurons and maintain Oct4 expression (green in right panels).
Figure 10. PB-iPS cell lines carry multiple copies of mFx transgenes. PB-derived iPS cell lines bear multiple random transposon insertions, as revealed by Southern analysis. Each clone tested was derived from treatment with a cocktail containing lOOng of each PB-TET-mFx transposon. Total genomic DNA from MEFs and derived PB-iPS cell lines was digested with BarήΑl, and screened with a probe detecting the entire neo coding region of βgeo. On average PB-iPS lines were found to contain a minimum of ~10 PB-TET transposon copies. Note that no fragmented transgene constructs (banding below the predicted lower limit of ~ 1.5kb) were detected. The red arrow indicates the band predicted from the neomycin resistance gene detected in contaminating multi-drug resistant feeder cells (MEF) used for PB-iPS maintenance (band not detected in rtTA- MEF).
Figure 11. Reprogramming factors delivered by PB-TAB are dox-dependent and permit contribution of induced cells to embryonic development, a) Contribution of the induced cell lines to chimaera formation supports the notion that PB-mediated factor delivery results in pluripotent reprogramming. Chimaeras with the strongest contribution of GFP-positive cells are shown beside their corresponding littermates which displayed no GFP signal (WT). Chimaeras from IB were dissected at 12.5dpc, while those from 6C were dissected at 15.5dpc. Scale bars shown are 2mm (IB) and 5mm (6C). b) iPS-derived fibroblasts from IB and 6C chimaeric embryos respond to dox treatment (24 hours) by reactivating βgeo expression. In the absence of dox, few cells stain positive for lacZ, implicating that at a low frequency some transgenes have residual expression. Scale bars are 50OuM.
Figure 12. PB vectors and colony- forming efficiencies using transposition of inducible mouse factors, (a) Schematic of the Gateway-compatible PB-CAG transposon vector used to deliver constitutively expressed mouse factors (mFx) by PB transposition. 5'/3'TR: PB 5' and 3' terminal repeats; CAG: pCAGGS promoter; Bl, B2: lambda attBl and attB2 sites; pA: rabbit β-globin polyadenylation signal, (b) Schematic of the Gateway- compatible PB-TET transposon vector used to deliver dox-inducible, βgeo-linked mouse factors (mFx) by PB transposition. tetO: dox-inducible promoter; IRES: internal ribosomal entry signal; βgeo: lacZ-neo fusion protein; pA: bovine growth hormone polyadenylation signal, (c) Induction cocktails were prepared containing lOOng of each of the four transposons and lOOng transposase plasmid. The dox concentration was varied in the media as indicated. Colonies were counted on the indicated days post-dox treatment. Total colony number per 10cm2 is indicated on the Y-axis. Transfections were performed and analyzed in triplicate. Error bars represent the standard error, (d) Morphologically distinct growth foci appeared from rtTA-MEFs 6-8 days following PB-TET-mFx cocktail transfection. For this study, colonies were picked on dl2 post-induction. Only those cell lines with typical ES cell morphology were capable of generating stable lines in culture. PB'ase: piggyBac transposase. Scale bars are 50μm.
Figure 13. Activation of pluripotency markers and characterization of transgene inducibility in PB-TET clonal cell lines, (a) Stable dox-independent PB-TET induced cell lines activate alkaline phosphatase (AP), SSEAl and Nanog, indicative of reprogramming. Representative images from a single cell line (IB) are shown for each marker analysis. Scale bars are lOOμm. (b) RT-PCR analysis of four stable dox-independent PB-TET-mFx reprogrammed clones reveals additional pluripotency marker gene expression. Rl ES cells and parental rtTA-MEFs serve as positive and negative controls, respectively. Amplification of the GAPDH housekeeping gene was used as an internal loading control. (c) Dox regulation of PB-delivered factors as monitored by transgene-specific RT-PCR analysis (gene-specific forward primer; reverse primer positioned between the attB2 site and IRES sequence, see Figure 12b). Reprogrammed cell lines, Rl-ES, and rtTA-MEF controls were grown in the presence (+) or absence (-) of dox for 2 passages (~96 hrs). Transgene expression is induced near to endogenous levels in the presence of dox. Some residual transgene expression is detected in certain cell lines in the dox minus state. The induction (Oct4, Sox2) or maintenance (c-Myc, Klf4) of corresponding endogenous gene expression was revealed using a gene-specific 3 'UTR-directed RT-PCR approach, (d) Residual transgene expression in reprogrammed cell lines was visualized as lacZ activity by the transcription-linked βgeo reporter gene (Figure 12b). The level and mosaicism of lacZ staining detected after 96 hours dox withdrawal correlates roughly with the basal level transcription detected by RT-PCR. Scale bars are 200 μm.
Figure 14. Seamless factor removal from iPS cells using transposase-stimulated PB excision, (a) Schematic of the MKOS (mouse reprogramming factors linked with 2A peptide sequences) containing PB-TET transposon. (b) Genomic integration site of the individual transposons in scBl and scC5 lines. The capital letters represent the flanking genomic sequences while the lower case letters are 5' and 3' transposon sequences. Both integrations occurred in the plus-strand orientation. [SEQ ID NOs 146-149] (c) Doxycycline inducibility of single-copy transposon iPS cell lines; scBl and scC5. Expression of the lacZ reporter in the presence of dox is lost in the factor- (transposon) removed sublines. Scale bar is lOOμm. (d) Sequence analyses revealed that no mutation was left behind following transposon-mediated removal in the majority of sublines (10 of 11). One single C5 subclone harbored a TTAA duplication at the excision site. [SEQ ID NOs 150-152] (e) Molecular demonstration (PCR) of the transposon removal. Lane 1: PCR for GFP shows that all the cell lines (excluding the Rl ES cell control) are derivatives of the original rtTA MEFs. Lane 2: PCR for βgeo detects the presence of the transposon regardless of its genomic insertion. Lane 3: ChI l- and Chlό-specific PCR across the TTAA tetranucleotide site of PB transposon integration. Note that scBl and scC5 are hemizygous for the transposon insertion and the PCR amplifies from the wild type allele. Lanes 4 and 5: Specific PCR for the 5' and 3' junction of transposon insertions, respectively. Lane 6: 3-primer simultaneous PCR amplification from the wild type allele and the transposon-genome junction, (f) RT-PCR analysis of the single transposon induced iPS cell lines and their factor-removed derivatives reveals maintenance of hallmark pluripotency gene expression. Rl ES cells and parental rtTA-MEFs serve as positive and negative controls, respectively. Amplification of the GAPDH housekeeping gene was used as an internal loading control.
Figure 15. Cell lines reprogrammed by PB-mediated factor transposition are pluripotent. (a) Contribution of PB-TET cell lines to embryonic development. Chimaeras dissected at 10.5dpc with the strongest contribution of PB-iPS-derived cells were easily detected as GFP positive (BF - bright field), (b) Those with weak GFP fluorescence were revealed as chimaeric following lacZ staining (βgeo reporter activation required in utero dox induction). Sectioning of whole mount stained embryos shows contribution of PB-iPS lacZ positive cells to derivatives of all three embryonic germ layers. Red arrowhead - neural tube (ectoderm); yellow arrowhead - dorsal aorta (mesoderm; black arrow foregut epithelium (endoderm). Scale bars are lOOμm. (c) Completely iPS cell-derived (IB) 13.5 dpc embryo generated by tetraploid embryo complementation. Note that the iPS cells were transgenic for GFP. Immunohistochemistry on sections shows the iPS cell contribution to germ cells (Vasa positive) populating the genital ridge, (d) Chimera obtained by aggregating IB PB-iPS cell with diploid eight cell stage ICR (albino) embryos. Scale bar is lOOμm. Figure 16. Properties of secondary fibroblast (20F) reprogramming. (a) FACS analysis establishing the dynamics of SSEAl activation in 2°F/1B and /6C cell lines. The inset shows colony formation as early as day 5. On day 6 of reprogramming gene induction, the cultures were also passed by standard trypsinization and analyzed in parallel to eliminate the negative effect of cell overgrowth, (b) Contribution of 20F/ IB and /6C derived cells to the chimaeric cell population in the function of factor induction time. The data was derived from Q2+Q4 of the FACS in (a), (c) Ratio of SSEAl positive cells in the 2°F/1B and /6C reprogramming fibroblasts. The data was obtained from 100*Q2/(Q2+Q4) in the FACS in (a), (d) Semi-quantitative RT-PCR to compare the level of exogenous reprogramming factor expression in 20F on days 2 and 12 in three different mouse iPS cell lines.
Figure 17. Colony-forming efficiencies using PB transposition of constitutively expressed mouse factors in mouse embryonic fibroblasts, (a) Cocktails were prepared containing 10, 100, or 400ng each of the four transposons, while the transposase plasmid amount was kept constant at lOOng. MEFs were transfected 24hrs after seeding at 1.25x105 cells/well of a 6-well dish (10cm2). Distinct changes in morphology were noted in the fibroblasts within 3 days post-transfection, with noticeable foci forming over d4-6, and the first ES cell-like colonies arising as early as d6. Colonies were counted on the indicated days post-transfection. Transfection with lOOng of each PB-CAG -mFx transposon resulted in the highest colony numbers for the titration range, while IOng of each resulted in a nearly 50-fold decrease in colony formation; a density suitable for clonal isolation. Increasing plasmid quantities to 400ng each had a negative effect on colony formation, possibly revealing errors in reprogramming associated with high-copy PB transposition resulting in factor overexpression or saturation mutagenesis. Diminution of the overall colony number using dox-inducible versus constitutive-expression PB vectors for reprogramming (compare Figure 7c with Figure 12a) may be the result of fundamental differences between the tetO2 and CAG promoters (such as sensitivity to genomic position effects) or core PB vector design differences (such as transcript length or the effect of IRES function). Transfections were performed and analyzed in triplicate. Error bars represent the standard error. Note that the scale is logarithmic. The panel on the right shows representative alkaline phosphatase staining of the resulting colonies on a 10cm2 surface 20 days post-transfection. Individual colonies were picked from induction fields on dl2 on the basis of morphology, and maintained on mitomycin-inactivated fibroblasts. Approximately 61% (54/88) resulted in established clonal lines which retained ES cell-like growth properties. On dl6, all 54 clones stained positive for AP, and an examined subset of eight clones tested positive alongside ES cell controls in Nanog immunostaining assays (data not shown), (b) Representative pictures of colonies obtained with PB mediated reprogramming. Essentially two classes of clones were noted: those with ES cell-like morphology (A-C), and those with a rough, disorganized periphery and interior D-F). Colonies with abnormal morphology were not able to generate pure ES cell- like cultures upon initial cloning and passage, and were not considered during numeration as described above. The underlying cause of clonal diversity is unknown, but may be directly related to over/underexpression of certain factors reflecting position effects or insertion copy number. Differences may also represent various stages in reprogramming, however no obvious progression through morphological classes was noted.
Figure 18. Reprogramming induced in human embryonic fibroblasts with PB- CAG transposons expressing mouse factors, (a) Typical cell morphology changes and colony formation observed over two weeks in the transfected fibroblast culture and following expansion of a representative clone (#39). (b) Three established PB-CAG lines were selected to demonstrate the activation of endogenous pluripotency marker genes using human-specific (with the exception of DNMT3b) RT-PCR primers. Lines #10 and #39 are alkaline phosphatase (AP) positive while #40 is AP negative. CAl and CA2 human ES cell lines 1 served as positive controls, while parental human fibroblasts and mouse fibroblasts served as negative controls. The AP negative cell line #40 appeared to activate only endogenous hTert. Line #10 activated most endogenous genes except for hTert. Line #39 was most similar to the human ES cell controls, (c) Immunohistochemistry revealed the expression of Nanog, SSEA-4 and TRA- 160 in the AP positive line #10.
Figure 19. Autonomous reprogramming is a stochastic process. PB-TET -mFx generated cell lines picked as early as dlO were split 1:3 on dl2 and two replica wells were subjected to dox withdrawal, (a) Line 5C was used to represent clones where 24hrs of dox removal was sufficient to revert colony morphology and slow growth (middle panel). Many of these cells were still in an intermediate reprogramming state as a certain proportion may be rapidly rescued (within 48hrs) upon re-addition of dox (right panel), (b) lacZ staining under such culture conditions revealed that these cells, which still retain residual lacZ activity, have reverted to a fibroblast-like morphology (middle panel).
Figure 20. PB transposon copy number and mobilization in reprogrammed cells, (a) Diagnostic Southern analysis of seven clonal PB-TET-mFx induced lines using BarήΑl digestion and a neo probe to determine the number of transposon integrations. The neo probe detects all PB-TET transgene insertions, regardless of the mFx transgene delivered at the insertion site. The estimated copy number for each line is indicated below the image. Note that no fragmented transgene constructs (banding below the predicted lower limit of ~1.5kb) were detected. Asterisk (*) indicates the neo band resulting from inactivated G418-resistant mouse embryonic fibroblast feeder DNA contamination (this band was not detected in G418-sensitive parental rtT A-MEFs). (b) Individual mobilization frequency of the nine transposase insertions in IB, determined by analyzing subclones derived after transient expression of the transposase. Southern blot analysis of 38 subclones (Figure 16) revealed high frequency mobilization and loss of transposons. Figure 21. Southern blot for detecting transposon insertions in 38 IB subclones that underwent transient transposase expression. 12 remained unchanged regarding the parental transposons (12/38=32%; ex. G2 and H2). From the total number of transposon insertions (38x9=351), 52 (15%) underwent mobilization (cut) of which only 24 were transposed (pasted). Therefore the probability loss after cut is 0.54 (28/52) and the absolute transposon removal rate is 7%. One particular transposon insertion (band #8 in Figure 9b) was transposed only once (1/38=2.5%) while another was cut 11 times (band #9; 11/38=29%).
Figure 22. Differentiation of PB-induced iPS cells into complex tissues, (a) Whole mount immunohistochemistry of the genital ridge of a chimaeric embryo obtained by aggregating IB iPS cells with C57BL/6 eight cell stage embryos. The Vasa and GFP double positive cells are iPS cell-derived germ cells. Scale bars are 40 μm. (b) PB-TET clones were capable of differentiating in teratoma formation assays, generating tissues derived from all three embryonic germ layers. Sections from clone IB are shown as representatives. Scale bars are 50μm. Figure 23. Reprogramming induction by PB-TET-mFx inducible factors in normal human embryonic fibroblasts by co-transfection with PB-CAG-rtTA. (a) lacZ activity in the presence and absence (two weeks) of dox revealed tight regulation of the reprogramming transgenes in all the four human iPS cell lines. Scale bars are 200 μm. (b) RT-PCR on individual transgenes and the corresponding endogenous gene expression in the presence and absence of dox further specifies the tight regulation of transgenes and proves the activation of the endogenous counterparts. CAl and CA2 human ES cell lines [Adewumi, O. et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol 25, 803-16 (2007)] served as positive controls, while parental human fibroblasts, Rl mouse ES cells and mouse fibroblasts acted as additional controls, (c) RT-PCR demonstrates the activation of endogenous pluripotency marker genes using human-specific primers. Figure 24. Immunohistochemistry results reveal the expression of SSEA4,
NANOG, TRA- 1-160, and Tra-1-81 in iPS cells induced from human MEFs by PB-TET- mFx transfection. Cells staining positive only for DAPI are fibroblast feeders. Scale bars are 50 μm.
Figure 25. Immunohistochemistry to detect in vitro differentiated human iPS cell- derivatives positive for markers representing each of the three embryonic germ layers. A- SMA: alpha smooth muscle actin; vWF: Von Willebrand factor; GFAP: glial fibrillary acidic protein; AFP: alpha-fetoprotein. Scale bars are 50 μm.
Figure 26. Onset of Nanog expression during the process of reprogramming of secondary fibroblast (20F). 2°F/1B and /6C were cultured in the presence of dox for 5, 9 and 13 days and then stained for Nanog (red). Many of the 2°F/1B cells expressed Nanog at day9, while 2°F/6C only showed robust Nanog expression at day 13. The bottom row shows magnified images of the inserts in the upper panels. Scale bars are 100 μm. DETAILED DESCRIPTION OF THE INVENTION Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and systems similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and systems are now described. It must be noted that as used herein and in the appended claims, the singular forms
"a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a reprogrammed cell" includes reference to one or more reprogrammed cells and equivalents thereof, and so forth.
The term "reprogramming" refers to changing the differentiation ability of a somatic cell including a somatic stem cell (i.e. making the cell pluripotent or increasing the degree of pluripotency - this is often now termed "induced pluripotency"), or being able to change the fate of the cell, for example changing a skin cell to a pancreatic cell. This may be achieved, for example, by expressing multiple genes which are specific for a particular cell type, such as pancreatic specific genes, in a cell of another cell type.
The term "reprogrammed cell" refers to a somatic cell that has undergone reprogramming or de-differentiation in accordance with a method of the invention. A reprogrammed cell may be subsequently induced to re-differentiate. A reprogrammed cell may express one or more cell specific markers, morphology and/or biological function that are not characteristic of the cell prior to reprogramming, de-differentiation or re- differentiation. In aspects of the methods of the invention, the reprogrammed cell expresses an embryonic stem cell marker not expressed in the somatic cell, in particular Nanog, Daxl, Eras, Fbxo, FoxD3, Rexl and/or Zfp296.
The term "differentiation" refers to the development process of lineage commitment. A "lineage" refers to a pathway of cellular development in which progenitor cells undergo progressive physiological changes to become a particular cell type with a characteristic function such as neural, muscle, endothelial or pancreatic cells. Differentiation can be determined by assaying for the presence of, or increases in one or more cell specific markers compared to their expression in corresponding undifferentiated control cells.
The term "somatic cell" refers to a cell that generally is obtained from a tissue of a subject generally at a post-natal stage of development (e.g. infant, child, adult). A somatic cell may be a differentiated somatic cell or a unipotent or multipotent somatic stem or progenitor cells. In aspects of the methods of the invention, the somatic cells are embryonic fibroblast cells, more particularly human fibroblast cells.
A "non-viral based vector system" refers to a system comprising non-viral sequence(s) that are capable of expressing proteins necessary for reprogramming somatic cells. The system may include promoters, excision or cleaving sequences, an IRES site, a polyadenylation signal, an origin of replication and/or an antibiotic selection marker gene. In aspects of the invention, the system may include promoters, excision sequences etc. of viral origin but viral sequences are generally excised from the reprogrammed cell or they are selected so that they cannot be re-activated when the cells differentiate.
The term "reprogramming factor" refers to a factor which can be used to induce reprogramming of somatic cells in particular induce reprogramming of differentiated cells to establish a pluripotent stem cell, more particularly an inducible pluripotent stem cell having similar pluripotency and growing ability to those of an embryonic stem cell. A reprogramming factor may be produced from a single copy or multiple copies of reprogramming sequences or genes or in the form of a fusion protein. A "fusion protein" comprises all or part (preferably biologically active) of a reprogramming sequence operably linked to a heterologous polypeptide (i.e., a polypeptide other than the reprogramming sequence). Within the fusion protein, the term "operably linked" is intended to indicate that reprogramming sequence and the heterologous polypeptide are fused in-frame to each other. The heterologous polypeptide can be fused to the N-terminus or C-terminus of the reprogramming sequence. Examples of heterologous polypeptides that can be incorporated in a fusion protein include green fluorescence protein (GFP) or glutathione-S-transferase (GST). Fusion proteins can be produced by standard recombinant DNA techniques.
A "reprogramming sequence(s)" refers to a sequence or gene encoding a reprogramming factor. In particular the term refers to a sequence encoding a reprogramming factor that can induce reprogramming of differentiated cells to establish a pluripotent stem cell, in particular an inducible pluripotent stem cell, having similar pluripotency and growing ability to those of an embryonic stem cell. A reprogramming sequence may be introduced into a vector system and in particular a transposon region using methods known to a skilled artisan (see, for example, the methods described in the Examples herein).
The original work by Takahashi and Yamanaka [9] proposed that four genes or reprogramming sequences: Oct3/4, Sox2, c-Myc and Klf4 were necessary for reprogramming to occur. However, this has since been amended and others have proposed that different genes may be used for reprogramming. The present invention is therefore directed to the use of any combinations of genes/proteins (i.e. reprogramming factors and reprogramming sequences) which are identified as being of use in reprogramming. Such genes or reprogramming sequences may include without limitation members of the Oct, Sox, Myc, KIf, and Nanog families, as well as LIN28 and suitable combinations thereof. [For example, see published PCT application WO07069666 or US 20090068742.] Additionally, factors related to differentiation, development, proliferation and the like may also be useful for reprogramming. However, this should not be construed as limiting and should other genes or preprogramming sequences be identified as being of use in reprogramming somatic cells or somatic stem cells, they may find application in the present invention.
Examples of the Oct family gene include, for example, Oct4, OctlA, Oct6, and the like. The use of Oct4 is preferred. Oct4 is a transcription factor belonging to the POU family, and is reported as a marker of undifferentiated cells (K. Okamoto et al., Cell, 60, pp461-72, 1990). Oct4 is also reported to participate in the maintenance of pluripotency (J. Nicholas et al., Cell, 95, pp379-91, 1998). Examples of the KIf family gene include KIf 1, Klf2, Klf4, Klf5 and the like. The use of Kif4 is preferred. Klf4 (Kruppel like factor -4) is reported as a tumor repressing factor (A.M. Ghaleb et al., Cell Res., 15, pp92-6, 2005). Examples of the Myc family gene include c-Myc, N-Myc, L-Myc and the like. The use of c-Myc is preferred. c-Myc is a transcription control factor involved in differentiation and proliferation of cells (S. Adhikary, M. Eilers, Nat. Rev. MoI. Cell Biol. 6, pp.635-45, 2005), and is also reported to be involved in the maintenance of pluripotency (P. Cartwright et al., Development, 132, pp.885-96, 2005). Examples of the Sox family gene include, for example, Soxl, Sox3, Sox7, Soxl5, Soxl7 and Soxl8, and a preferred example includes Sox2. Sox2, expressed in an early development process, is a gene encoding a transcription factor (A.A. Avilion et al., Genes Dev., 17, pp.126-40, 2003). Nanog and Lin28 have been proposed as other inducing factors which may be used (Yu J, et al. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells, Science 2007 Nov 23;318(5854)).
The NCBI accession numbers of representative genes of families that can be employed in the present invention are listed in the following Table: Accession Numbers of Representative Reprogramming Sequences/Genes
Figure imgf000027_0001
In aspects of the invention a reprogramming factor may be replaced with a cytokine or the vector system may include a cytokine. In aspects of the invention utilizing a Myc family gene product, the gene product may be replaced with a cytokine. Examples of cytokines that may be used in the present invention include without limitation basic fibroblast growth factor, stem cell factor or the like.
Some of the reprogramming sequences or genes may be replaced by small molecules such as BIX as shown by Y. Shi, et. al. (Y. Shi, et. al. Cell Stem Cell, 2, pp. 525-528, 2008).
The present invention is not however limited to use of the aforementioned mouse or human genes; other mammalian homologues to these genes are known in the databases and can easily be used, as will be appreciated by the skilled reader. For example, if one wished to reprogram a rat somatic cell or somatic cell progenitor, it may be preferred to use a rat homologue of the aforementioned genes/proteins.
In addition to wild-type genes, mutant genes including substitutions, insertions, and/or deletions of several (for example, 1 to 10, preferably 1 to 6, more preferably 1 to 4, still more preferably 1 to 3, and most preferably 1 or 2) amino acids and having similar function to that of the wild-type gene/protein can also be used. For example, as a gene product of c-Myc, a stable type product (T58A) may be used as well as the wild-type product. All this is well within the understanding of the skilled reader and does not depart from the scope of the present invention.
In aspects of the invention, preferred combinations of reprogramming factors comprise one or more gene products of each of the following families: Oct family, KIf family and Sox family.
In aspects of the invention, preferred combinations of reprogramming factors can include at least a gene product of an Oct family member and a Sox family member, optionally with a KIf and/or Myc family member.
In aspects of the invention, preferred combinations of reprogramming factors comprise one or more gene products of each of an Oct gene family, a KIf gene family and a Myc gene family. In aspects of the invention, preferred combinations of reprogramming factors can include one or more gene products of each of an Oct gene family member, a Sox gene family, a KIf gene family and a Myc gene family.
In embodiments of the invention the reprogramming factors include products of the following genes: Oct 3/4, Klf4, and Sox2. In aspects of the invention, the reprogramming factors are one or more Yamanaka factors.
In embodiments of the invention the reprogramming factors include products of the following genes: c-Myc, Oct 3/4, Klf4, and Sox2.
The term "transposon" refers to a short piece of nucleic acid bounded by inverted repeat sequences. Active transposons encode enzymes that facilitate the insertion of the nucleic acid into DNA sequences. A vector system with a transposon may comprise regulatory elements including without limitation promoters, enhancers, or polyadenylation signals. A transposon may also comprise a sequence encoding a transposase. Any suitable transposon can be used in the present invention including a DNA transposon or retrotransposon sequence or a sequence substantially similar to that of any known retrotransposon or DNA transposon. In aspects of the invention the transposon is a DNA transposon including without limitation those substantially similar to the TcI family of DNA transposons (Plasterk, R. H., Curr Top Microbiol Immunol. 1996, 204: 125-43; Plasterk, R. H. et al, Trends Genet. 1999, 15(8):326-32). DNA transposons comprise inverted or direct repeat sequences flanking the sequence to be integrated, sequences encoding a transposase which catalyzes the excision of the transposon from its original location and promotes its reintegration elsewhere, and a promoter sequence operably linked to the transposase encoding sequence. Such transposons include those with sequences substantially similar to naturally occurring transposons such as Mariner (Gueiros-Filho and Beverly, 1997, Science 276(5319): 1716- 9) as well as those substantially similar to natural sequences such as Sleeping Beauty (Ivies, Z. et al., 1997, Cell. 91(4):501-10).
In a particular aspect of the invention the transposon is a piggyBac (PB) transposable element from the cabbage looper moth, Trichoplusia ni [1; Cary et al., Virology, Volume 161, 8-17, 1989]. In embodiments of the invention the transposon is a PB transposable element with a CAG promoter (CMV enhancer and β-actin promoter), optionally terminated with a β-globin polyadenylation sequence. In another embodiment of the invention, the transposon is a PB transposable element with an inducible promoter, for example a tetC^ tetracycline/doxycycline inducible promoter. In particular aspects of the invention, the transposon is a PB-CAG or PB-TET described in the examples herein.
It will be appreciated that a transposon region may be in combination with a helper plasmid which expresses a transposase enzyme. In an embodiment of a method of the invention using a PB transposable element, expression of the transposase enzyme is provided in trans from a helper plasmid.
A transposon region comprises one or more reprogramming sequences, i.e. a sequence encoding a reprogramming factor which is integrated into the cell along with the transposon during the process of transposon integration. The reprogramming sequence can also be operably linked to a promoter, polyadenylation signal, and other sequences in order to facilitate its expression in the host cell. "Means to enable excision of the reprogramming sequences" refers to sequences that may be included in a non-viral based vector system of the invention which are capable of excising the reprogramming sequences from a reprogrammed cell. Suitable means to enable excision of the reprogramming sequences include site-specific recombination systems such as the cre/lox P system. Other such means may comprise excision/self- processing or cleaving sequences. In an aspect the transposon includes excision or cleaving sequences which facilitate the excision of the transposon from a reprogrammed cell. Examples of cleaving sequences include sequences encoding the self cleaving peptides including 2A peptides from Aphthoviruses , a typical example of which is foot and mouth virus, or 2A like sequences from other viruses in particular Picornaviridae like the Equine rhinitis A virus as well as the insect Thosea asigna virus, and Porcine teschovirus- 1 (Donnelly et. al. J Gen Virol (2001) 82, 1027-41.) [See also, for example, SEQ ID NOs. 153-155.] In aspects of the invention, a non-viral based vector system comprises a transposon region and an excision or cleaving sequence comprising one or more transcription factor open reading frames and/or self-cleaving sequences such as 2A peptide sequences. In an embodiment, an excision sequence comprises a MKOS sequence (c-Myc, Klf4, Oct4 and Sox2 ORFs linked with 2A peptide sequences).
The term "subject" or "patient" refers to a vertebrate, in particular a mammal. Preferably, the term refers to a human. The term also includes domestic animals bred for food, sport, or as pets, including horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo animals. The methods herein for use on subjects contemplate prophylactic as well as curative use. Typical subjects for treatment include persons susceptible to, suffering from or that have suffered a condition, disorder, disease and/or injury. Compositions, Methods and Kits The invention features compositions, methods and kits that are useful for reprogramming somatic cells without the disadvantages of viral transfection systems.
Somatic cells may be obtained from a number of sources such as cell lines, biopsies or autopsies using conventional methods. In aspects of the invention the somatic cells are allogenic cells or autologous cells isolated by biopsy from a subject. Cells may also be obtained from other donors of the same species using techniques known to those skilled in the art. By way of example, a tissue or organ can be mechanically disaggregated (e.g. using grinders, blenders, sieves, homogenizers, pressure cells or sonicators), or treated with digestive enzymes (e.g. trypsin, chymotrypsin, collagenase, elastase, hyaluronidase, DNase, pronase and dispase) and/or chelating agents that weaken connections between adjacent cells to generate a suspension of individual cells. While the example below describes embodiments where fibroblast cells are reprogrammed, the invention is not so limited. Cell suspensions can be fractionated into subpopulations using standard techniques such as positive selection or negative selection. Selection techniques may utilize for example fluorescence-activated cell sorting, electrophoresis, cell agglutination, freeze-thaw procedures, differential adherence properties of cells, filtration, conventional and zonal centrifugation, unit gravity separation, and countercurrent distribution. [See for example, Freshney, Culture of Animal Cells, A Manual of Basic Techniques, 2d Ed, A.R. Liss, Inc., New York, Chapters 11 and 12, 1987.]
A non-viral based vector system that can be used in the present invention to reprogram somatic cells may comprise an integrating vector. In such an instance, preferably the vector includes means to enable excision of a major portion (e.g. at least the genes themselves) of the vector from a host cell's genome, following cell reprogramming. Such means may be a site-specific recombination system such as the cre/lox P system. However, the multiple protein expression vector system described herein, may be applied to episomal vector systems such as transposon based systems and Epstein-Barr virus based episomal vector, pCEP4 (Invitrogen), which can avoid integration of the vector into genome.
Typically, the non-viral vector of the present invention is a plasmid vector which may or may not integrate into a host's genome. If an integrating vector is used, the vector may comprise sequences specifically designed to facilitate integration and may for example comprise sequences designed to cause integration at a specific locus or loci. The expression of the genes encoding the reprogramming factors (e.g. at least two genes or reprogramming sequences) is generally under the control of a promoter, in particular a single promoter. The promoter may be a ubiquitous promoter rather than inducible promoter, but an inducible promoter may be used in certain aspects of the invention. Examples of suitable promoters include a tetracycline dependent expression regulatory system (Clontech http://www.clontech.com/products/detail.asp?product_id =157247&tabno=2. Urlinger, S., et al. (2000) Proc. Natl. Acad. ScL USA 97(14):7963- 7968.), RheoSwitch Mammalian Inducible Expression System (NEB, http://www.neb.com/nebecomm/products/ producte3000.asp. Palli, S. R., Kapitskaya, M.Z., Kumar, M.B. and Cress, D.E. (2003) Eur. J. Biochem. 270, 1308-1315). The complete control® mammalian expression system (Stratagene http://www.stratagene.com/products/displayProduct. aspx?pid=247) the Q-mate™ Inducible Expression System (Krackeler Scientific, Inc. http://www.krackeler.com/ products/fid/2755). A particularly preferred promoter is the CAG promoter (Niwa, H., Yamamarua, K. & Miyazaki, K. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193-9 (1991)). The CAG promoter is a composite promoter that combines the human cytomegalovirus immediate-early promoter and a modified chicken beta-actin promoter and first intron.
The skilled addressee will appreciate that the vectors of the present invention may include other features that are not essential, but nevertheless may be desirable/advantageous. Thus, other preferred features vectors of the present invention may include one or more of the following an IRES (internal ribosome entry site), antibiotic resistance marker gene, polyadenylation signal sequence, origin of replication site and one or more site-specific recombination sites.
An IRES site and/or polyadenylation signal sequence may facilitate expression and/or processing of two or more reprogramming sequences or genes.
An origin of replication site and/or antibiotic selection marker gene may facilitate production and/or maintenance of the vector in an in vitro culture prior to its use in transfecting somatic or somatic cell precursors.
Site-specific recombination sites can assist with removal of the vector sequence, especially two or more reprogramming genes, once reprogramming has occurred. A variety of site-specific recombination systems are well known to those skilled in the art, including Cre/Lox, Dre/rox, Att/λintegrase, frt/Flp, gamma delta resolvase, Tn3 resolvase and 0C231 integrase (see Gover et al, 2005, to which the skilled reader is directed).
Prior to transfection into the somatic cell or somatic stem cell, the vector may be linearised by, for example, cleavage using a restriction endonuclease.
Preferred vectors in accordance with the present invention are identified in Figures 4, 10, 12, 14 and 20.
Transfection of the non-viral vector may be carried out by any suitable means including electroporation, liposome mediated transfection, dendrimers, calcium phosphate mediated transfection and the like, all well known to those skilled in the art (see for example Nature Methods 2, 875 - 883 (205)). A particularly preferred method is nucleofection developed by Amaxa which is now part of Lonza Group, (http ://www. amaxa. com/products/technology/) (Efficient transfection method for primary cells. Hamm A, Krott N, Breibach I, Blindt R and Bosserhoff AK. Tissue Eng (2002) 8(2): 235-245; Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte-derived dendritic cells.Lenz P, Bacot SM, Frazier-Jessen MR and Feldman GM.FEBS Lett (2003) 538(1-3): 149-154; and Rapid and highly efficient gene transfer into natural killer cells by nucleofection.Trompeter HI, Weinhold S, Thiel C, Wernet P and Uhrberg M. J Immunol Methods (2003) 274: 245-256).
A transposon region or multiple transposon regions may be produced and introduced into a somatic cell using methods known to those skilled in the art. In particular, one or more reprogramming factors may be placed via cloning (e.g. Gateway cloning) into a transposon (e.g. PB transposon plasmid) and transposon regions containing one or more of the factors may be delivered by standard transfection techniques into somatic cells. If the transposon region does not contain a transposase sequence the cells are transfected in conjunction with a transposase vector or plasmid.
In the methods of the invention, the somatic cells are grown under suitable conditions, for example, embryonic stem cell media. Reprogrammed cells generated using the methods of the invention may be identified using methods known to a skilled artisan including methods based on cell morphology or expression of cell markers, gene products, antigens, nucleic acids (DNA, cDNA, RNA, antisense RNA, microRNA) and portions thereof. For example, iPS cells may be identified based on morphology and/or expression of ES cell markers such as SSEAl, ES cell pluripotency markers (e.g. Daxl, Eras, Fbxo, FoxD3, Nanog, Rexl and/or Zfp296), and endogenous c-Myc, Oct 3/4, Klf4 and/or Sox2. A reprogrammed cell can also be identified by down-regulation of markers characteristic of the somatic cells from which the reprogrammed cell is induced. By way of example, Thyl, a fibroblast cell marker, should be down-regulated in a reprogrammed cell induced from a fibroblast cell. A reprogrammed cell produced by a method of the invention may be cultured with at least one agent that induces differentiation i.e. a differentiation agent. In an aspect, the agent induces differentiation of reprogrammed cells to cardiomyocytes and is for example one or more of LIF, BMP-2, retinoic acid, trans-retinoic acid, dexamethasone, insulin and indomethacin. In another aspect, the agent induces differentiation of reprogrammed cells into endothelial cells and is for example fibronectin and/or fetal bovine serum (e.g. 10% fetal bovine serum). In another aspect, the agent induces differentiation of reprogrammed cells into neuronal cells and is for example all-trans-retinoic acid. In another aspect, the agent induces differentiation of reprogrammed cells into adipocytes and is for example retinoic acid, dexamethasone, insulin, and indomethacin. The invention thus provides cells differentiated or derived from reprogrammed cells of the invention. Differentiated cells can be used to prepare a cDNA library relatively uncontaminated with cDNA preferentially expressed in cells from other lineages, and they can be used to prepare antibodies that are specific for particular markers of non-hematopoietic cells.
In a preferred embodiment illustrated in the examples and Figures herein, non-viral transfection with a single multiprotein expression vector, comprising the coding sequences of c-Myc, KIf 4, Oct4 and Sox2 linked with intervening 2A peptides, in which peptide bond formation is impaired [HA], can generate iPS cells from non-genetically modified mouse embryonic fibroblasts. This one vector system allows the generation of iPS cells by a single vector integration and subsequently complete elimination of the exogenous reprogramming factors by Cre-mediated excision. These non-viral iPS cells show robust expression of pluripotent markers and genuine pluripotency was confirmed by in vitro differentiation assays and formation of adult chimeric mice. This non-viral single vector system minimizes genome modification and eliminates the unpredictable reactivation of reprogramming factors, providing iPS cells more applicable to regenerative medicine, reliable drug screening and establishment of trustworthy disease models.
In another preferred embodiment illustrated in the examples and Figures herein, murine and human embryonic fibroblasts were reprogrammed using tetracycline inducible reprogramming factors delivered by transposition. The stable iPS cells generated with this transposon vector approach expressed hallmark pluripotency markers and performed in a series of rigorous differentiation assays. Additionally, the traceless removal of the reprogramming factors joined with excision sequences and delivered by a single transposon was demonstrated.
Cells described herein, in particular reprogrammed cells can be modified by introducing mutations into genes in the cells (or the cells from which they are obtained) or by introducing transgenes into the cells. Insertion or deletion mutations may be introduced in a cell using standard techniques. A transgene may be introduced into cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated trans fection, lipofection, electroporation, or microinjection. Suitable methods for transforming and transfecting cells can be found in Sambrook et al. (Sambrook, Fritsch, & Maniatis, DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985)), and other laboratory textbooks. By way of example, a transgene may be introduced into cells using an appropriate expression vector including but not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses). Transfection is easily and efficiently obtained using standard methods including culturing the cells on a monolayer of virus-producing cells (Van der Putten, 1985, Proc Natl Acad Sci U S A.;82:6148-52; and Stewart et al. 1987, EMBO J. 6:383-388).
A selection marker gene may be integrated into cells described herein. For example, a gene which encodes a protein such as β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or a fluorescent protein marker may be integrated into the cells. Examples of fluorescent protein markers are the Green Fluorescent Protein (GFP) from the jellyfish A. victoria, or a variant thereof that retains its fluorescent properties when expressed in vertebrate cells. (For example, the EGFP commercially available from Clontech Palo Alto, CA).
Another aspect of the present invention relates to genetically engineering the cells described herein in such a manner that they or cells derived or differentiated therefrom produce, in vitro or in vivo, polypeptides, hormones and proteins not normally produced in the cells in biologically significant amounts, or produced in small amounts but in situations in which regulatory expression would lead to a therapeutic benefit. For example, the cells could be engineered with a gene that expresses a molecule that specifically inhibits bone resorption, but does not otherwise interfere with osteoclasts binding to bone, or the cells could be engineered with a gene that expresses insulin at levels compatible with normal injected doses. Alternatively the cells could be modified such that a protein normally expressed will be expressed at much lower levels. These products would then be secreted into the surrounding media or purified from the cells. The cells formed in this way can serve as continuous short term or long term production systems of the expressed substance.
Thus, in accordance with an aspect of the invention, reprogrammed cells can be modified with genetic material of interest. The modified cells can be cultured in vitro under suitable conditions so that they are able to express the product of the gene expression or secrete the expression product. These modified cells can be administered to a target tissue where the expressed product will have a beneficial effect.
In aspects of the invention, reprogrammed cells may be engineered to express a gene of interest whose expression promotes survival, proliferation, differentiation, maintenance of a cellular phenotype, or otherwise enhances the engraftment of the cell. Expression of a gene of interest in reprogrammed cells of the invention may also promote repair or regeneration of a tissue or organ having a deficiency in cell number or excess cell death. Examples of proteins that may be expressed in reprogrammed cells include without limitation, growth factors such as BMP, FGF, VEGF, TGFβ, PD-ECGF, PDGF, TNF, HGF, IGF, erythropoietin, CSF, M-CSF, and fragments or variants thereof. Reprogrammed cells may also comprise a component of the extracellular matrix, such as collagen and elastin; proteins having specialized functions such as fibrillin, fibronectin, and laminin, and proteoglycans such as hyaluronan, chonodroitin sulphate, dermatan sulphate, heparin sulphate, heparin, keratin sulphate and aggrecan. The reprogrammed cells and cells differentiated or derived therefrom can be used in a variety of methods (e.g. transplantation) and they have numerous uses in the field of medicine. They may be used for the replacement of body tissues, organs, components or structures which are missing or damaged due to trauma, age, metabolic or toxic injury, disease, idiopathic loss, or any other cause. Transplantation or grafting, as used herein, can include the steps of producing reprogrammed cells or cells differentiated or derived therefrom and transferring cells in the preparation into a patient, in particular a mammal. Transplantation can involve transferring the cells into a patient by injection of a cell suspension into the patient, surgical implantation of a cell mass into a tissue or organ of the patient, or perfusion of a tissue or organ with a cell suspension. The route of transferring the cells may be determined by the requirement for the cells to reside in a particular tissue or organ and by the ability of the cells to find and be retained by the desired target tissue or organ. Where the transplanted cells are to reside in a particular location, they can be surgically placed into a tissue or organ or simply injected into the bloodstream if the cells have the capability to migrate to the desired target organ.
The invention may be used for autografting (cells from an individual are used in the same individual), allografting cells (cells from one individual are used in another individual) and xenografting (transplantation from one species to another). Thus, the cells described herein may be used in autologous or allogenic transplantation procedures to improve a cell deficit or to repair tissue.
In an aspect of the invention, the newly created reprogrammed cells and cells differentiated or derived therefrom, can be used in both cell therapies and gene therapies aimed at alleviating disorders and diseases. The invention obviates the need for human tissue to be used in various medical and research applications.
The cell therapy approach involves the use of transplantation of the newly created cells as a treatment for injuries and diseases. The steps in this application include: (a) allowing reprogrammed cells or cells derived or differentiated therefrom to form functional connections either before or after a step involving transplantation of the cells. The gene therapy approach also involves reprogrammed cells or cells derived or differentiated therefrom, however, the newly created cells are transfected with an appropriate vector containing a cDNA for a desired protein, followed by a step where the modified cells are transplanted.
In either a cell or gene therapy approach, therefore, reprogrammed cells, or cells or tissues differentiated from the cells can be transplanted in, or grafted to, a patient in need. Thus, the cells can be used to replace particular cells in a patient in a cell therapy approach which will be useful in the treatment of tissue injury and diseases. These cells can be also used as vehicles for the delivery of specific gene products to a patient. One example of how these newly created cells or cell differentiated therefrom can be used in a gene therapy method is in treating the effects of Parkinson's disease. For example, tyrosine hydrolase, a key enzyme in dopamine synthesis, may be delivered to a patient via the transplantation of reprogrammed cells that can differentiate into neuronal cells, or transplantation of neuronal cells differentiated from the reprogrammed cells, which have been transfected with a vector suitable for the expression of tyrosine hydrolase. The invention provides methods of using reprogrammed cells of aspects of the invention to repair or regenerate diseased or damaged tissues and organs. In aspects of the invention, the reprogrammed cells are used to increase the number of cells in a tissue or organ having a deficiency in cell number or an excess in cell death. Methods of the invention may also stabilize a damaged tissue or organ in a subject. Methods of the invention may be carried out in vitro, in vivo or ex vivo and they may include prophylactic treatment.
The invention contemplates methods of treating a disease and/or disorder or symptoms thereof characterized by a deficiency in cell number or excess cell death comprising administering a therapeutically effective amount of reprogrammed cells or pharmaceutical compositions of aspects of the invention to a subject in need thereof, in particular to a mammal such as a human. In aspects of the invention, the invention provides a method of treating a subject suffering from or susceptible to a disease characterized by a deficiency in cell number or excess cell death or symptom or disorder thereof. In particular aspects of the invention, the disease, disorder or condition is a heart attack, heart failure, stroke, a neurodegenerative disease such as Parkinson's disease or Alzheimer's disease, diabetes, an inflammatory disease such as arthritis, or cancer.
In an aspect the invention provides a method for stem cell therapy comprising: (a) isolating and collecting somatic cells from a patient; (b) inducing reprogramming of the somatic cells by contacting or transfecting the cells with a vector system of the invention; (c) inducing differentiation of the reprogrammed cells; and (d) transplanting the differentiated cells into the patient or another patient.
The treatment methods of the invention may be administered to subjects (e.g. humans) suffering from, susceptible to, or at risk for a disease characterized by a deficiency in cells or increase in cell death, disorder or symptom thereof. A subject at risk may be identified by any objective or subjective determination of a subject or health care professional using a diagnostic method or test including without limitation genetic tests, biological markers, family history and the like.
Therapeutic efficacy may be determined by measuring, for example, the biological function of the treated tissue or organ (see for example, the Textbook of Medical Physiology, Tenth Edition, Guyton et al, W.B. Saunders Co., 2000). A method of the invention may increase the biological function of a tissue or organ by at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, or 200%. Therapeutic efficacy can also be measured by assaying the increase in cell number in the treated tissue or organ compared to a control that did not receive treatment. Cell number may be increased by at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, or 200% relative to a control. Cell proliferation can be assayed using methods known to a skilled artisan (see for example, Bonifacino et al, Current Protocols in Cell Biology Loose-leaf, John Wiley and Sons, Inc., San Francisco, Calif).
The invention provides pharmaceutical compositions comprising reprogrammed cells of the invention or cells derived or differentiated therefrom and a pharmaceutically acceptable carrier, excipient or vehicle. On this basis, the compositions include, albeit not exclusively, the reprogrammed cells in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. A pharmaceutical composition may be provided as a sterile liquid preparation (e.g. isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions). The pharmaceutical compositions herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective amount of the reprogrammed cells are combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in the standard texts Remington: The Science and Practice of Pharmacy (21st Edition. 2005, University of the Sciences in Philadelphia (Editor), Mack Publishing Company), and in The United States Pharmacopeia: The National Formulary (USP 24 NF 19) published in 1999.
Reprogrammed cells of the invention may be administered directly or indirectly to a tissue or organ, and preferably engraft within the tissue or organ. The cells of the invention may be provided to a site where an increase in the number of cells is desired, for example, due to disease, damage, injury or excess cell death. Reprogrammed cells can also be indirectly administered to a tissue or organ by administration into the circulatory system. Cells may be delivered to a portion of the circulatory system that supplies the target tissue or organ. Administration may be autologous or heterologous. Cells obtained from one subject may be administered to the same subject or a different compatible subject. Agents including expansion and/or differentiation agents may be provided prior to, during or after administration of reprogrammed cells to increase, maintain, or enhance production or differentiation of the cells in vivo.
Cells can be administered to subjects using a variety of means apparent to those of skill in the art. Suitable methods include injection of the cells into a target site in a subject. Cells may be inserted into a delivery device to facilitate injection or implantation into the subjects. Examples of delivery devices include tubes, e.g., catheters, for injecting cells and fluids into the body of a subject. Cells can be prepared for delivery in a variety of different forms. For example, the cells may be suspended in a solution or gel, or mixed with a pharmaceutically acceptable carrier, excipient, or diluent in which the cells remain viable. Pharmaceutically acceptable carriers, excipients, and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. The solution is generally sterile, and will often be isotonic. A solution of cells is preferably selected that is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
Modes of administration of cells include without limitation systemic intracardiac, intracoronary, intravenous, intradermal, or intra-arterial injection and injection directly into the tissue or organ at the intended site of activity, or in proximity to the site of activity. A cell preparation can be administered by any convenient route, for example by infusion or bolus injection and can be administered together with other biologically active agents. Administration in some aspects is preferably systemic.
In aspects of the invention, reprogrammed cells and compositions of the invention are administered by catheter administration, systemic injection, localized injection, intravenous injection, intramuscular, intracardiac injection or parenteral administration. In aspects of the invention, a pharmaceutical composition is formulated in a unit dosage injectable form (solution, suspension, or emulsion).
Reprogrammed cells of the invention may be supplied along with additional reagents in a kit. Kits may include instructions for the treatment regime or assay, reagents, equipment (test tubes, needles, syringes, etc.) and standards for calibrating or conducting the treatment or assay. The instructions in a kit may be directed to suitable operation parameters in the form of a label or a separate insert. A kit may additionally comprise standard or control information to compare the results of a test sample.
Reprogrammed cells or cells differentiated or derived therefrom, and instructions for use of the cells in tissue repair or regeneration may be supplied in a kit form. Kits for transplantation of reprogrammed cells or cells derived or differentiated therefrom may also be supplied comprising a flask with medium and the cells or compositions of the invention.
Kits are also provided for carrying out methods of the invention to generate reprogrammed cells of the invention. Such kits may comprise non-viral based vector systems of the invention, and optionally somatic cells (e.g. control mouse, canine or human fibroblasts), transfection reagents for the vector system, and/or characterization reagents such as antibodies to the pluripotent markers and the like. A kit may also contain culture media products to carry out the reprogramming methods of the invention.
In an aspect of the invention, the kit comprises one or more transposon region and transposon expression plasmids. In an aspect, the kit comprises a transposon region or components thereof in a package.
Kits are also contemplated for rational drug design comprising reprogrammed cells and cells derived or differentiated therefrom obtained by a process of the invention. Such kits may comprise reprogrammed cells and instructions for their use in toxicity assays or instructions for their use in an absorption assay.
Kits of the invention preferably comprise the vectors identified in Figures 4, 10, 12, 14 and 20 and the Examples.
The invention also contemplates methods for identifying modulators which enhance or inhibit reprogramming of somatic cells. Screening methods may involve identification of an agent that modulates the generation of reprogrammed cells, or increases proliferation or survival of reprogrammed cells, or progenitors or cells derived therefrom. These methods may involve contacting somatic cells with a non-viral based vector of the invention and a test agent and quantitating the number of reprogrammed cells produced, and comparing to an untreated control. An increase in the number of reprogrammed cells relative to a control may be indicative of a desired activity. These methods may be used to evaluate a physiological function or toxicity of a test compound. To identify agents/compounds that could replace one or more of the reprogramming factors, iPS cells from which one or more of the reprogramming factors are removed by transient PB transduction, can be generated. Such cells can be used for screening of small molecules/siRNA libraries, for example, in order to identify molecules which could replace the one or more reprogramming factors. Differentiated cells generated from the one or more reprogramming factor(s) removed from the iPS cells, may be cultured and then exposed to the small molecules/siRNA together with doxycycline, to induce the remaining factor(s). Efficiency of reprogramming can be measured by alkaline phosphate staining, immunofluorescense, or morphological criteria. The following non-limiting examples are illustrative of the present invention:
Example 1
The following materials and methods were used in the studies described in the example. Materials and Methods Vectors, pCAGMKOSiE and pCAG2LMKOSimO, which has c-Myc, Klf4, Oct4 and Sox2 coding region linked with 2A peptide sequence under CAG promoter, were constructed as described in Methods. Two to ten micrograms of the vectors were introduced into MEFs using Nucleofector II (Amaxa) according to manufacturers' protocols and cells were cultured in the presence or absence of G418 for 4 weeks after Nucleofection. Colonies showing ES cell-like morphology were picked and cultured either on feeders or gelatin after trypsinization. Gene expression, integration number/sites of the vector in the established cell lines were analyzed by quantitative PCR, Southern blotting and inverse PCR, respectively. The reprogramming cassette was excised by Cre transient transfection in the presence or absence of a Fgf receptor inhibitor PD173074 (100 ng/ml), and pluripotency of the cell lines was examined in vitro (embryoid body formation, neural differentiation) and in vivo (teratoma formation, blastocyst injection). Plasmid construction
Sequences of all primers used for plasmid construction are listed in Table 4. The c-Myc coding region plus F2A 5' 50 bp, F2A 3' 54 bp plus Klf4 coding region plus T2A, Oct4 coding region plus E2A 5' 50 bp, E2A 50 bp plus Sox2 coding region fragments were amplified by PCR using mouse ES cell cDNA as a template, separately. The PCR was performed with Phusion (Finnzymes) with supplement of 3-10% dimethyl sulfoxide. The fragments containing c-Myc, Oct4 and Sox2 coding region were amplified with the following conditions, 95°C for 3 min xl cycle, 95°C for 20 sec - 55°C for 20 sec - 72°C for 1 min x 5 cycles, 95°C for 20 sec - 68°C for 20 sec - 72°C for 1 min x 25 cycles, 72°C for 5 min xl cycle. The fragment containing Klf4 coding region was amplified with 95°C for 3 min xl cycle, 95°C for 20 sec - 46°C for 20 sec - 72°C for 1 min x 5 cycles, 95°C for 20 sec - 700C for 20 sec - 72°C for 1 min x 30 cycles, 72°C for 5 min xl cycle, c- Myc-F2A-Klf4-T2A, Oct4-E2A-Sox2 fragments are amplified by PCR using their flanking primers used in the first PCR after annealing and extension take advantage of complimentary 2A peptide regions of the first PCR products. More specifically purified fragments containing c-Myc and Klf4 with overlapping F2A sequence, Oct4 and Sox2 with overlapping E2A sequence were mixed and subjected to the PCR (95°C for 20 sec - 72°C for 1 min x30 cycles) without primers. The resulting c-Myc-F2A-Klf4-T2A, Oct4- E2A-Sox2 fragments were amplified using EcoRI Koz 5'-Myc and Klf4-3' GSG T2A Xho primers, Xho 5'-Oct4 and Sox2-3' EcoRI Xho primers, respectively, under a PCR condition, 95°C 20 sec - 700C 20 sec - 72°C 90 sec x30 cycles, 72°C for 5 min xl cycle. The fragments were subcloned into pTOPO-bluntll (Invitrogen), individually, and Oct4- E2A-Sox2 fragment was transferred into Xhol site locating after c-Myc -F2A-Klf4-T2A, resulting in c-Myc-F2A-Klf4-T2A Oct4-E2A-Sox2 (MKOS) in pTOPO-bluntll. pCAGMKOSiE is constructed by inserting the MKOS fragment into EcoRI site of pCIG2, which is generated by replacing CMV promoter of pIRES2-EGFP (BD biosciences Clontech) with CAG promoter, ires fragment was amplified by NotlRES, mOrangeIRES primers using pIRES2-EGFP as a template (95°C for 3 min x 1 cycle, 95°C 20 sec - 500C for 20 sec - 72°C for 30sec x5 cycles, 95°C 20 sec - 65°C for 20 sec - 72°C for 30sec xlO cycles, 72°C for 5 min xl cycle). mOrange fragment was amplified by IRESmOrange, XbaBamloxPmOrange primers using pmOrange (clontech) as a template using the same PCR cycles as the ires fragment, ires mOrange loxP fragment (imOloxP) was amplified using these fragments and the flanking primers at 95°C for 3 min x 1 cycle, 95°C 20 sec - 700C for 20 sec - 72°C for 30sec x30 cycles without primers, and then subsequently 95°C 20 sec - 72°C for 45 sec xlO cycles, 72°C for 5 min xl cycle with NotlRES and Xba Bam loxP mOrange primers, and subcloned into pTOPO-bluntll. MKOS was inserted into EcoRI site of pcDNA3 (Invitrogen), and then one loxP site made by annealing KpnBamloxPBamX F/R oligos, and imOloxP was inserted before c-Myc and after Sox2, respectively (pCMV2LMKOSimO). pCAG2LMKOSimO is constructed by transferring a BamHI-BamHI fragment containing MKOSimO flanked by 21oxP sites from pCMV2LMKOSimO into pCAGDNA3 vector, which is made by replacing its CMV promoter with CAG promoter. Cell culture and blastocyst injection
MEFs from 13.5-14.5 dpc 129 mice embryos were generated following a standard method. MEFs from C57BL/6 strain TNG mice [19A] (TNG MEFs) were kindly provided by I. Chambers. MEFs and HEK293 cells were cultured in GMEM containing 10% FCS, penicillin/streptomycin, L-glutamine, β-mercaptoethnol and nonessential amino acids. Leukemia inhibiting factor (LIF) (1000 U/ml) was added for iPS cell derivation and maintenance. ES cell-like colonies derived from 129 MEFs were picked 4 weeks after nucleofection, trypsinised with 0.25% Trypsin, 0.1% EDTA in PBS, and seeded on irradiated MEF or directly on gelatin in 4 well plates as the first passage. A wild type ES cell line, E14Tg2a, and established iPS cells were passaged on gelatin-coated dishes every 2-3 days. Cell lines, imO2Ec3, imO3Ec5 and imO7Ec3, were generated by excising the reprogramming cassette from imO2El, imO3El and imO7El which has random integration of EGFP cassette under CAG promoter. Embryoid body formation and neural differentiation was performed used for RT-PCR and immunofluorescence as described before [19, 22A]. Chimeras were produced by microinjection into C57BL/6 blastocysts Immunoblotting
Nuclear extracts were prepared as described before [19]. Transfected HEK293 cells were harvested 48 hrs post transfection. Antibodies against c-Myc (N-262), Klf4 (H- 180) Oct4 (N- 19), Sox2 (Y- 17) and α-tubulin (B-7) were purchased from Santa Cruz. Southern blotting
Ten micrograms of genomic DNA digested with AIfII or Kpnl was separated in 0.8% of agarose gel in TAE buffer, and hybridized with radioisotope-labeled probes in PerfectHyb Plus Hybridization Buffer (Sigma) at 65°C after transferred onto Hybond-XL (GH Healthcare). Expected band size from tandem integration is; same orientation: < 13.0 kb, opposite orientation: < 13.8 kb or < 12.2 kb in AfIII digestion, same orientation: <6.4 kb, opposite orientation: < 9.8 kb or < 5.1 kb in Kpnl digestion using either CAG or
Orange probe.
Nucleofection and reprogramming efficiency estimation
MEF Nucleofector Kit 2 (Amaxa) and program T-20 was used for Nucleofection following manufacturer's instruction. 2xlO6 MEFs were transfected with 2-10 μg of linearized DNA, and 105 cells were seeded in 6 well plates on either irradiated MEFs or gelatin in the presence of LIF. Forty-eight hours later living cells were harvested from one of gelatin wells, and after counting cell number mOrange positive population was measured by flow cytometry (CyAn ADP, Dako). mOrange positive cell numbers per well were estimated using the results in each experiment as shown in Table 1. The rest of the wells were kept for 4 weeks changing medium every 2-3 days. Nanog-GFP positive colonies from TNG MEFs were picked and seeded on irradiated MEFs after trypsinization. Colonies from 129 MEFs were fixed with 4% PFA and stained with anti-Nanog antibody (Abeam) followed by Alexa Fluor 488 conjugated anti-rabbit IgG antibody (Molecular Probes).
Inverse PCR
Genomic DNA extracted from imOl, imO3, and imO7 was digested with AIuI, Spel or Taql. After self-ligation for 18 hours at 16°C, the DNA was used as PCR templates for primers in Table 5. Amplified fragments were purified, cloned into pCRII- TOPO vector (Invitrogen) and sequenced. Integration site of imOl and imO7 was identified with invAluF/invAluR2 primers in AIuI digested genome and Spel digested genome, respectively. Integration site of imO3 was identified with invAluF/invAluRl primers in AIuI digested genome. inv3TaqFl/inv3TaqR primers identified concatemer in Taql digested imOl and imO3 genome. The integration site and concatemer were verified using specific primers listed in Table 5. Reprogramming cassette excision
Cells were cultured in the presence and absence of 100 ng/ml PD 173074 (Sigma) for 24 hours before Cre transfection with Lipofectamine2000 (Invitrogen). Forty-eight hours after Cre transfection cells were harvested and seeded at 103 cells per 10 cm dishes with gelatin in the presence or absence of PD 173074. Undifferentiated mOrange negative colonies were marked 5 days later and morphology of the colonies was monitored to day 9. Colonies were picked between day 12-14, propagated in 96 well plates, and reprogramming cassette excision in each clone was verified by genomic PCR using CAG F, ires mOrange and BGH R primers (Figure 8b, c, Primer sequences are in Table 5). Reprogramming cassette positive colonies by genomic PCR, which had undetectable mOrange expression by microscopy, were eliminated from the total number. Differentiated colonies, which failed to grow after being picked, were considered as reprogramming cassette-excised colonies. Quantitative PCR
Each quantitative PCR used 1% of cDNA made from 1 μg of total RNA in Light
Cycler 480 system with SYBR Green (Roche). See Table 6 for the gene specific primers. Data was shown as relative expression to an ES cell line, E14Tg2a, after normalized with TBP expression. Data represent one of two independent experiments performed in duplicate. Error bars represent standard deviation. Results and Discussion
Since Yamanaka and Takahashi had identified the four critical reprogramming factors, c-Myc, Klf4, Oct4 and Sox2, in the original excellent work [19], all direct reprogramming have so far employed viral gene delivery systems. Although viral gene transduction is efficient, the consequent multiple vector integrations could result in proto- oncogene activation. Although c-Myc, is dispensable for reprogramming [16, 22], it is of concern that levels of the reprogramming factor reactivation, which can occur in iPS cell- derived somatic cells, differ in different iPS cell lines and types of tissues [18] and ectopic expression of either Oct4 or Klf4 induces dysplasia [12A, 13A]. To solve the problems non-viral reprogramming was challenged using a multiprotein expression system.
While the internal ribosomal entry site (IRES) is widely used to achieve multicistronic expression, translation of downstream genes is relatively poor compared to the upstream genes [14A, 15A]. Recently efficient multiprotein expression using the 2 A peptide sequence of foot-and-mouth disease virus (F2A), or 2A-like sequence from other viruses has been reported in various types of cells including human ES cells [HA, 14A- 16A]. The F2A sequence is proposed to modify the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNA(Gly) ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows a ribosomal 'skip' from one codon to the next without the formation of a peptide bond [17A]. First the ability of 2 A peptide- mediated multiprotein expression to achieve robust expression of four reprogramming factors, c-Myc, Klf4, Oct4 and Sox2, under the CAG promoter was tested [18A] (Figure 1 and Figure 4). When the vector, pCAGMKOSiE, is transfected into HEK293 cells, efficient 'translational skip' and robust expression of Klf4, Oct4 and Sox2 was detected by western blotting, although endogenous c-Myc expression in HEK293 cells is too high to identify exogenous c-Myc (Figure 5a). Clear nuclear localization of exogenous Oct4 and Sox2 with comparable expression level to ES cells was also observed in the transfected HEK 293 cells (Figure 5b). The optimal amount and balance of four reprogramming factors for reprogramming is not known, except that c-Myc is dispensable [22]. Nevertheless, when pCAGMKOSiE is introduced into mouse embryonic fibroblasts (MEFs), some transfected cells showed ES cell-like morphology with large nucleus at day 5-6, and some colonies contained alkaline phosphatase (ALP) positive cells by day 9 (Figure Ib, c). Based on morphology, ES cell-like colonies were picked between days 20- 30 and successfully propagated on either feeders or gelatin keeping their ES cell-like morphology (Figure Id). Using a second vector, pCAG2LMKOSimO, where loxP sites flank the four factors and ires mOrange, allowing for the complete removal of the reprogramming cassette and reporter gene (Figures Ia, 4), the reprogramming efficiency was estimated using Nanog reactivation as a maker of reprogramming [26, 30]. MEFs from TNG mice, which has EGFP reporter gene under the Nanog promoter [19A], and from non-genetically modified 129 mice, were transfected with the pCAG2LMKOSimO plasmid and cultured on either irradiated MEFs or gelatin. The number of Nanog-EGFP positive colonies from TNG MEFs and Nanog positive colonies from 129 MEFs at day 28 is summarized in Table 1. Robust numbers of Nanog-GFP/Nanog positive colonies appeared in the absence of selection drug (Figure Ie). G418 selection during the culture dramatically reduced number of colonies (data not shown). This may have occurred because the neo-resistance gene was under the control of the SV40 promoter, which was not suitable for iPS cell derivation in this system. Five of nine Nanog-GFP positive colonies from TNG MEFs, and eight of twelve colonies picked based on ES like-morphology from 129 MEFs in independent experiments successfully propagated as stable cell lines. All cell lines grow without feeders. To estimate the reprogramming efficiency, stable transfection efficiency in MEFs was measured using a vector that derives EGFP expression by CAG promoter without drug selection (Figure 6). Two days after nucleofection, over 86% were EGFP positive, but the number of fluorescent cells dropped to about 3% by day 20. Considering this (3% of 86% = 3.6%) as stable transfection efficiency in MEFs using nucleofection, efficiency of Nanog positive colony induction in each experiment is estimated at on average 2% (Table 2). This relatively high efficiency compared to that with viral system (-0.1% [2A, 18, 30]) may depend on several factors. Firstly the robust expression of 4 reprogramming factors by the multiprotein expression system. Secondly the use of the CAG promoter that is less sensitive to silencing, and thirdly the different estimation method. Importantly this level of efficiency can be achieved by single site vector integration (Figure 2e).
Next the expression of pluripotent markers was examined in 8 cell lines, imOl- imO8, generated from wild type 129 MEFs. All cell lines showed robust reactivation of endogenous Oct4 and Sox2, comprising the majority of total Oct4 and Sox2 mRNA transcript (Figure 2a). Endogenous c-Myc expression, which is higher in MEFs than ES cells, became similar to ES cells in the cell lines, while there is no large difference in endogenous Klf4 expression level among all cell types here (Figure 2a). In general the CAG promoter derives strong gene expression, but contribution of exogenous Oct4 and Sox2 is very low in all the cell lines, which are derived and maintained in the absence of drug selection. It suggests a possibility that the CAG promoter was subject to moderate silencing, consistent with low and heterogeneous EGFP and mOrange expression (Figure Id and data not shown). The total amount of the four reprogramming factor proteins is also comparable to ES cells in all cell lines examined (Figure 2c). Abundant exogenous Klf4 (Figure 2c, asterisk) was detected in most of cell lines, however, the c-Myc protein level does not agree with higher expression mRNA expression relative to ES cells as shown by Takahashi et. al. [9] (Figure 2a and c). This suggests that post- transcriptional/translational regulation of the reprogramming factors occurs in iPS cells and/or during the process of reprogramming. In addition to Oct4 and Sox2, 13 additional pluripotent markers, which are highly enriched in both ES cells and iPS cells [9, 2OA, 26], are reactivated in all cell lines (Figure 2b), indicating efficient reprogramming had occurred. The number of vector integration sites was analyzed in the 8 129 iPS cell lines (imOl-imO8), as well as cell lines derived from TNG MEFs (TNGim01-im05) (strategy shown in Figure 2d). Of the 13 cell lines, Southern blotting strongly suggested that imO7 and TNGimO5 have a single vector integration in one site, indicating that a single copy of the non-viral expression vector is sufficient to mediate reprogramming (Figure 2e). The integration sites of imOl, imO3 and imO7, as well as the tandem repeats in imOl and imO3, were identified by inverse PCR and confirmed by genomic PCR (Figure 2f). All integration sites of the cell lines are different (Figure 7), which agrees with Aoi et. α/. 's indication that reprogramming is independent from viral vector integration at specific loci [8A].
To remove the exogenous reprogramming factors, transient Cre transfection was performed. Surprisingly many of the mOrange negative colonies started to differentiate after Cre tranfection (Figure 8a). This reprogramming excision-mediated differentiation was successfully prevented by culturing the cells in the presence of Fgf receptor inhibitor, PD 173074, which inhibits mouse ES cell differentiation [21A] (Figure 3a). The percentage of differentiated colonies after Cre-mediated excision is different among clones, possibly reflecting different stability of the reprogrammed status (Figure 3b). All tested reprogramming cassette excised clones generated from imO2, imO3, imO7 and TNGimO5 in the presence of PD 173074 could maintain an undifferentiated morphology for at least 5 passages after removal of PD 173074 (data not shown). The endogenous gene expression of c-Myc, Klf4, Sox2, and Oct4 decreased slightly after the reprogramming cassette was excised (Figure 3 c), and some pluripotency markers also changed their level of expression, however overall expression of all genes tested was sustained (Figure 3d). This is the first evidence that reprogramming can remain stable even after the irreversible removal of exogenous reprogramming factors.
The pluripotency of the cell lines was examined before and after reprogramming cassette excision in vitro, using embryoid body (EB) formation and a monolayer neural differentiation protocol [19, 22A]. Cre-treated cell lines imO3c8 and imO7c8 up-regulated markers of all three germ layers in the embryoid bodies, and generated β-tubulin positive neurons efficiently, while differentiation of their parental cell lines, imO3 and imO7, was less efficient like iPS cells maintaining reprogramming factors expression using lentiviral doxycycline-inducible system [13] (Figure 9). It is surprising that imO3 and imO7 do not differentiate efficiently even though the proportion of exogenous mRNA in total Oct4 and Sox2 expression was low (Figure 2A). This may be explained by exogenous Klf4 expression, which is about half of total expression in imO3 and imO7 (Figure 2A), as overexpression of Klf4 in mouse ES cells prevents differentiation in embryoid bodies, suggesting that Klf4 contributes to ES cell self-renewal [25A]. Both imO7 and imO7c8 produce teratomas under the kidney capsule, although teratomas derived from imO7 contained more undifferentiated cells and unknown cell types than those from imO7c8, consistent with in vitro differentiation results (Figure 3e). Finally the reprogramming cassette-free iPS cells were injected into C57BL/6 blastocysts (results summarized in Table 3). Two independent cell lines, imO3Ec5 and imO7Ec3, gave rise to high contribution in embryonic chimera with high frequency (imO3Ec5; 4 in 5 embryos at 10.5 dpc; imO7Ec3; 4 in 8 embryos at 11.5 dpc) (Figure 3f). Two additional cassette-free iPS cell lines, imθlc5 and imO7c8, give rise to live chimeras, indicating iPS cells derived with the non-viral multiprotein expression vector are genuinely pluripotent (Figure 3g). To address the reprogramming ability of the non-viral single-vector system in human cells, stable transfection efficiencies were enhanced using a piggyBac (PB) transposon gene delivery system [5]. Co-transfection of two PB transposons carrying a doxycycline (doc) inducible MKOS-ires-geo cassette and a constitutively active CAG- rtTA transactivator construct was applied to human embryonic fibroblasts [see Examples 2 and 3]. In this design, upon genomic integration, the two transposons allow dox-inducible activation of MKOS expression in wildtype cells. iPS-like colony formation was observed 14 days post transfection (dpt) when the cells were maintained in hES cell culture conditions supplemented with dox. In total, fifteen colonies were picked from 14-25dpt from four wells of 6-well plates, initially containing either 3.2xlO4 or 6.4xlO4 fibroblasts/well. When dox was withdrawn on 32dpt, three clones successfully propagated as stable cell lines, while the exogenous factor expression was uninduced by negative staining for lacZ activity. All clonal lines displayed human ES cell morphology and were positive for alkaline phosphatase. Robust expression of endogenous pluripotency markers, SSEA4, NANOG, TRA- 160 and TRA-181 was confirmed in all the three cell lines. These results demonstrate that the non-viral single-vector can reprogram human fibroblasts and strongly suggests that it can also be applied to the production of exogenous factor-free, non-viral human iPS cells.
Generation of iPS cells with this non-viral single vector system has many advantages. Firstly, making iPS with only one integration site extensively reduces risks of unexpected effects by multiple viral vector integrations, which is easily more than 20 in iPS cells derived from MEFs [8A]. Secondly, the reprogramming factors can be completely removed, and which is independent from an unstable silencing mechanism or another expression regulatory system like a doxycycline-inducible system. Complete elimination of unpredictable reactivation of exogenous reprogramming factors is important not only for regenerative medicine but also for drug screening since some of small molecules could affect epigenetic genome modification [26A]. Although a part of the vector remains in the integration site after Cre-mediated excision in the current system, this single vector system has high potential to improve the reprogramming technology further, such as targeting of the reprogramming cassette into well-characterized loci [27A], combining with episomal vector systems [28A] or transposon systems. The study demonstrated the single-vector reprogramming system combined with PB transposon delivery system for human cell reprogramming. PB transposons are completely removable from the integration site without any residual change in the original DNA sequence. This PB-based single-vector reprogramming system will enable the generation of non- genetically modified human iPS cells as shown in the mouse (see Examples 2 and 3). Example 2 Transgenic expression of key genes involved in maintaining embryonic stem (ES) cell self-renewal has proven sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble ES cells in their properties and have potential to differentiate into a spectrum of adult cells types. For the first time, a possibility of generating a broad range of cell types from individual patients has become reality, promising a powerful tool for generating new disease models and eliminating concerns associated with the risk of immune rejection in future cell therapies of degenerative diseases. However, the current method to achieve somatic cell reprogramming requires viral delivery of the defined factors. Such an approach raises concerns over the unpredictable effects of random and irreversible transgene insertion, and the potential for tumourigenesis should these potent transgenes remain active in the derivatives of iPS cells. For future clinical applications of iPS cells to be realized, traceless removal of the transgenes must be achieved. The study described in this Example demonstrates successful and highly efficient non-viral reprogramming of murine embryonic fibroblasts using tetracycline inducible 'Yamanaka factors' delivered by piggyBac (PB) transposition. Stable iPS cells generated with this PB transposon vector approach express predicted pluripotency markers and form chimaeras when they are introduced to embryonic development. Finally, the natural propensity for precise PB excision offers the possibility of removing genomic modifications from iPS cell lines prior to downstream applications. This virus-independent simplification of iPS cell production has many applications in cell-based therapies.
To explore the utility of PB as a vector for somatic cell reprogramming, the 'Yamanaka' factors (mFx: c-Myc, Oct3/4, Klf4, and Sox2) [9] were placed via Gateway cloning into the PB-CAG transposon plasmid (Figure 12a) where they were under transcriptional control of the CAG promoter (CMV enhancer + chicken β-actin promoter) [10], and terminated by the rabbit β-globin polyadenylation signal [H]. Here, the expression cassette was oriented in the 5 '-^ 3' direction with respect to the defined terminal repeat regions minimally required to transpose flanked elements from a donor plasmid to genomic DNA [12]. Circular PB-CAG-mFx plasmid DNA 'cocktails' containing each of the four factors were delivered in conjunction with a PB transposase expression plasmid [5] using Fugene HD transfection of 15.5dpc-derived mouse embryonic fibroblasts (MEFs) as described below in Methods. From the time of transfection onwards, MEFs were maintained in ES cell culture conditions.
Distinct changes in morphology were noted in the fibroblast induction field within 3 days post-transfection (d3). Noticeable foci began to form over d4-6, with the first ES cell-like colonies arising as early as d6. No colony formation was observed in fibroblasts transfected with any single PB-CAG-mFx transposon. As shown in Figure 17a, the amount of transposon DNA used in the preparation of each transfection cocktail had a discernable effect on the number of ES cell-like colonies which formed over a ~3 week period. Morphologically verified colonies were scored on the indicated days, and later stained in situ for alkaline phosphatase (AP) activity [13] for visualization and as an additional confirmation of early stage reprogramming (Figure 17a). Transfection of 1.25x10 MEFs with lOOng of each PB-CAG-mFx transposon resulted in the highest colony numbers for the titration range (>1000 colonies on a 10cm2 surface by dl6), while IOng of each resulted in a nearly 50-fold decrease in colony formation; a density suitable for clonal isolation at late stages of induction (Figure 17a). Increasing PB-CAG-mFx plasmid quantities to 400ng each had a detrimental effect on colony formation, possibly revealing errors in reprogramming associated with high-copy PB transposition resulting in factor overexpression. Apart from the formation of colonies with characteristic ES cell morphology, colonies were also observed which had a rough, disorganized periphery or interior (Figure 17b), similar to non-reprogrammed variants illustrated in previous reports using viral induction [13]. Such colonies were not able to generate pure ES cell-like cultures upon initial cloning and passage, and were not considered during numeration. Individual colonies were picked from induction fields on dl2 on the basis of morphology, and maintained on mitomycin-inactivated fibroblasts. Of the 88 clones picked, 15 were intentionally chosen which initially displayed and later preserved non- compact growth, 9 clones failed to establish, and 10 clones resulted in altered morphology upon passaging. Approximately 61% (54/88) clones resulted in established cell lines which retained ES cell-like growth properties. On dl6, all of these 54 clones stained positive for AP, and an examined subset of eight clones tested positive alongside ES cell controls in Nanog immunostaining assays (data not shown).
Although it was evident that PB transposition could be effectively harnessed to deliver factors and initiate early reprogramming of somatic cells, transposons are not purposefully subjected to silencing processes which diminish retroviral expression. It was therefore necessary to achieve effective transgene silencing to demonstrate the reprogrammed cells' capacity for autonomous maintenance and subsequent differentiation. Reprogramming with inducible piggyBacs
In order to gain temporal expression control of the reprogramming factors, the tetθ2 tetracycline/doxycycline inducible promoter 14] was used to regulate factor expression in PB-TET (Figure 12b). The mFx coding regions in PB-TET are linked to an IRES-βgeo-pA cassette, which may be used as a reporter to assess the approximate factor expression level in both the induced (doxycycline (dox) plus) and non-induced (dox minus) states. The inducible expression unit in PB-TET is placed in the 3'->5' TR orientation to minimize any effects of the 5 'TR promoter/enhancer activity [4] on the basal level expression from tetθ2, as determined prior to using tetθ2 test constructs (Table 7). The reverse tetracycline transactivator protein (rtTA) was provided by parental MEFs (rtTA-MEFs) established from 15.5dpc Rosa26 rtTA-IRES-GFP knock-in [15] transgenic embryos. Based on results employing PB-CAG-mFx, lOOng of each PB-TET-mFx vector was used in the transfection cocktail. As with PB-CAG-mFx transposition, colonies produced by PB-TET-mFx cocktails were scored on the basis of morphology, followed by staining of post-induction plates for AP activity. Colony formation followed a similar progression to that observed with PB-CAG-mFx, however was typically delayed by 2 days. Foci formation was noted on days 6-8 with colonies forming around d8-10. Colonies formed by PB-TET-mFx were mostly ES cell-like, but also displayed diverse morphologies similar to those described for PB-CAG-mFx (Figure 17b).
To address the question of factor expression level effects on the initiation of reprogramming, a dox concentration range in replica inductions was used (Figure 12c). Under the standard dox treatment regiment (1.5μg/mL ES media) described herein the resulting PB-reprogrammed colonies were similar in number to that achieved with 1 Ong- mixtures of PB-CAG-mFx (compare the graph in Figure 12c with that of Figure 17a). Adjusting the dox concentration above or below the standard resulted in subtle declines (150ng/mL) or drastic decreases (15ng/mL or 15ug/mL) in colony numbers, indicating that factor expression level was induced within the required range for reprogramming induction using standard drug treatment. In the absence of dox-treatment, absolutely no reprogramming was noted, nor were any cells found positive for AP staining (data not shown). Diminution of the overall colony number using dox-inducible PB vectors for reprogramming may be the result of fundamental differences between the tetC^ and CAG promoters (such a sensitivities to genomic position effects) or core PB vector design differences (such as transcript length or the effect of IRES function). Despite the cause of this overall decrease in colony number from that obtained with CAG-driven factors, the maximum yield using PB-TET-mFx vectors did not differ greatly from previous reports using viral delivery [16]. PB-TET-mFx transposition provided ample, easily isolated colonies of high morphological quality and growth stability for subsequent applications. Endogenous pluripotency gene activation To establish stable cell lines, 48 PB-TET-mFx induced colonies were picked between dlO and dl2 from fibroblast induction fields, and passaged routinely (-1 :6 every 48hrs) on inactivated wildtype MEF feeder layers (Figure 12d). Clones were maintained in 1.5ug/mL dox during establishment, until found to be dox independent in duplicate wells. Dox independence occurred as early as dl2 for 3 lines (4D, 4E, 6C), but was most often achieved between dl6-18. All clones isolated which could be expanded became dox- independent by d22-24. During establishment, cultures which had not achieved autonomous maintenance of reprogramming rapidly flattened and returned to a fibroblast- like state upon dox withdrawal (Figure 19) as observed in a prior report [13]. Dox- independence was sustained for at least an additional 12 passages, with no major disturbance of growth characteristics as compared to replica cultures maintained in 1.5ug/mL dox. Growth characteristics with respect to doubling time and morphology for dox- inducible cell lines were indistinguishable from that of the Rl ES cell clones.
It has been shown that stable reprogramming by exogenous factor expression results in a sequential activation of ES cell markers [13, 17]. Alkaline phosphatase is an early but non-definitive marker of reprogramming which is followed by SSEA-I expression, and finally the more determinate activation of endogenous Nanog expression [18]. On dl6 (p2-3) all dox-independent lines tested passed the AP staining criterion (Figure 13a). Seven clones were chosen for further analysis, and on d20-22 all clones tested positive for the cell-surface marker SSEA-I and nuclear-localized Nanog protein (Figure 13a). Staining was similar to that of an Rl control ES cell line grown in parallel, while un-induced rtT A-MEFs were negative for both markers (data not shown). Further in-depth analysis into the characteristics of dox-independent cell lines by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed the expression of ES cell pluripotency markers [19, 21], such as Daxl, Eras, Fbxo, FoxD3, Nanog, Rexl and Zfp296 (Figure 13b). These genes were expressed in both Rl-ES cells and reprogrammed fibroblasts, but lacked detectable expression in parental rtT A-MEFs. In all cases, the clones tested expressed these pluripotency markers at a level similar to that found in Rl-ES cells. Using 3'UTR-specific reverse primers (Table 9), the endogenous counterparts of the four mFx transgenes were also shown to be fully active (Figure 13). Multiple PB insertions in induced cells
Genomic Southern analysis was used to determine the copy number in selected dox-independent PB-induced cell lines (Figure 10). The neo probe used detects all PB- TET transgene insertions, regardless of the mFx transgene delivered at the insertion site (Figure 10). The average estimated PB transposon copy number was 10, although co- migrating bands which may obscure the exact total for each cell line could not be ruled out using this analysis. Considering the necessity of 4 independent transgene insertions (3 if one assumes c-Myc is dispensable [22]), it is likely that most reprogrammed cell lines carry 2 or more copies of each mFx reprogramming factor. An interesting exception is line 3D which contained 6 transposon insertions. Since all four PB-TET-mFx transgenes were represented (see transgene-specific RT-PCR data in Figure 10) successful reprogramming of this line suggests that at least 2 of the four factors are sufficient in single copy. Induced and uninduced niFx expression
PB-TET-reprogrammed clones, cultured in the presence and absence of doxycycline were screened for lacZ expression (Figure 13d) to determine general transgene activity under either condition. LacZ expression is an indicator of all or any of the transgenes being activated, while only RT-PCR can distinguish which of the four factors remains active in the absence of dox (Figure 13c). Four clones tested by RT-PCR revealed that transgene expression levels are near that of their corresponding endogenous transcripts in the presence of dox. Interestingly, there was variable low level of expression of some transgenes in the uninduced state (dox-). Oct3/4 transgene expression could be detected in IB and to a lesser extent in 3B, and c-Myc was maintained at minimal levels in both IB and 6C (Figure 13c, lane 4). For clones IB and 3D Sox2 expression could be detected, while Klf4 appeared silent in all lines except for IB. Note that RT-PCR detection of mFx transcripts reflects the expression level for the population as a whole, and does not reflect the possibility of mosaic expression (as seen in Figure 13d), where there is only a small population of cells maintaining factor expression, while in others, the expression remains silent. PB reprogrammed cells are iPS cells From the panel of dox-independent, PB -reprogrammed cell lines, IB and 6C were chosen to test pluripotency in aggregation chimaeras with wildtype 8-cell stage mouse embryos. These lines showed differential responses to dox withdrawal, where 6C showed minimal expression of the PB-mFx transgenes (Figure 13c) and minor lacZ staining (Figure 13d), and line IB displayed residual expression of all four transgenes by RT-PCR (Figure 13c) and strongest lacZ staining of all cell lines tested (Figure 13d). Embryos resulting from aggregations were dissected on 12.5dpc (IB) and 15.5dpc (6C), and scored for GFP fluorescence indicating contribution of reprogrammed Rosa26-rtTA-IRES-GFP parental MEFs (Table 8). Chimaeras were obtained with high-level contribution from both lines (Figure Ha). Clone 6C produced a single strong chimaera as determined by GFP fluorescence. For clone IB, three out of seven embryos demonstrated high-level chimaeric contribution despite the residual mFx factor expression detected (Figure 13 c) suggesting a threshold level of tolerance which permits differentiation of these cells in the embryonic environment. To further demonstrate contribution, fibroblasts from the resulting GFP positive embryos were derived. Strong lacZ staining obtained 24 hours after dox induction (Figure l ib) proved the presence of reprogrammed cell derivatives, while the lacZ negative cell population indicated the chimaeric nature of the embryonic fibroblast cell lines. Sporadic faint lacZ positive cells in uninduced cells were also observed suggesting residual expression of some transgenes. The chimaeras obtained from cell lines were clear testaments that the PB transposon-mediated reprogramming generated iPS cells. Discussion
The results reported herein demonstrate that piggyBac transposition is a valid alternative for the delivery of reprogramming factors to somatic cells, achieving non-viral genesis of induced pluripotent stem (iPS) cells. As with select viral reports [22,23], PB- iPS colony selection was performed exclusively on the basis of morphology. Although it is difficult to make a direct comparison of efficiency between the two delivery methods, PB transposition provided iPS colonies at high frequency using small-scale cell culture. The high induction level observed using PB-CAG vectors is suitable for in vitro studies of the reprogramming process itself, during which robust factor expression is a requirement. Supplemented with small-molecule augmentation of iPS biogenesis [24], PB transposition may achieve the induction efficiency required for high-throughput peptide or chemical screens. Regulable factor expression by PB-TET transposition, on the other hand, permits further study of post-reprogramming processes. The Rosa26 knocked in rtTA in MEF provided convenience in these experiments, but it was not essential. Reprogramming with co-transfection of PB-TET-mFx and PB-CAG-rtTA expresser transposons has also been initiated (not shown).
Successful transposon-based reprogramming represents a number of significant improvements over current viral methods of delivery. First and foremost, PB-transposition allows technical simplification of reprogramming methodology. Without a viral vector intermediate, there is no need for specialized biohazard containment facilities or the production of high-titer, limited-lifetime [25], viral stocks. PB transposition makes use of simple plasmid DNA preparation, and vectors may be introduced into cells directly using commercial transfection products. Thus, accessible somatic cell types are limited by the effective range of the transfection reagent employed, rather than decreased susceptibility of certain cells to viral infection [26]. These practical simplifications will make reprogramming technology more accessible. Second, PB holds the potential for high- fidelity post-reprogramming transgene removal, once exogenous factor expression becomes dispensable. This approach is more thorough than FIp- or Cre-mediated deletion [27], since the PB terminal repeat elements themselves are removed during the excision process and no footprint remains in 95% of insertion sites [2, 5-8]. In mouse ES cells, PB transposase expression results in transposon mobilization in 1% of cells, with reinsertion occurring at a rate of 40%. Therefore, transposon loss may be expected to occur in more than 0.5% of cells, a rate which is amenable to negative selection schemes and would allow cells to be seamlessly cured of genomic modifications. The removal process could be further improved by first minimizing the initial number of transposon insertions into target somatic cells. Strategies such as factor consolidation using 2A peptide cleavage signals [28] into a single vector system allow single-copy transgene insertion while maintaining high efficiency iPS production. Finally, PB-mediated delivery allows the option of xeno-free production of human iPS cells contrary to current viral production protocols which use xenobiotic conditions. In combination with high-efficiency PB transposition, factor consolidation should allow somatic cell reprogramming to be achieved with single-copy, removable, multi-transgene expression, opening the door to potential clinical applications of iPS cells and their derivatives. Methods Summary PB-CAG and PB-TET expression vectors were generated using standard cloning procedures. Reprogramming factors were shuttled into PB plasmid vectors from retroviral sources using Gateway cloning (Invitrogen).
Fibroblasts (rtT A-MEFs) for PB transposition were isolated from 15.5dpc embryos resulting from heterozygous breeding of Rosa26 knock-in rtTA-IRES-GFP mice [15]. Passage three MEFs were seeded in ES media at 1.25xl05cells/10cm2 and transfected with PB-mFx transposon vectors and PB transposase expression plasmid using Fugene HD (Roche) 24hrs later. For PB-TET-mFx transfections, expression was induced with 1.5μg/mL dox after 24hrs. After 48hrs transfection, cells were fed with fresh ES media daily without passage. The resulting colonies were allowed to develop and were counted, or were picked on dlO-12 for establishment and analysis. iPS clones and ES cell controls were maintained on inactivated feeders, or gelatin for DNA and RNA isolations. lacZ staining was performed on 0.2% glutaraldehyde fixed cells overnight. AP staining was carried out according to the manufacturer's specifications (Vector Labs). Immunostaining used cells grown and fixed on chambered slides (Nunc) and primary antibodies directed to SSEAl (MC-480, Developmental Hybridoma Bank) or Nanog (AB5731, Chemicon). RT-PCR used cDNA prepared with the QuantiTect Reverse Transcription Kit (Qiagen). Southern blotting was carried out using standard methods and DIG-labeled probes for immunodetection (Roche).
PB-iPS cell lines were grown on feeders in the absence of dox for at least three passages prior to aggregation with 8 cell stage ICR host embryos [29]. The resulting embryos were dissected on the indicated days, scored for GFP, and dissociated to establish fibroblasts and confirm chimaerism via lacZ staining. Full Methods
Plasmid vector construction. PB-CAG was constructed by exchanging the 573 'TR- flanked neo cassette of PB-PGK-neo-bpA [5] digested with MeI (polished with T4 DNA polymerase) and HmdIII with a CBA-Rf A-rβgpA cassette released by OHI and HindIII digestion from a constitutive gene expression plasmid. For PB-TET, the PGK-neo cassette was replaced with a blunt Notl/HindUI fragment containing tetCh via digestion with MeI and HindIII (both polished), resulting in PB-tetO2. The MeI and Sail (polished) IRES- βgeo-bpA element from pIFS was ligated into PB-tetθ2 prepared by MeI and Smal digestion to yield PB-tetCh-IRES-βgeo. Finally, a Gateway RfA cassette (Invitrogen) maintained in the polished Notl site of pBluescriptKS+ (Stratagene), was inserted by digestion with Sacll and Spel into PB-tetO2-IRES-βgeo digested with Sacll and MeI.
The four reprogramming factors were shuttled from retroviral backbones (Addgene) into the PB-CAG and PB-TET transposon vectors using their flanking attBl/2 Gateway sites and pDONR221 (Invitrogen) as an intermediate. Sequence confirmation at the intermediate stage and restriction analysis of the final PB-mFx constructs was used to ensure correct recombination events.
Fibroblast isolation. 15.5dpc Rosa26-rtTA-IRES-GFP embryos were decapitated and eviscerated, washed in PBS, macerated and treated thrice with 0.25% trypsin, 0.1% EDTA to achieve a fine suspension which was plated on untreated dishes in MEF medium (DMEM, 10% FBS, penicillin-streptomycin, glutamax) and grown for 3 days at 37°C, 5% CO2. Primary rtTA-MEFs were passaged once for expansion and frozen in aliquots (p2). MEFs were used within p3 for this study. To obtain fibroblasts from iPS cells, chimaeric embryos were produced as described below and subjected to the above fibroblast preparation.
PB transfection and cell culture. Defrosted MEFs were allowed to reach 90% confluency (36-48hrs at 37°C, 5% CO2). Cells (1.25xl05/10cm2) were seeded in ES cell medium (DMEM, 15% FBS, penicillin-streptomycin, glutamax, β-mercaptoethanol, sodium- pyruvate, non-essential amino acids, 2 x leukemia inhibitory factor) to a gelatinized (0.1%) 6-well dish. After 18-24hrs growth, cells were transfected with a cocktail consisting of IOng, lOOng, or 400ng of each mFx transposon, and lOOng of pCyL43 PB transposase plasmid [5] (empty pBluescriptKS+ was used to normalize each cocktail to 2μg total DNA) in fresh ES media. To generate transfection complexes 2μg DNA cocktails suspended in lOμL sterile water were diluted with lOOμL DMEM, mixed with Fugene HD (Roche) at a Fugene:DNA ratio of 8μL:2μg, incubated at room temperature for 15-20min, and added to the media (day zero for PB-CAG inductions). For PB-TET -mFx transfections, each well was supplemented at 24hrs with dox at the appropriate concentration (dθ). 48hrs post-transfection, the media was replaced with fresh ES media. Cells were fed daily at the appropriate dox concentrations until analyzed.
Colonies were picked in 96-well format on mitomycin-c arrested feeder layers over dlO-12. Routine cultivation (1 :6 passage with trypsin every 2 days) of clones and control Rl ES cells was performed with maintenance in ES media on a feeder layer, while preparation for DNA or RNA required preplating on an untreated surface to deplete feeders (30-40min) and growth on gelatin (0.1%). For PB-TET induced clones, dox treatment was maintained until dl6-24, or removed earlier if replica dox- free cultures appeared stable for at least 48hrs (one passage). /αcZ/AP staining and immunofluorescence. lacZ staining was performed on cells fixed in situ with 0.2% glutaraldehyde, and stained overnight (~16hrs) in lacZ staining solution: 2OmM MgCl2, 5mM K4Fe(CN)6, 5mM K4Fe(CN)6 and lmg/mL X-gal in PBS.
Staining for alkaline phosphatase activity was performed on cells without fixation using the Vector Red Kit (Vector Labs), according to the manufacturer's specifications. Immunostaining used cells grown on a feeder layer plated on gelatin-coated
(0.1%), Lab-Tek chambered borosilicate glass slides (Nunc). Cells were washed with PBS, fixed in 4% PF A/PBS for lOmin at 25°C, washed, and permeabilized with 0.3% Triton X- 100 in PBS for 10 min at 25°C. After blocking with 5% goat serum (lhr), primary Ab was added overnight at 4°C (Nanog: AB5731, Chemicon, used at 1 : 1000; and SSEA-I : MC- 480, Developmental Hybridoma Bank, used at 1 :200). Samples were washed in PBS and secondary Ab (Nanog: mouse anti-rabbit secondary IgG, cy3 linked, used at 1: 1000; SSEA-I : mouse anti-mouse IgM, Alexa488 linked, used at 1:400) was added for lhr at 25°C. After washing samples were mounted in Vectashield with DAPI (Vector Labs). Images were acquired by confocal microscopy. Southern blotting. Genomic DNA from Rl ES cells, PB-iPS lines grown in feeder- depleted conditions or rtTA-MEFs was collected and purified. 10μg of DNA was digested with BarήΑl overnight, resolved by gel electrophoresis, and transferred to Hybond N+ (GE Healthcare). The neo probe was isolated by PCR amplification of the neomycin resistance gene coding region from plasmid, gel purified, and labeled with DIG High Prime DNA Labeling and Detection Kit II (Roche). Transposon insertions were detected with the neo DIG-labeled probe (~25ng/mL hybridization solution), processing the membrane and performing DIG immunodetection according to the manufacturer's instructions. RT-PCR. RNA was collected using RNeasy Mini Kit (Qiagen) from Rl ES, PB-iPS and rtTA-MEF cells grown in the presence or absence of dox. RNA was quantified and treated with gDNA WipeOut prior to preparation of cDNA with QuantiTect Reverse Transcription Kit (Qiagen) according to the guidelines provided by the manufacturer. For each reaction, l-2uL of cDNA was used. RT-PCR primers used in this study (Table 9) were obtained through Operon. Standard PCR conditions were: 94°C for 30 sec, 58-62°C for 30sec, 72°C for 15-30sec; x30 cycles. Reaction products were resolved using standard gel electrophoresis. Diploid aggregations and chimaera production. On the day before aggregation, dox- independent iPS cells were preplated and passaged at low density on gelatinized dishes. The following day ES cell clumps of -8-12 cells were collected by gentle trypsinization and suspended in ES cell medium. Diploid ICR host embryos were collected ~20hrs prior to aggregation and maintained overnight in KSOM medium to delay development. Aggregation with iPS cells (lines IB and 6C, 120 embryos each) was performed using standard techniques [29]. The resulting embryos were transferred after 24hrs in groups of 20 to recipient pseudopregnant mothers. Example 3
Transgenic expression of just four defined transcription factors is sufficient to reprogram somatic cells to a pluripotent state [9, 18, 23, 30]. The resulting induced pluripotent stem (iPS) cells resemble ES cells in their properties and potential to differentiate into a spectrum of adult cells types. Current reprogramming strategies involve retroviral [9], lentiviral [13], adenoviral [31] and plasmid [32] transfection to deliver reprogramming factor transgenes. The latter two approaches are seriously hampered by limited efficiency while the other viral systems evoke obvious limitations and concerns for both in vitro and in vivo utilization of these cells. Here successful and efficient non-viral reprogramming of murine and human embryonic fibroblasts is illustrated using tetracycline inducible 'Yamanaka factors' delivered by piggyBac (PB) transposition [I]. Stable iPS cells generated with this PB transposon vector approach express hallmark pluripotency markers and perform in a series of rigorous differentiation assays. Additionally, by taking advantage of the natural propensity of PB for seamless excision [6], the traceless removal of reprogramming factors joined with 2A sequences (see Example 1) and delivered by a single transposon is demonstrated. By the same mechanism individual factors can also be removed in a combinatorial manner. Such controlled reduction of reprogramming factors from established iPS cell lines is an invaluable tool.
Despite efforts to generate iPS cell lines without transgene integrations [31,32], to date only integrating viral delivery systems have been able to provide sufficient simultaneous introduction of multiple reprogramming factors into somatic cells in order to achieve efficient iPS cell establishment in mouse and human. The PB transposon/transposase system is an effective transgene delivery system, which requires only the inverted terminal repeats (5 'TR and 3'TR) flanking a transgene and transient expression of the transposase enzyme [I]. PB transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines [2, 3], including mouse embryonic stem (ES) cells [4,5].
To explore the utility of PB as a vector for somatic cell reprogramming, the 'Yamanaka' mouse factors (mFx: c-Myc, Oct4, Klf4, and Sox2) [9] were transferred into the PB-CAG transposon plasmid (Figure 12a) under the transcriptional control of the CAG promoter [10]. Human (HEF) and mouse (MEF) embryonic fibroblasts were transfected with circular PB-CAG -mFx plasmid DNA 'cocktails' containing each of the four factors in conjunction with a PB transposase expression plasmid [5]. From the time of transfection onwards, fibroblasts were maintained in relevant ES cell culture conditions without passage before picking. Both human and mouse fibroblast cells underwent ES cell-like colony formation, which resulted in the derivation of self-renewing cell lines displaying key characteristics of reprogramming (for details see Figures 17 and 18). The mouse factors effectively initiated reprogramming of human cells underlining the functional conservation of these transcription factors between the two species.
Although PB-CAG transposition was effectively harnessed to deliver factors and initiate early reprogramming, PB transposons are not purposefully subjected to the same silencing process that diminish retroviral (and less extensively lentiviral) expression [33]. It was therefore necessary to achieve temporal expression control and transgene silencing to demonstrate the reprogrammed cells' capacity for autonomous maintenance and subsequent differentiation. The tetCh tetracycline/doxycycline (dox) inducible promoter [14] was used to regulate factor expression in PB-TET (Figure 12b). The mFx coding regions in PB-TET are linked to an IRES-βgeo-pA cassette, which may be used as a reporter to approximate factor expression in both the induced (dox +) and non-induced (dox -) states. The inducible expression unit in PB-TET is placed in the 3'->5' TR orientation to minimize any effects of the 5 'TR promoter/enhancer activity [4] on the basal level expression from tetθ2, as determined prior to using tetθ2 test constructs (Table 7). The reverse tetracycline transactivator (rtTA) protein was provided by parental MEFs (rtTA-MEFs) established from 15.5dpc ROSA26 rtTA-IRES-GFP knock-in [15] transgenic embryos.
Colonies produced by PB-TET-mFx cocktails were scored on the basis of morphology, with foci formation noted on days 6-8 and colonies forming around d8-10. These were mostly ES cell-like, while a handful displayed diverse morphologies similar to those described for PB-CAG-mFx (Figure 17b). Adjusting the dox concentration above or below the standard of 1.5μg/mL resulted in subtle declines (150ng/mL) or drastic decreases (15ng/mL or 15μg/mL) in colony numbers (Figure 12c) supporting the notion that factor expression level affects reprogramming rates. In the absence of dox, no reprogramming was noted, nor were any cells found positive for AP staining (data not shown). Equal efficiency of colony formation was achieved by lOOng-mixtures of PB- TET-mFx and IOng-mixtures of PB-CAG-mFx (compare the graph in Figure 12c with that of Figure 17a), possibly due to more consistent expression levels from the CAG promoter.
Fourty-eight PB-TET-mFx induced colonies were picked from fibroblast induction fields and passaged on inactivated fibroblast feeder layers (Figure 12d). Surviving clones were maintained in dox during establishment, until found to be dox independent in duplicate wells. Dox independence occurred as early as dl2 for 3 lines (4D, 4E, 6C), and was achieved by most clones by d24 (7/39=18% on dl5, 21/39=54% on dl9, 31/39=80% on d21, 33/39=86% on d23). During establishment, cultures which had not yet achieved autonomous maintenance of reprogramming rapidly flattened and returned to a fibroblast- like state upon dox withdrawal (Figure 19) as observed in a prior report [13]. Dox- independence was sustained for at least an additional 12 passages, with no apparent disturbance of growth characteristics compared to replica cultures maintained in 1.5μg/mL dox. Doubling time and morphology of PB-TET clones were indistinguishable from that of Rl mouse ES cells [34] (data not shown). It has been shown that stable reprogramming by exogenous factor expression results in the sequential activation of ES cell markers [13, 17, 18]. On dl6 (p2-3) all dox- independent lines tested passed the alkaline phosphatase (AP) staining criterion (Figure 13a). Seven clones analyzed further tested positive on d20-22 for the cell-surface marker SSEAl and the nuclear-localized Nanog protein (Figure 13a). Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed the expression of ES cell pluripotency markers [19, 21, 35], such as Daxl, ERas, Fbxo, FoxD3, Nanog, Rexl and Zfp296 (Figure 13b). Using 3'UTR-specific reverse primers (Table 9), the endogenous counterparts of the four mFx transgenes were also shown to be active (Figure 13 c).
PB-TET-reprogrammed clones were screened for lacZ expression to determine general transgene activity in the presence and absence of doxycycline (Figure 13d). RT- PCR, which allows further distinction of individual transgene activity, revealed variable trace expression levels of some transgenes in the uninduced (dox -) state (Figure 13 c). Oct4 transgene expression could be detected in IB and to a lesser extent in 3B, and c-Myc was maintained minimally in both IB and 6C. For clones IB and 3D Sox2 expression could be detected, while Klf4 appeared silent in all lines except for IB. Note that RT-PCR detection of mFx transcripts reflects average expression levels for each cell population, and does not expose mosaic expression patterns (as revealed in Figure 13d). Genomic Southern analysis was used to determine transgene copy number in selected dox-independent mouse PB-TET lines (Figure 20a). The average estimated PB transposon copy number was 9, although co-migrating bands which may obscure the exact total could not be ruled out. Line 3D contains only five transposon insertions. As all four PB-TET-mFx transgenes are represented in 3D (Figure 13c), three of the four factors are sufficient in single copy for reprogramming.
Transposons located at the ROSA26 locus in mouse ES cells are mobilized by additional transient transposase expression at a rate of 0.7% [5]. Furthermore, for -60% of these mobilization events reinsertion does not occur, resulting in loss of the transposable element. Clone IB, which contains an average number of insertions, was used to characterize the "cut" and "paste" transposition steps in reprogrammed cells (Figure 20, 21). Of 38 IB subclones established after transient expression of transposase, 12 remained unchanged (12/38=32%). From the total number of transposon insertions (38x9), -15% (52 transgenes) underwent mobilization of which 54% were not pasted back to the genome (28/52 transgene loss); an absolute transposon removal rate of -7%. A broad range was observed in the mobilization rate for each transposon insertion (Figure 20, 21); transposon #8 was excised only once (1/38=2.5%) while transposon #9 was excised 11 times (11/38=29%). Such high variability suggests that the excision frequency is integration site- dependent. The "cut" step of transposition was utilized to remove the transgenes following complete reprogramming. To simplify the removal event the MKOS sequence (c-Myc, Klf4, Oct4 and Sox2 ORFs linked with 2 A peptide sequences (see Example 1) was inserted into the PB-TET transposon (Figure 14a). Using culture conditions similar to four-factor reprogramming with PB-TET-mFx, induction of rtTA-MEFs with PB-TET- MKOS in the presence of dox yielded colonies after 12-14 days, at which point 48 iPS cell-like colonies were picked, expanded in the absence of dox, and subjected to diagnostic Southern analysis (not shown) to determine the absolute number of transposon insertions. The analysis revealed two single-copy cell lines, designated as scBl and scC5. The flanking genomic sequences of the single-copy transposon insertions were determined by splinkerette PCR [5]. PB inserted into the first intron of the Myold gene (Chrl 1) in csBl, and into an intragenic region of Chrl 6 in scC5 (Figure 14b); insertion events were also confirmed by locus-specific genomic PCR (Figure 14e). These lines showed typical ES cell morphology, and indirect measurement through the lacZ reporter revealed no expression of the MKOS reprogramming factors in the absence and high inducibility in the presence of dox (Figure 14c). For both scBl and scC5, transient transfection of PB transposase led to the removal of the linked reprogramming factors in greater than 2% of the exposed cells as estimated by the ratio of lacZ negative sublines in the presence of dox (Figure 14c). Sequence analysis across the original site of insertion revealed that in ten of the eleven subclones the original transposon integration site was reverted to wild type (Figure 14d). Complete loss of the PB-TET-MKOS transposon was further confirmed by genomic PCR revealing subclones which were negative for both genome-transposon junction and internal transposon specific PCRs (Figure 14e). Even after the exogenous factors were removed, the endogenous pluripotency genes remained actively expressed (Figure 14f).
Pluripotency of PB-reprogrammed cell lines (IB, 3D, 6C, scBl and scC5) was demonstrated by their contribution to chimaera development following aggregation with 8- cell stage ICR embryos (Figure 15a,b). The resulting embryos were dissected at 10.5dpc (IB and 3D), 12.5dpc (IB), 14.5 (scBl and scC5) and 15.5dpc (6C), and scored for GFP fluorescence indicating contribution of reprogrammed ROSA26-rtTA-IRES-GFP parental MEFs (Table 10). Chimaeras with high-level contribution were obtained (Figure 15a). To reveal chimaeric contribution at the cellular level, recipient dams were treated with dox 2Oh prior to dissection, and then embryos were stained for lacZ expression (Figure 15b, Table 9). Chimeras treated with dox displayed lacZ positive cells in tissues derived from all three germ layers upon sectioning (Figure 15b). Interestingly, for cell line IB, many embryos demonstrated high-level chimaeric contribution despite low-level residual factor expression (Figure 13c,d), suggesting a threshold that permits differentiation of these cells in the embryonic environment. Pluripotency achieved by PB reprogramming was stringently confirmed by the production of completely iPS cell-derived 13.5dpc embryos via tetraploid embryo complementation assay [34], including germ cell formation detected by Vasa expression in the genital ridge (Figure 15c). Germ cell formation from iPS cells was also detected in genital ridges of standard diploid chimaeric embryos (Figure 22a). Teratomas containing derivatives from all three embryonic germ layers are additional proof that these cells are pluripotent and able to differentiate to complex tissues (Figure 22b). The chimaeric contribution of iPS cells to postnatal animals (Figure 15d) is a clear testament to PB transposon-assisted complete reprogramming of fibroblasts to iPS cells (PB-iPS) with the capability of building functional adult tissues.
To avoid the necessity for rtTA transgenic HEF lines, a PB-CAG-rtTA transposon was successfully employed as a "fifth factor" in the reprogramming of human embryonic fibroblasts. Doxycycline tightly regulates the expression of the reprogramming factors; expression in the uninduced state was not detectable by lacZ staining for the factor-linked IRES-βgeo reporter (Figure 23a) and only trace amounts of transcription was detected in colonies by semi-quantitative RT-PCR (Figure 23b). Dox activation, however, evoked expression of the endogenous forms of the inducer genes to the level characteristic of human ES cells (Figure 23b) and activated hallmark pluripotency marker genes (Figure 23c) including nuclear-localized NANOG (Figure 24), eventually resulting in the derivation of dox independent human iPS cells. Spontaneous differentiation following bFGF withdrawal from the medium gave rise to cystic embryoid bodies and to various differentiated cell types, including AFP (endoderm), α— SMA and vWF (mesoderm) or Bill TUBULIN and GFAP (ectoderm) positive cells (Figure 25).
Chimeric embryos from three mouse iPS cell lines (IB, 6C and 3D) were used to derive chimeric fibroblasts. The iPS cell-derived secondary fibroblast component (2°F/1B, 2°F/6C and 2°F/3D) was tracked by the expression of GFP (Figure 15 and Figure 16a). Adding dox to the medium had a dramatic effect on 2°F/1B and 2°F/6C, which initiated early signs of reprogramming within two days, including cellular aggregation. Proliferation of 2°F-derived cells increased dramatically, (Figure 16a, b) as initial contribution of the GFP positive cells (10-18% on day 0) reached 90% by day 10 (Figure 16b). These physical changes were reflected at the molecular level by the activation of endogenous pluripotency gene expression, such as AP (not shown) and SSEAl (Figure 16a, c). Interestingly, Nanog activation occurred four days earlier (by day 9) in 2°F/1B than in 2°F/6C (Figure 26), highlighting clonal variation and flexibility in the reprogramming process. Time course FACS analysis for the appearance of the pluripotency marker SSEAl showed very rapid activation; detected as early as day 2 and expressed by nearly 50% of 2°F/1B and /6C cells after 4 days of dox treatment (Figure 16a, c). Reprogramming continued as the ratio of positive cells was greater than 80% on day eight. Interestingly, these PB-iPS cell derived fibroblasts displayed a much more rapid initial response to reprogramming factor induction than that previously reported for the dox inducible lentivirus system [37], which may reflect the high level of instability of lentivirus-inserted transgene expression. Moreover, single cell sorting of 2°F/6C cells into 96 well plates revealed that 56 of 192 single cells (29%) were capable of forming colonies. Thirty-nine out of fourty-four (89%) established single-cell clones evaluated for Nanog expression were found positive on day 13 of induction. In contrast, line 2°F/3D was inefficient at 2° iPS cell production, as few colonies formed under similar conditions to 2°F/1B and 2°F/6C and expression of AP and SSEAl was delayed (not shown). To explain this impotence, the level of reprogramming transgene activation by dox was tested by RT- PCR in all the three 20F cell lines. The PB-TET-Oct4 transgene was noticeably less active in 2°F/3D as compared to /IB and /6C (Figure 16d), signifying the need for a sufficient level of Oct4 expression even in secondary iPS cell induction. Interestingly no major change was observed in the levels of exogenous factor expression at the beginning (day 2) or later in the progress of reprogramming (day 12) (Figure 16d).
Successful transposon-based reprogramming of fibroblasts to iPS cells represents significant improvements over current viral methods of delivery. First, PB transposition permits technical simplification and improved accessibility of the reprogramming methodology by making use of effortless plasmid DNA preparation and commercial transfection products for delivery. This eliminates the need for specialized biohazard containment facilities or the production of high-titer, limited-lifetime viral stocks [25]. Secondly, the range of somatic cell types that could be used for reprogramming are not limited by the decreased susceptibility to viral infection [25]. Thirdly, PB-mediated delivery will allow the option of xeno-free production of human iPS cells contrary to current viral production protocols that use xenobiotic conditions. Finally, transgene removal through transposase expression has been demonstrated in other cell lines [2, 5, 6, 7, 8, 38] have harnessed this potential and show here that the reprogramming factors can be removed without a trace from iPS cells once exogenous expression becomes dispensable. These four key characteristics of PB transgenesis mark important advances towards achieving clinically acceptable methods of deriving reprogrammed cells. Complementary, secondary fibroblasts or alternate secondary cell types with a combinatorial removal of proven reprogramming transgene insertions may provide an enormously powerful tool for high throughput screening to further explore and eventually fully understand the mechanisms which play a pivotal role in the reprogramming process. Methods Summary
PB-CAG and PB-TET expression vectors were generated using standard cloning procedures. Reprogramming factors were shuttled into PB plasmid vectors from retroviral sources using Gateway cloning (Invitrogen). MEFs were isolated from 15.5dpc ROSA26 knock-in rtTA-IRES-GFP [15] embryos. HEFs were derived from 12 week abortuses. Fibroblasts were seeded in ES media at 1.25XIO5CeIIsZlOCm2 (MEFs) or 6.25XIO4CeIIsZlOCm2 (HEFs) and transfected with PB-mFx transposon vectors and PB transposase expression plasmid 24hrs later. For PB- TET-mFx transfections, expression was induced with 1.5μgZmL dox the following day. 48hrs after transfection, cells were fed with fresh ES media daily without passage. The resulting colonies were picked on dlO-12. iPS clones were maintained on inactivated feeders, or gelatin coated dishes. lacZ staining was performed overnight on 0.2% glutaraldehyde fixed cells. AP staining was done according to the manufacturer's specifications (Vector Labs). RT-PCR used cDNA prepared with the QuantiTect Reverse Transcription Kit (Qiagen). Southern blotting was carried out using standard methods and DIG-labeled probes for immunodetection (Roche). Immunostaining was performed on cells grown and fixed on chambered slides (Nunc) or 4-well dishes. Flow cytometry used live cells stained with SSEAl. Mouse PB-TET clones were grown in the absence of dox for at least three passages prior to aggregation with diploid or tetraploid ICR or diploid C57BLZ6 host embryos [29]. The prenatal embryos were dissected, scored for GFP and then further processed to perform IH or to derive secondary MEFs. Embryos prepared for lacZ staining and sectioning were treated with dox in utero 20hrs before dissection. Teratoma formation using PB-TET clones was performed as previously described [9]. Differentiation of human iPS was spontaneous on Matrigel following plating of EBs formed in AggreWell dishes. Full Methods Plasmid vector construction.
PB-CAG was constructed by exchanging the 5' /3' TR- flanked PGK-neo cassette of PB-PGK-neo-bpA [5] digested with MeI (polished with T4 DNA polymerase) and Hinάlll with a CBA-RfA-rβgpA cassette released by OHI and HindIII digestion from a constitutive gene expression plasmid (unpublished data). For PB-TET, the PGK-neo cassette was replaced with a blunt Notl/HindUI fragment containing tetO2 (provided by S. Mohammadi) via digestion with Nhel and HmdIII (both polished), resulting in PB-tetθ2. The Nhel and Sail (polished) IRES-βgeo-bpA element from pIFS (provided by J. Dixon) was ligated into PB-tetθ2 prepared by Nhel and Smal digestion to yield PB-tetθ2-IRES- βgeo. Finally, a Gateway RfA cassette (Invitrogen) maintained in the polished NM site of pBluescriptKS+ (Stratagene), was inserted by digestion with Sacll and Spel into PB-tetCV IRES-βgeo digested with Sacll and NAeI.
The four reprogramming factors were Gateway cloned from retroviral backbones (Addgene) into the PB-CAG and PB-TET transposon vectors using pDOΝR221 (Invitrogen) as an intermediate. The MKOS element from pCAG2LMK0Sim0 (Kaji et al.) was cloned into pENTR2B using EcoRl prior to Gateway shuttling. The Tet transactivator was amplified from pTet-On Advanced (Clontech) using attBl/2 primers (Table 9). Fibroblast isolation. 15.5dpc ROSA26-rtTA-IRES-GFP embryos (from Gt(ROSA)26Sortml [
(rtτA,EGFP)Na g y ^anSg6nJ0 crosses) were decapitated and eviscerated, dissociated with 0.25% trypsin, 0.1% EDTA, and plated on uncoated dishes in MEF medium (DMEM, 10% FBS, penicillin-streptomycin, glutamax). Primary rtTA-MEFs were passaged once for expansion and frozen (p2). ΗEFs were isolated from 12 week-old abortion material, with maintenance and expansion in ΗEF media (DMEM, 15% human serum, IOng/mL bFGF, penicillin- streptomycin, glutamax, β-mercaptoethanol, sodium-pyruvate, non-essential amino acids). PB transfection and cell culture.
MEFs were grown to 90% confluency (36-48hrs at 37°C, 5% CO2) and seeded in mouse ES cell medium (DMEM, 15% FBS, penicillin-streptomycin, glutamax, β- mercaptoethanol, sodium-pyruvate, non-essential amino acids, LIF) to a gelatinized (0.1%) 6-well dish (p3; 1.25x10 cells/lOcm ). After 24hrs growth, FugeneΗD (Roche) was used to transfect cells with IOng, lOOng, or 400ng of each mFx transposon (25ng, 50ng, or lOOng for PB-TET-MKOS) plus lOOng of pCyL43 PB transposase plasmid [5] (normalized to 2μg total DNA with empty pBluescriptKS+) at a Fugene:DNA ratio of 8uL:2μg (day zero for PB-CAG inductions). For PB-TET inductions, media was supplemented at 24hrs with dox (dθ). 48hrs post-transfection, the media was replaced with fresh ES media. Cells were fed daily at the appropriate dox concentration (standard 1.5ug/mL, unless otherwise indicated). MEF-derived colonies were picked in 96-well format over dlO-14 and routinely cultivated on mitomycin-c arrested fibroblast feeders. For PB-TET induced clones, dox treatment was typically maintained until dl6-24. iPS cells for DNA or RNA preparation were grown on gelatin. Established iPS cells were passaged 1:6 every 48 hours.
Transfection of HEFs was performed similarly, except fibroblasts were initially seeded in DMEM supplemented with 15% human serum, IOng/mL bFGF, penicillin- streptomycin, glutamax, non-essential amino acids at a density of 6.25x104cells/l 0cm2, and grown in HEScGRO (Millipore) 48 hours after transfection. Doxycycline (1.5 μg/ml) was added 24h post transfection to the medium. From four wells of a six- well plate 17 colonies were picked between day 14 and 29, five of them were AP positive and four of those grew doxycycline independently to a permanent cell line. Doxycycline was withdrawn a week after picking. The cells were initially passaged using mechanical cutting. Subsequent passaging was done with TripLE Select (Invitrogen) at a rate of 1:2 and later 1 :4 every 7days. Human iPS cells were maintained on inactivated MEF feeders in human ES medium (KO-DMEM, 20% serum replacement, IOng/mL bFGF, penicillin- streptomycin, glutamax, non-essential amino acids). Southern blotting.
Ten micrograms of genomic DNA from Rl ES cells, PB-iPS lines or rtTA-MEFs was digested with BamHI overnight, resolved by gel electrophoresis, and transferred to Hybond N+ (GE Healthcare). The neo probe was isolated by PCR amplification of the neomycin resistance gene from plasmid, gel purified, and labeled with DIG High Prime DNA Labeling and Detection Kit II (Roche). Transposon insertions were detected with the neo DIG-labeled probe (~25ng probe/mL hybridization solution) after processing the membrane and performing DIG immunodetection according to the manufacturer's instructions. Splinkerette, Genomic, and RT-PCR. Splinkerette PCR to determine PB genomic integration sites was performed as described [5]. PCR products TA-cloned and sequenced bidirectionally with Ml 3 forward and reverse primers. PB insertion loci were determined using BLAST. Genomic PCR on factor-removed PB-iPS lines was performed using primer sets described in Table 9. Approximately lOOng of genomic template DNA was amplified using Qiagen Taq (Qiagen) with the inclusion of Q-Solution. Due to highly repetitive sequence, chromosome 16 amplification required the use of nested PCR. Three-primer PCR amplification used PB-3F in conjunction with the chromosome-specific primer set. Standard PCR conditions were: 95°C for 30 sec, 55°C for 30sec, 72°C for 45sec; x35 cycles. Reaction products were resolved using standard gel electrophoresis.
RNA was collected using RNeasy Mini Kit (Qiagen) from the indicated cell lines grown in the presence or absence of dox. RNA was quantified and treated with gDNA WipeOut prior to preparation of cDNA with QuantiTect Reverse Transcription Kit (Qiagen) according to the guidelines provided by the manufacturer. For each reaction, 1 - 2μL of cDNA was used. RT-PCR primers used in this study (Table 9) were obtained through Operon. Standard PCR conditions were: 94°C for 30 sec, 58-62°C for 30sec, 72°C for 15-30sec; x30-35 cycles. Reaction products were resolved using standard gel electrophoresis.
PB transgene removal.
Stable PB-TET clones were treated transiently with pCyL43 transposase plasmid (2μg DNA: 8uL FugeneHD). Non-transfected cells were eliminated by treatment with puromycin (lμg/mL) for 3 days. Viable cells were counted and plated at clonal density ^SOO-lOOOcells/όOcm2) on inactivated feeders. Clones were picked after 5 days for expansion, lacZ staining and DNA preparation. lacZ/AP staining. lacZ staining was performed on cells or embryos fixed in situ with 0.25% glutaraldehyde, and stained overnight (~16hrs) in lacZ staining solution: 2OmM MgCl2, 5mM K3Fe(CN)6, 5mM K4Fe(CN)O and lmg/mL X-gal in PBS. Embryos were rinsed in wash buffer (2mM MgCl2, 0.01% Sodium deoxycholate, and 0.02% Nonidet-P40 in PBS) prior to whole-mount lacZ staining. Embryos were embedded in paraffin, sectioned using Microm Ergostar HM200 microtome, and counterstained with neutral red.
Staining for alkaline phosphatase activity was performed on cells without fixation using the Vector Red Kit (Vector Labs), according to the manufacturer's specifications. Immunostaining and flow cytometry. Immunostaining used cells grown on an inactivated feeder layer plated on gelatin- coated Lab-Tek borosilicate glass slides (Nunc) or 4-well dishes containing gelatinized or Matrigel-treated glass coverslip inserts. Cells were washed with PBS, fixed in 4% PFA/PBS for lOmin at 25°C, and permeabilized with 0.3% Triton X-IOO in PBS for 10 min at 25°C. After blocking (5% goat serum for lhr), primary Ab was added overnight at 4°C (mNanog, AB5731, Chemicon, 1: 1000; hNanog, 0002P-F, ReproCell, 1:200; SSEAl, SSEA4, Tral-60, Tral-81, 1:5, provided by P. Andrews; Muscle Actin, M0635, DakoCytomation, 1: 100; βlll-Tubulin, TU-20, Millipore, 1 : 100; HNF-3β, sc-9187, Santa Cruz, 1 : 100; GFAP, Z0334, DakoCytomation, 1 :200; AFP, MAB 1369, R&D Systems, 1 :200)). Samples were washed in PBS and secondary Ab (cy3 IgG, 1 : 1000; Alexa488 IgG or IgM, 1 :400; Alexa594 IgG, 1 :200) was added for lhr at 25°C. After washing, samples were mounted in Vectashield with DAPI (Vector Labs).
Genital ridges were pre-fixed with 4% paraformaldehyde in PBS for 1 hour at 25°C, then embedded and cryosectioned at 30μm thickness. Sections were washed, blocked (5% goat serum for lhr), and incubated overnight at 4°C with rabbit anti- primordial germ cell marker (DDX4/MVH, abl3840, abeam, 1 :400). Sections were washed in PBS and secondary Ab (cy3 IgG, 1:500) was added for 2hrs at 25°C. All immunofluorescence was visualized and obtained using a Zeiss LSM 510 confocal microscope equipped with UV, argon and helium-neon lasers (Zeiss). Dox-induced fibroblast pools from PB-iPS chimaeric mice for FACS analysis were diluted to -15% GFP positive 2°F/1B and /6C representation with wildtype ICR MEFs prior to seeding. Cells were harvested on the indicated day, washed once in PBS containing 5% FBS, and incubated with anti-SSEAl antibody (1 :200) for 30min on ice. Cells were washed twice in PBS:5% FBS, and incubated with Alexa647 conjugated secondary antibody for 30min on ice. Cells were washed twice and resuspended in PBS:5% FBS for analysis on a FACS-Calibur. Single cell plating from PB-iPS chimeric MEF pools was performed using a FACS-Aria, gating on the GFP positive cell fraction. In vitro differentiation assays.
Human PB-iPS lines were dissociated and used to generate embryoid bodies (EBs) by aggregation in AggreWell 400 plates (StemCell Technologies) in 15% FBS DMEM with all additives except LIF and bFGF, in the absence of dox. After 14 days growth, EBs were collected and permitted to attach on Matrigel coated cover slips or 4 chamber slides in the same medium. After 10 days outgrowth the resulting cultures were analyzed by immunohistochemistry.
Teratoma formation.
Cell lines were suspended in DMEM containing 10% FBS, and lOOuL (IxIO6CeIIs) injected subcutaneously into both dorsal flanks of nude mice (CByJ. Cg-Foxnlnu/J) anesthetized with isoflurane. Six weeks after injection, teratomas were dissected, fixed overnight in 10% buffered formalin phosphate and embedded in paraffin. Sections were stained with hematoxylin and eosin. Generation of chimaeras. On the day before aggregation, dox-independent PB-TET clones were passaged at low density on gelatinized dishes. The following day PB-iPS cell clumps of ~8-12 cells were collected by gentle trypsinization and suspended in mouse ES cell medium. For standard diploid chimeras, ICR or C57BL/6 embryos were collected at 2.5dpc, aggregated with PB-iPS cell clumps, cultured overnight and transferred into pseudopregnant recipients using standard techniques [29]. Embryos were dissected in cold F 12 media plus HEPES and examined for GFP on the indicated day, or left to term to verify iPS contribution by coat color. For lacZ detection in embryos, pregnant dams were treated with dox (1.5μg/mL dox; 5% sucrose in water) 20hrs prior to dissection. For tetraploid embryo complementation, the blastomeres of two-cell stage embryos (1.5dpc) from superovulated ICR females were electro fused by using a CF- 150B Pulse Generator (BLS). The fused embryos were cultured overnight at 37°C in 5% CO2 in KSOM medium (Specialty Media). Two tetraploid embryos at the four-cell stage were aggregated with a clump of ~8- 15 ES cells. The following day, embryos were transferred into pseudopregnant recipient ICR females [29]. Embryos were dissected at 13.5dpc and contribution of GFP-positive cells (iPSC-derived) was visualized by using Leica MZ 16 FA stereomicroscope.
While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Figure imgf000076_0001
Figure imgf000077_0001
Reprogramming efficiency was estimated using number of Nanog-GFP/Nanog positive colonies, mOrange positive cell number at day 2 in Table 1, and stable transfection efficiency (3.6% of transient transfected cells as estimated in Figure 5). -; no data.
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
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Claims

We Claim:
1. A method of reprogramming a somatic cell comprising: a) providing a non-viral based vector system which is capable of expressing one or more reprogramming factors necessary to reprogram said somatic cell; b) transfecting said vector into said somatic cell; and c) expressing said reprogramming factors, such that reprogramming of said somatic cell can take place.
2. The method of claim 1 wherein the non-viral based vector system is capable of expressing at least two or more reprogramming factors.
3. The method of either of claims 1 or 2 wherein the non-viral based vector system comprises a single vector comprising reprogramming sequences encoding said reprogramming factors.
4. The method of claim 3 wherein said reprogramming factors are expressed from a single promoter.
5. The method of claim 4 wherein the sequences encoding said reprogramming factors are each separated by a sequence encoding a self cleaving peptide.
6. The method according to claim 5 wherein said self cleaving peptide is the 2 A peptide from foot and mouth virus, or a 2A like peptide from another virus.
7. The method of any preceding claim wherein the non-viral based vector system comprises one or more transposon region(s) each comprising a transposon and one or more reprogramming sequences encoding reprogramming factors.
8. The method of claim 7 wherein the non-viral based vector system comprises a plurality of transposon regions each comprising a transposon and a reprogramming sequence.
9. The method of claim 7 wherein the non-viral based vector system comprises a single transposon region comprising a transposon and at least two, three or four reprogramming sequences.
10. The method of claim 7 wherein the non-viral based vector system comprises four separate transposon regions each comprising a transposon and a reprogramming sequence.
1 1. The method of any one of claims 7 to 10 wherein the transposon region comprises a piggyBac transposable element.
12. The method of any of claims 3 to 1 1 wherein the reprogramming sequences are a combination of genes from an Oct gene family, a KIf gene family, a Sox gene family, and optionally a Myc gene family.
13. The method of claim 9 or 10 wherein each reprogramming sequence in the non- viral based vector system is different and is selected from each of the Oct, KIf, Sox; and optionally Myc gene families.
14. The method of claim 13 wherein the reprogramming sequences each encode Oct 3/4, Klf4 or Sox2.
15. The method of claim 14 further comprising a reprogramming sequence which encodes c-Myc.
16. The method of claim 9 wherein the transposon region comprises a transposon and two or more reprogramming factors which are products of an Oct gene family, a KIf gene family, and a Sox gene family.
17. The method of claim 9, wherein the transposon region comprises a transposon and Oct 3/4, Klf4 and Sox2, and optionally c-Myc.
18. The method of any preceding claim wherein the non-viral based vector system comprises a promoter and optionally polyadenylation signal.
19. The method according to any preceding claim wherein the non-viral based vector system is integrated into the genome of the somatic cell for expression of said reprogramming factors, or is not integrated and the reprogramming factors are episomally expressed.
20. The method of claim 19 wherein the non-viral based vector system is to be integrated and comprises means for excising the reprogramming sequences from the genome of the reprogrammed cell.
21. The method according to any preceding claim further comprising differentiating said reprogrammed cell to a desired cell type.
22. The method according to claim 21 wherein the desired cell type is a cardiac cell, neural cell, hepatic cell, haemopoietic cell, lymphoid cell, bone cell, endothelial cells, epithelial cell, kidney cell, pancreatic cell, or muscle cell type.
23. The method of any preceding claim further comprising providing the reprogrammed cell or differentiated cell to a subject for the repair or regeneration of a tissue or organ.
24. A reprogrammed cell produced by a method of claims 1 - 20.
25. A non-viral based vector system as defined in any of claims 2 - 20.
26. A non-viral based vector system comprising the vector identified in Figure 4, 10, 12, 14 or 20.
27. A kit for carrying out a method of any of claims 1 - 23 or comprising a non-viral based vector system of claims 25 or 26.
28. A reprogrammed cell comprising a non-viral based vector system which is capable of expressing reprogramming factors necessary to reprogram said somatic cell.
29. The reprogrammed cell according to claim 28 wherein the non-viral based vector system is as defined in any of claims 2 - 20.
30. The reprogrammed cell according to claim 29 wherein one or more of said reprogramming sequences has been excised from the reprogrammed cell, such that one or more of said other reprogramming sequences remains in the reprogrammed cell.
31. The reprogrammed cell according to claim 30 wherein all of said reprogramming sequences have been excised from the reprogrammed cell.
32. A reprogrammed cell comprising excision sequences or portions of excision sequences remaining in the genome of the cell following excision of the non-viral based vector system.
33. The reprogrammed cell according to claim 32 wherein the excision sequences are LoxP site.
34. Use of the reprogrammed cell according to claims 28-333, or reprogrammed or differentiated cell prepared by the method of any of claims 1 - 23 in the manufacture of a medicament for the repair or regeneration of a tissue or an organ in a subject.
35. Use of the reprogrammed or differentiated cell prepared by the method according to claims 1 - 23, for drug screening and/or toxicity testing.
36. Use of a reprogrammed cell according to claim 28 for identifying an agent that mimics or can replace one or more of said reprogramming factors excised from the reprogrammed cell.
37. A method for stem cell therapy comprising: (a) isolating and collecting somatic cells from a patient; (b) inducing reprogramming of the somatic cells by contacting or transfecting the cells with a non-viral based vector system defined in any preceding claim (c) inducing differentiation of the reprogrammed cells; and (d) transplanting the differentiated cells into the patient or another patient.
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