WO2015140005A1

WO2015140005A1 - Method of generation of pluripotent cells

Info

Publication number: WO2015140005A1
Application number: PCT/EP2015/054946
Authority: WO
Inventors: Vincenzo Costanzo
Original assignee: Ifom Fondazione Istituto Firc Di Oncologia Molecolare
Priority date: 2014-03-19
Filing date: 2015-03-10
Publication date: 2015-09-24

Abstract

A method is disclosed for the generation of pluripotent cells from non-pluripotent cells.

Description

Method of Generation of Pluripotent Cells

Field of the Invention

The present invention relates to the use of SSRP1 in the generation of pluripotent cells.

Background to the Invention

The stable states of differentiated cells are now known to be controlled by dynamic mechanisms that can easily be perturbed. An adult cell can therefore be reprogrammed, altering its pattern of gene expression, and hence its fate, to that typical of another cell type. This has been shown by three distinct experimental approaches to nuclear reprogramming: nuclear transfer, cell fusion and transcription-factor transduction (Yamanaka and Blau, 2010). Using these approaches, nuclei from 'terminally differentiated' somatic cells can be induced to express genes that are typical of embryonic stem (ES) cells, which can differentiate to form all of the cell types in the body. This remarkable discovery of cellular plasticity has important medical applications. In particular, the transcription factor transduction approach can be used to form induced pluripotent stem (iPS) cells, which have similar properties to ES cells and can be generated from almost any cell type in the body (to generate cells for tissue repair or replacement while avoiding the ethical and immunological issues that are inherent in the use of ES cells) through the introduction of four genes (Oct4, Sox2, Klf4 and c-Myc - the OKSM factors') by using retroviruses (Yamanaka and Blau, 2010).

Although the elegant fusion experiments by Tada, Surani and colleagues (Tada et al., 997) clearly showed that ES cells and embryonic germ cells contain factors that can induce reprogramming and pluripotency in somatic cells, attempts by many investigators to identify master regulators of the ES-cell state have failed. As a result, the prevailing view until about four years ago was that nuclear reprogramming to a pluripotent state is a highly complex process that might entail the cooperation of up to 100 factors.

S.Y. and colleagues (Yamanaka and Blau, 2010) used retroviral vectors to introduce into mouse embryonic and adult fibroblasts a mini-complementary-DNA library of 24 genes expressed by ES cells and these genes were then tested for their collective ability to induce pluripotency. Rather than determining the contribution of each factor singly or in subgroups, factors were progressively eliminated from the pool one at a time. As a result, the authors identified four key factors that sufficed to induce pluripotency in fibroblasts— OCT4, SOX2, KLF4 and c-MYC— and they named these induced pluripotent cells iPS cells (Takahashi and Yamanaka, 2006). iPS cells are similar to natural pluripotent stem cells, such as ES cells. ES cells are among the first cells to be generated following fertilization. These cells have the remarkable property of giving rise to all the different types of cells of an adult individual. iPS cells are similar to ES in many aspects: they express similar stem cell genes and proteins, have similar DNA methylation pattern and chromatin modification status patterns, divide with similar doubling time, give rise to embryoid body formation, induce formation of teratoma, a tumor with embryonic features and importantly give rise to a viable chimera when injected into a mouse blastocyst (Yamanaka and Blau, 2010). The features collectively show that iPS cells have the potential ability to give rise to all adult differentiated tissues similar to ES cells. For therapeutic and research applications, iPS cells are particularly advantageous, for three main reasons. First, diseases can readily be modelled using iPS cells derived from patients, overcoming the ethical issues and problems with immunological rejection that are inherent in obtaining human ES cells for studying disease. Skin fibroblasts can be readily obtained from the skin of an individual with a particular heritable disease, induced to become pluripotent in vitro and then induced to undergo differentiation to become the cell type of interest (for example a specific kind of cardiac cell). The pathways underlying a disease state (that is, gene expression and signaling) can thus be studied in cells that are not easily accessible in living humans. Second, drug screening can be carried out in vitro using these iPS-cell-based disease models to determine whether therapeutic drug candidates ameliorate or correct aberrant pathways. Third, for certain diseases, cell therapy might soon be used to regenerate or replace defective tissues, with the caveat that the tumorigenic potential, which is in part due to viral vector integration, must be overcome. Work in the past few years has highlighted several potential issues that might underly the potential danger of using iPS for therapeutic purposes. Most importantly reprogramming can induce mutations independently of the vectors used to transduce the iPS inducer genes known so far. Few groups sequenced the genomes of iPS cells and found changes in single DNA bases (Gore et al., 201 1 ), DNA rearrangements called copy-number variations (Hussein et al., 201 1 ) and differences in chromosome number (Mayshar et al., 2010). The mutations in iPS cells were not just inherited from the parent cells— some seemed to result from the reprogramming and culture process giving rise to DNA damage (Marion et al., 2009). The induction of DNA damage and mutations could underly the issues related to the fact that reprogrammed cells not always develop into some cell types and are not always a good model for disease. Most importantly, these mutations could contribute to generation of genomic instability typical of cancer cells. As one of the factor used to generate iPS, namely c-Myc, is an oncogene itself, the use of iPS could be hampered by safety issues such as their tumorigenic potential.

Gaining a better understanding of the reprogramming process and how epigenetic memory is established could reduce the chances of generating tumorigenic cells and therefore enhance the usefulness of iPS cells, providing safer sources for cell therapy in the future.

The four factors that were initially identified can be substituted with different factors or with certain small molecules. But the original finding— that a set of factors is required— held true, and certain key factors, such as OCT4, were thought not to be omittable. The original isolation of iPS cells was based upon retrovirus-mediated transduction of oncogenes and on drug-dependent selection for Fbx15, Oct4, or Nanog activation. These two experimental requirements seriously hinder the eventual application of the in vitro reprogramming approach for therapeutic use in humans because mice derived from iPS cells frequently developed cancer (Okita et al, 2007) and because the isolation of human iPS cells cannot be based on genetically modified donor cells. Some of these limitations have been overcome in recent experiments. First, in an effort to reduce the risk of tumors in iPS cell-derived chimeras, more recent experiments showed that c-myc is dispensable for reprogramming (Nakagawa et al, 2008, Werning et al, 2008, Yu et al, 2008), though the reprogramming process was significantly delayed and less efficient in the absence of this oncogene.

A key issue in the generation of iPS is the generation of DNA damage during the reprogramming (Marion et al., 2009). It is unclear why DNA damage is generated (Blasco et al., 2011 ). It is possible that replication of DNA during reprogramming is impaired. High level of transcription during reprogramming might create blocks to replication fork progression that could results in DNA breakage (Tudu et al., 2009). It is known that genomic regions with high tendency to DNA breakage have low density of replication origins (Letessier et al., 201 1 ). Somatic nuclei replicate their DNA from fewer replication origins than their embryonic counterparts. Inefficient reconfiguration of replication origins in somatic cells that fail to adapt to the fast replication pace of embryonic division could contribute to inefficient reprogramming of somatic nuclei. Therefore, reconfiguration of replication origin pattern during nuclear reprogramming might be necessary to promote efficient formation of DNA damage free iPS cells. Since the initial discovery, the technology has been improved in several ways. For example, pluripotency factors can now be delivered into the cell without the use of retroviral vectors, which integrate randomly in the genome and cause deregulation of nearby endogenous genes that may contribute to tumour formation. Non-integrating viruses, stabilised RNAs or proteins, as well as episomal plasmids, are now used for integration-free delivery of the pluripotency genes. In certain cell types fewer than four factors are required to induce pluripotency, for example adult mouse neural stem cells only require Oct4 for iPS cell induction (Kim et al., 2009). Similarly, small molecules have been shown, in certain cellular contexts, to substitute for some of the pluripotency factors (Li et al., 2009).

Summary of the Invention

The present invention concerns the identification of SSRP1 as a nuclear reprogramming factor. The present invention provides methods of nuclear reprogramming a cell which involve increasing the level of SSRP1 protein, and/or nucleic acid encoding SSRP1 , in the cell. This may involve induction of expression, e.g. overexpression, by the cell of endogenous SSRP1 , or the introduction into the cell of exogenous SSRP1 protein or exogenous nucleic acid encoding SSRP1.

As such, in some embodiments the use of SSRP1 protein, or nucleic acid(s) encoding SSRP1 , is provided for use in nuclear reprogramming.

In some preferred embodiments the method of nuclear reprogramming comprises producing pluripotent cell(s) from non-pluripotent cell(s). The pluripotent cells may be induced pluripotent stem cells.

In one aspect of the present invention a method for generating a nuclear reprogrammed cell from a non-pluripotent cell is provided, the method comprising (i) introducing into a non-pluripotent cell nucleic acid encoding SSRP1 or an effective amount of SSRP1 , or (ii) expressing in a non-pluripotent cell an effective amount of SSRP1 or nucleic acid encoding SSRP1. The nuclear reprogrammed cell may be a pluripotent cell, preferably an induced pluripotent stem cell. As such, the method may be part of a method of producing an induced pluripotent stem cell or may be part of a method of reprogramming of a non- pluripotent cell to a pluripotent cell. The method may be one which reduces the level of DNA damage to the cell, e.g. compared to reprogramming using OKSM factors.

In another aspect of the present invention there is provided a method of producing a pluripotent cell, the method comprising (i) contacting a non-pluripotent cell with nucleic acid encoding SSRP1 or an effective amount of SSRP1 , or (ii) expressing in a non- pluripotent cell an effective amount of SSRP1 or a nucleic acid encoding SSRP1.

In another aspect of the present invention there is provided a method establishing an induced pluripotent stem cell, the method comprising the step of increasing the amount of SSRP1 in a non-pluripotent cell in a nuclear reprogramming step of said non-pluripotent cell. The method may comprise the step of expressing in a non-pluripotent cell an effective amount of SSRP1 or a nucleic acid encoding SSRP1.

In some embodiments method according to the present invention may further comprise culturing the nuclear reprogrammed or pluripotent cell(s) obtained under conditions to produce a population of cells, which may be isolated cells. Preferably, substantially all of the cells in the population will have the same nuclear programming characteristic and/or be pluripotent. This may be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, or substantially 100%. In some embodiments the method further comprises isolating one or more of the nuclear reprogrammed or pluripotent cell(s) obtained from non-pluripotent cell(s). Accordingly, an isolated cell or isolated population of cells, preferably in vitro, produced by a method according to the present invention is provided. Cells obtained by methods according to the present invention may be placed under conditions that cause or induce the differentiation of the nuclear reprogrammed or pluripotent cell(s) into a specific cell type, preferably a specific somatic cell type.

Accordingly, the use, preferably in vitro, of a nuclear reprogrammed or pluripotent cell obtained by a method according to the present invention is provided for producing a somatic cell. As such, a somatic cell generated by differentiation of a nuclear

reprogrammed cell or pluripotent cell according to the present invention is also provided.

In another aspect of the present invention a somatic cell or non-pluripotent cell, preferably isolated, into which nucleic acid encoding SSRP1 or a homologue thereof has been introduced so as to be expressed in the cell is provided. The cell may overexpress SSRP1 or nucleic acid encoding SSRP1.

In a further aspect of the present invention a somatic cell or non-pluripotent cell, preferably isolated, induced to overexpress endogenous SSRP1 or endogenous nucleic acid encoding SSRP1 is provided.

In a further aspect of the present invention a somatic cell or non-pluripotent cell, preferably isolated, into which exogenous SSRP1 has been introduced is provided.

In another aspect of the present invention the use, preferably in vitro, of nucleic acid encoding SSRP1 or SSRP1 for nuclear reprogramming of non-pluripotent cell(s), producing pluripotent cell(s) from non-pluripotent cell(s), or improving induced pluripotent stem cell establishment is provided.

Improvement in induced pluripotent stem cell establishment may comprise reducing DNA damage during establishment of the induced pluripotent stem cell(s), e.g. as compared with establishment of induced pluripotent stem cell(s) using one or more of the OKSM factors, and reducing the number of factors required compared to OKSM.

In another aspect of the present invention an inducer from a non-pluripotent cell to a nuclear reprogrammed cell is provided, the inducer comprising nucleic acid encoding SSRP1 or SSRPl The inventor also found that embryos obtained by nuclear transfer in the presence of SSRP1 protein were of higher quality. Accordingly, in another aspect of the present invention, SSRP1 protein is provided for use in a method of nuclear transfer. In one aspect of the present invention the use, in vitro, of SSRP1 in a method of nuclear transfer is provided. In a related aspect of the present invention a method of nuclear transfer is provided, the method comprising providing an enucleated egg cell, and in the presence of exogenous SSRP1 protein transferring the nucleus of a somatic cell into the enucleated egg cell. The enucleated egg cell is preferably an oocyte. Preferably it is unfertilised.

The SSRP1 protein may be present at a concentration of about less than 100 ng, e.g. one of about 50-100 ng, 1 -50ng, 1-40ng, 1-30ng, 1-20ng, 1-10ng, 1-5ng, or 5-10ng. The SSRP1 may be provided as recombinant SSRP1 protein. The method may further comprise one or more of the steps of (i) stimulating the cell to divide, (ii) culturing the dividing cells, optionally to form an embryo, (iii) isolating individual cells from the developing embryo, optionally pluripotent cells, (iv) maintaining a viable embryo under in vitro culture conditions. These steps may all be performed in vitro. The egg cell and somatic cell nucleus may be from any human or animal. Preferably they are from the same type of animal, but for generation of an animal hybrid may be from different types of animal. In some preferred embodiments neither cell is from a human. In some preferred embodiments neither cell is from a primate. The cells may be from non- human cells, e.g. rabbit, guinea pig, rat, mouse or other rodent (including cells from any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle, horse, non-human primate or other non-human vertebrate organism; and/or non-human mammalian cells.

The method of nuclear transfer may normally be used for research purposes in non- human animals where the method is used to generate pluripotent cells, the embryo being destroyed before further development, e.g. beyond the blastula.

The method of nuclear transfer may be used in a method of animal cloning, which method may further comprise one or more of (i) implanting the developing embryo into the uterus of a corresponding animal, (ii) allowing the embryo to develop into a fetus, (iii) delivering the newborn animal.

Methods of the invention may optionally further comprise introduction of one or more of the OKSM factors, e.g. by introducing to the cell nucleic acid(s) encoding one or more or all of Oct4, Sox2, Klf4 and c-Myc. In some preferred embodiments of the invention none of the OKSM factors are introduced into the non-pluripotent cell and/or the normal level of expression, or level of, the OKSM factors in the non-pluripotent cell is not directly modified. In some embodiments of the invention SSRP1 is the only nuclear

reprogramming factor which is introduced into the non-pluripotent cell, or is the only nuclear reprogramming factor whose expression, or level, in the non-pluripotent cell is modified.

In further aspects of the present invention the nuclear reprogrammed or pluripotent cell(s) obtained according to the present invention is/are used in methods of screening for the effect of a candidate agent or stimulus on the nuclear reprogrammed or pluripotent cell(s). In one aspect the use, preferably in vitro, of a nuclear reprogrammed or pluripotent cell obtained according to the present invention in a method of determining the effect of a candidate agent or stimulus on the nuclear reprogrammed or pluripotent cell is provided. In another aspect, a method, preferably in vitro, is provided comprising contacting a nuclear reprogrammed or pluripotent cell according to the present invention with a candidate agent or stimulus and determining the effect of the candidate agent or stimulus on said cell. By way of example, the candidate agent may be a compound or substance (e.g. a drug candidate) and the stimulus may be an environmental condition, such as light, heat or other radiation.

Description

The inventor has identified SSRP1 as an essential factor to establish embryonic vertebrate replication origins and embryonic pluripotency state by regulating the assembly of pre-replication complex on chromatin, being able to drive alone cell reprogramming with significantly reduced levels of DNA damage.

Stem Cells

In the present specification "stem cells" may be cells that are obtained or produced by the methods of the present invention, and are normally created from non-pluripotent cells such as somatic cells, and may therefore be referred to as "induced pluripotent stem cells".

As used in this document, the term "stem cell" refers to a cell that on division faces two developmental options: the daughter cells can be identical to the original cell (self- renewal) or they may be the progenitors of more specialised cell types (differentiation). The stem cell is therefore capable of adopting one or other pathway (a further pathway exists in which one of each cell type can be formed). Stem cells are therefore cells which are not terminally differentiated and are able to produce cells of other types.

In general, reference herein to cells (plural) may include the singular (stem cell), and vice versa. In particular, methods of culturing and differentiating stem cells may include single cell and aggregate culturing techniques. In the present invention stem cell cultures may be of aggregates or single cells.

Stem cells can be described in terms of the range of cell types into which they are able to differentiate, as discussed below. The stem cells obtained or produced by the methods of the present invention are preferably at least pluripotent. Optionally, they are multipotent. "Totipotent" stem cells refers to a cell which has the potential to become any cell type in the adult body, or any cell of the extraembryonic membranes (e.g., placenta). Thus, normally, the only totipotent cells are the fertilized egg and the first 4 or so cells produced by its cleavage.

"Pluripotent" stem cells are true stem cells, with the potential to make any differentiated cell in the body. However, they cannot contribute to make the extraembryonic

membranes which are derived from the trophoblast. Embryonic Stem (ES) cells are examples of pluripotent stem cells, and may be isolated from the inner cell mass (ICM) of the blastocyst, which is the stage of embryonic development when implantation occurs.

"Multipotent" stem cells are true stem cells which can only differentiate into a limited number of cell types. For example, the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but not to other types of cells. Multipotent stem cells are found in adult animals, and are sometimes called adult stem cells. It is thought that every organ in the body (brain, liver) contains them where they can replace dead or damaged cells. Methods of characterising stem cells are known in the art, and include the use of standard assay methods such as clonal assay, flow cytometry, long-term culture and molecular biological techniques e.g. PGR, RT-PCR and Southern blotting.

In addition to morphological differences, human and murine pluripotent stem cells differ in their expression of a number of cell surface antigens (stem cell markers). Markers for stem cells and methods of their detection are described elsewhere in this document (under "Maintenance of Stem Cell Characteristics"). The present invention includes techniques for the generation of pluripotent cells from non- pluripotent cells. As such, the "stem cells" generated by the methods of the present invention have not been obtained by a method that causes the destruction of an embryo. In particular, the pluripotent cells of the present invention have been obtained by a method that does not cause the destruction of a human or mammalian embryo. As such, methods of the invention may be performed using cells that have not been prepared exclusively by a method which necessarily involves the destruction of human embryos from which those cells may be derived. Indeed, cells obtained from embryos are not required to perform methods according to the present invention. This optional limitation is specifically intended to take account of Decision G0002/06 of 25 November 2008 of the Enlarged Board of Appeal of the European Patent Office.

Culture of Stem Cells

Any suitable method of cultu ng cells, including induced pluripotent stem cells, may be used in the methods and compositions described here. Any suitable container may be used to propagate cells according to the methods and compositions described here. Suitable containers include those described in US Patent Publication US2007/0264713.

Containers may include bioreactors and spinners, for example. A "bioreactor", as the term is used in this document, is a container suitable for the cultivation of eukaryotic cells, for example animal cells or mammalian cells, such as in a large scale. A typical cultivation volume of a regulated bioreactor is between 20 ml and 500 ml.

The bioreactor may comprise a regulated bioreactor, in which one or more conditions may be controlled or monitored, for example, oxygen partial pressure. Devices for measuring and regulating these conditions are known in the art. For example, oxygen electrodes may be used for oxygen partial pressure. The oxygen partial pressure can be regulated via the amount and the composition of the selected gas mixture (e.g., air or a mixture of air and/or oxygen and/or nitrogen and/or carbon dioxide). Suitable devices for measuring and regulating the oxygen partial pressure are described by Bailey, J E.

(Bailey, J E., Biochemical Engineering Fundamentals, second edition, McGraw-Hill, Inc. ISBN 0-07-003212-2 Higher Education, (1986)) or Jackson A T. Jackson A T.,

Verfahrenstechnik in der Biotechnologie, Springer, ISBN 3540561900 (1993)).

Other suitable containers include spinners. Spinners are regulated or unregulated bioreactors, which can be agitated using various agitator mechanisms, such as glass ball agitators, impeller agitators, and other suitable agitators. The cultivation volume of a spinner is typically between 20 ml and 500 ml. Roller bottles are round cell culture flasks made of plastic or glass having a culture area of between 400 and 2000 cm². The cells are cultivated along the entire inner surface of these flasks; the cells are coated with culture medium accomplished by a "rolling" motion, i.e. rotating the bottles about their own individual axis.

Alternatively, culture may be static, i.e. where active agitation of the culture/culture media is not employed. By reducing agitation of the culture aggregates of cells may be allowed to form. Whilst some agitation may be employed to encourage distribution and flow of the culture media over the cultured cells this may be applied so as not to substantially disrupt aggregate formation. For example, a low rpm agitation, e.g. less than 30rpm or less than 20rpm, may be employed.

Co-Culture and Feeders

Culture methods for nuclear reprogrammed, pluripotent cells or stem cells may comprise culturing cells in the presence or absence of co-culture. The term "co-culture" refers to a mixture of two or more different kinds of cells that are grown together, for example, stromal feeder cells. The two or more different kinds of cells may be grown on the same surfaces, such as particles or cell container surfaces, or on different surfaces. The different kinds of cells may be grown on different particles. Feeder cells, as the term is used in this document, may mean cells which are used for or required for cultivation of cells of a different type. In the context of stem cell culture, feeder cells have the function of securing the survival, proliferation, and maintenance of cell pluripotency. Cell pluripotency may be achieved by directly co-cultivating the feeder cells. Alternatively, or in addition, the feeder cells may be cultured in a medium to condition it. The conditioned medium may be used to culture the stem cells.

By way of example, the inner surface of the container such as a culture dish may be coated with a feeder layer of mouse embryonic skin cells that have been treated so they will not divide. The feeder cells release nutrients into the culture medium which are required for pluripotent cell growth. The stem cells growing on particles may therefore be grown in such coated containers.

Arrangements in which feeder cells are absent or not required are also possible. For example, the cells may be grown in medium conditioned by feeder cells or stem cells. Media and Feeder Cells

Media for isolating and propagating pluripotent cells can have any of several different formulas, as long as the cells obtained have the desired characteristics, and can be propagated further. Suitable sources are as follows: Dulbecco's modified Eagles medium (DMEM),

Gibco#1 965-092; Knockout Dulbecco's modified Eagles medium (KO DMEM),

Gibco#10829-018; 200 mM L-glutamine, Gibco#15039-027; non-essential amino acid solution, Gibco 11140-050; beta-mercaptoethanol, Sigma#M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco#13256-029. Exemplary serum-containing embryonic stem (ES) medium is made with 80% DMEM (typically KO DMEM), 20% defined fetal bovine serum (FBS) not heat inactivated, 0.1 mM non-essential amino acids, 1 mM L-glutamine, and 0.1 mM beta-mercaptoethanol. The medium is filtered and stored at 4 degrees C for no longer than 2 weeks. Serum-free embryonic stem (ES) medium is made with 80% KO DMEM, 20% serum replacement, 0.1 mM non-essential amino acids, 1 mM L-glutamine, and 0.1 mM beta-mercaptoethanol. An effective serum replacement is Gibco#10828-028. The medium is filtered and stored at 4 degrees C for no longer than 2 weeks. Just before use, human bFGF is added to a final concentration of 4 ng/mL (Bodnar et al., Geron Corp, International Patent Publication WO 99/20741 ).

The media may comprise Knockout DMEM media (Invitrogen-Gibco, Grand Island, New York), supplemented with 10% serum replacement media (Invitrogen- Gibco, Grand

Island, New York), 5ng/ml FGF2 (Invitrogen-Gibco, Grand Island, New York) and 5ng/ml PDGF AB (Peprotech, Rocky Hill, New Jersey).

Feeder cells (where used) may be propagated in mEF medium, containing 90% DMEM (Gibco#11965-092), 10% FBS (Hyclone#30071 -03), and 2 mM glutamine. mEFs are propagated in T150 flasks (Coming#430825), splitting the cells 1 :2 every other day with trypsin, keeping the cells subconfluent. To prepare the feeder cell layer, cells are irradiated at a dose to inhibit proliferation but permit synthesis of important factors that support human embryonic stem cells (about 4000 rads gamma irradiation). Six-well culture plates (such as Falcon#304) are coated by incubation at 37 degrees C. with 1 mL 0.5% gelatin per well overnight, and plated with 375,000 irradiated mEFs per well. Feeder cell layers are typically used 5 h to 4 days after plating. The medium is replaced with fresh human embryonic stem (hES) medium just before seeding pluripotent stem (pPS) cells. Conditions for culturing other pluripotent cells are known, and can be optimized appropriately according to the cell type. Media and culture techniques for particular cell types referred to in the previous section are provided in the references cited.

Serum Free Media

The methods and compositions described here may include culture of cells in a serum- free medium.

The term "serum-free media" may comprise cell culture media which is free of serum proteins, e.g., fetal calf serum. Serum-free media are known in the art, and are described for example in US Patents 5,631 ,159 and 5,661 ,034. Serum-free media are commercially available from, for example, Gibco-BRL (Invitrogen).

The serum-free media may be protein free, in that it may lack proteins, hydrolysates, and components of unknown composition. The serum-free media may comprise chemically- defined media in which all components have a known chemical structure. Chemically- defined serum-free media is advantageous as it provides a completely defined system which eliminates variability, allows for improved reproducibility and more consistent performance, and decreases possibility of contamination by adventitious agents.

The serum-free media may comprise Knockout DMEM media (Invitrogen-Gibco, Grand Island, New York).

The serum-free media may be supplemented with one or more components, such as serum replacement media, at a concentration of for example, 5%, 10%, 15%, etc. The serum-free media may be supplemented with 10% serum replacement media from Invitrogen- Gibco (Grand Island, New York).

The serum-free medium in which the dissociated or disaggregated embryonic stem cells are cultured may comprise one or more growth factors. A number of growth factors are known in the art, including FGF2, IGF-2, Noggin, Activin A, TGF beta 1 , HRG1 beta, LIF, S1 P, PDGF, BAFF, April, SCF, Flt-3 ligand, Wnt3A and others. The growth factors) may be used at any suitable concentration such as between 1pg/ml to 500ng/ml.

Propagation with Passage

The nuclear reprogrammed cells, pluripotent cells or stem cells produced or obtained in accordance with the present invention may be maintained in cell culture. Such culture may comprise passaging, or splitting during culture. The methods may involve continuous or continual passage. The term "passage" may generally refer to the process of taking an aliquot of a cell culture, dissociating the cells completely or partially, diluting and inoculating into medium. The passaging may be repeated one or more times. The aliquot may comprise the whole or a portion of the cell culture. The cells of the aliquot may be completely, partially or not confluent. The passaging may comprise at least some of the following sequence of steps: aspiration, rinsing, trypsinization, incubation, dislodging, quenching, re-seeding and aliquoting. The protocol published by the Hedrick Lab, UC San Diego may be used (http://hedricklab.ucsd.edu/Protocol/COSCell.html).

The cells may be dissociated by any suitable means, such as mechanical or enzymatic means known in the art. The cells may be broken up by mechanical dissociation, for example using a cell scraper or pipette. The cells may be dissociated by sieving through a suitable sieve size, such as through 100 micron or 500 micron sieves. The cells may be split by enzymatic dissociation, for example by treatment with collagenase, Trypsin or TrypLE™ harvested. The dissociation may be complete or partial. Cells in culture may be dissociated from the substrate or flask, and "split", subcultured or passaged, by dilution into tissue culture medium and replating. The dilution may be of any suitable dilution. The cells in the cell culture may be split at any suitable ratio. For example, the cells may be split at a ratio of 1 :2 or more, 1 :3 or more, 1 :4 or more or :5 or more. The cells may be split at a ratio of 1 :6 or more, 1 :7 or more, 1 :8 or more, 1 :9 or more or 1 :10 or more. The split ratio may be 1 :10 or more. It may be 1 :11 , 1 :12, 1 :13,

1 :14, 1 :15, 1 :16, 1 :17, 1 :18, 1 :19 or 1 :20 or more. The split ratio may be 1 :21 , 1 :22, 1 :23, 1 :24, 1 :25 or 1 :26 or more.

Thus, cells may be passaged for 1 passage or more. For example, stem cells may be passaged for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 passages or more. In principle, cells may be propagated indefinitely in culture.

Maintenance of Stem Cell Characteristics

The nuclear reprogrammed cells, pluripotent cells or stem cells produced or obtained in accordance with the present invention may retain at least one characteristic of a stem cell. The cells may retain the characteristic after one or more passages. They may do so after a plurality of passages.

The characteristic may comprise a morphological characteristic, immunohistochemical characteristic, a molecular biological characteristic, etc. The characteristic may comprise a biological activity. For example, stem cells may display increased expression of Oct4 and/or SSEA-1 and/or TRA-1-60. Stem cells may display defined morphology. For example, in the two dimensions of a standard microscopic image, human pluripotent stem cells display high nuclear/cytoplasmic ratios in the plane of the image, prominent nucleoli, and compact colony formation with poorly discernable cell junctions.

Expression of Pluripotency Markers

Stem cells may also be characterized by expressed cell markers. The biological activity that is retained may comprise expression of one or more pluripotency markers.

Stage-specific embryonic antigens (SSEA) are characteristic of certain embryonic cell types. Antibodies for SSEA markers are available from the Developmental Studies

Hybridoma Bank (Bethesda Md.). Other useful markers are detectable using antibodies designated Tra-1 -60 and Tra-1 -81 (Andrews et al., Cell Lines from Human Germ Cell Tumors, in E. J. Robertson, 1987, supra). Human embryonic stem cells are typically SSEA-1 negative and SSEA-4 positive. hEG cells are typically SSEA-1 positive.

Differentiation of primate pluripotent stem cells (pPS) cells in vitro results in the loss of SSEA-4, Tra-1 -60, and Tra-1-81 expression and increased expression of SSEA-1. pPS cells can also be characterized by the presence of alkaline phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.).

Embryonic stem cells are also typically telomerase positive and OCT-4 positive.

Telomerase activity can be determined using TRAP activity assay (Kim et al., Science 266:201 1 , 1997), using a commercially available kit (TRAPeze.RTM. XK Telomerase Detection Kit, Cat. s7707; Intergen Co., Purchase N.Y.; or TeloTAGGG.TM. Telomerase PCR ELISA plus, Cat. 2,013,89; Roche Diagnostics, Indianapolis). hTERT expression can also be evaluated at the mRNA level by RT-PCR. The LightCycler TeloTAGGG.TM. hTERT quantification kit (Cat. 3,012,344; Roche Diagnostics) is available commercially for research purposes.

Any one or more of these pluripotency markers, including FOXD3, alkaline phosphatase, OCT-4, SSEA-4, and TRA-1-60 etc, may be retained by the propagated stem cells.

Detection of markers may be achieved through any means known in the art, for example immunologically. Histochemical staining, flow cytometry (FACS), Western Blot, enzyme- linked immunoassay (ELISA), etc may be used. Flow immunocytochemistry may be used to detect cell-surface markers.

Immunohistochemistry (for example, of fixed cells or tissue sections) may be used for intracellular or cell-surface markers. Western blot analysis may be conducted on cellular extracts. Enzyme-linked immunoassay may be used for cellular extracts or products secreted into the medium.

For this purpose, antibodies to the pluripotency markers as available from commercial sources may be used.

Antibodies for the identification of stem cell markers including the Stage-Specific

Embryonic Antigens 1 and 4 (SSEA-1 and SSEA-4) and Tumor Rejection Antigen 1-60 and 1 -81 (TRA-1-60, TRA-1 -81 ) may be obtained commercially, for example from

Chemicon International, Inc (Temecula, CA, USA). The immunological detection of these antigens using monoclonal antibodies has been widely used to characterize pluripotent stem cells (Shamblott M.J. et. al. (1998) PNAS 95: 13726-13731 ; Schuldiner M. et. al. (2000). PNAS 97: 1 1307 - 1 1312; Thomson J.A. et. al. (1998). Science 282: 1 145-1 47; Reubinoff B.E. et. al. (2000). Nature Biotechnology 18: 399-404; Henderson J.K. et. al. (2002). Stem Cells 20: 329-337; Pera M. et. al. (2000). J. Cell Science 1 13: 5-10.).

The expression of tissue-specific gene products can also be detected at the mRNA level by Northern blot analysis, dot-blot hybridization analysis, or by reverse transcriptase initiated polymerase chain reaction (RT-PCR) using sequence-specific primers in standard amplification methods. Sequence data for the particular markers listed in this disclosure can be obtained from public databases such as GenBank (URL

www.ncbi.nlm.nih. qov:80/entrez). See U.S. Pat. No. 5,843,780 for further details.

In some embodiments substantially all cells in a culture, or a substantial portion of them, may express one or more of the marker(s). For example, the percentage of cells that express the marker or markers may be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, or substantially 100%.

Cell Viability

The biological activity may comprise cell viability, e.g. after a stated number of passages. Cell viability may be assayed in various ways, for example by Trypan Blue exclusion.

A protocol for vital staining follows. Place a suitable volume of a cell suspension (20-200 μΙ_) in appropriate tube, add an equal volume of 0.4% Trypan blue and gently mix, let stand for 5 minutes at room temperature. Place 10 μΙ of stained cells in a hemocytometer and count the number of viable (unstained) and dead (stained) cells. Calculate the average number of unstained cells in each quadrant, and multiply by 2 x 10⁴ to find cells/ml. The percentage of viable cells is the number of viable cells divided by the number of dead and viable cells.

The viability of cells in a culture may be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, or substantially 100%.

Karyotype The propagated cells may retain a normal karyotype during or after propagation. A "normal" karyotype is a karyotype that is identical, similar or substantially similar to a karyotype of a parent stem cell from which the stem cell is derived, or one which varies from it but not in any substantial manner. For example, there should not be any gross anomalies such as translocations, loss of chromosomes, deletions, etc. Karyotype may be assessed by a number of methods, for example visually under optical microscopy. Karyotypes may be prepared and analyzed as described in McWhir et al. (2006), Hewitt et al. (2007), and Gallimore and Richardson (1973). Cells may also be karyotyped using a standard G-banding technique (available at many clinical diagnostics labs that provide routine karyotyping services, such as the Cytogenetics Lab at Oakland Calif.) and compared to published stem cell karyotypes.

The methods of the present invention have been found to reduce DNA damage.

Therefore, the nuclear reprogrammed or pluripotent cells produced or obtained in accordance with the present invention may retain a normal karyotype. In particular, all or a substantial portion of propagated cells in a culture may retain a normal karyotype. This proportion may be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, or substantially 100%.

Pluripotency

The nuclear reprogrammed or pluripotent cells produced or obtained in accordance with the present invention may retain the capacity to differentiate into all three cellular lineages, i.e., endoderm, ectoderm and mesoderm. Methods of induction of stem cells to differentiate to each of these lineages are known in the art and may be used to assay the capability of the propagated stem cells. All or a substantial portion of propagated cells in a culture may retain this ability. This may be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, 99% or more, or substantially 100% of the propagated stem cells. Differentiation / Embryoid Bodies

Cultured stem cells, including pluripotent cells, may be differentiated into any suitable cell type by using differentiation techniques known to those of skill in the art.

We describe a process for producing differentiated cells, the method comprising propagating a stem cell by a method as described herein, and then differentiating the stem cell in accordance with known techniques. For example, we provide for methods of differentiating to ectoderm, mesoderm and endoderm, as well as to cardiomyocytes, adipocytes, chondrocytes and osteocytes, etc. We further provide embryoid bodies and differentiated cells obtainable by such methods. Cell lines made from such stem cells and differentiated cells are also provided. Methods of differentiating stem cells are known in the art and are described in for example Itskovitz-Eldor (J Itskovitz-Eldor, M Schuldiner, D Karsenti, A Eden, O Yanuka, M Amit, H Soreq, N Benvenisty. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med. 2000 Feb ;6 (2):88-95) and Graichen et al (Ralph Graichen, Xiuqin Xu, Stefan R Braam, Thavamalar Balakrishnan, Siti Norfiza, Shirly Sieh, Set Yen Soo, Su Chin Tham, Christine Mummery, Alan Colman, Robert Zweigerdt, Bruce P Davidson. Enhanced

cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK. Differentiation 2007 (Nov 15)), Kroon et al (Kroon et al. (2008) Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol Apr;26(4):443-52.) and Hay et al (Hay et al. (2008). Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signalling. PNAS Vol. 105. No.34 12310-12306), WO 2007/030870, WO 2007/070964, Niebrugge et al (Niebrugge et al (2009). Generation of Human Embryonic Stem Cell-Derived Mesoderm and Cardiac Cells Using Size-Specified

Aggregates in an Oxygen-Controlled Bioreactor. Biotechnology and Bioengineering. Vol.102, no.2, February 1 , 2009), R Passier et al. (R Passier et al. 2005. Serum free media in cocultures (FBS inhibits cardiomyocytes differentiation). Curr Opin Biotechnol. 2005 Oct;16(5):498-502. Review. Stem Cells. 2005 Jun-Jul;23(6):772-80), P W Burridge et al. (P W Burridge et al. 2006. Defined Medium with polyvinyl alcohol (PVA), Activin A and bFGF. Stem Cells. 2007 Apr;25(4):929-38. Epub 2006 Dec 21 ), M A Laflamme et al. (M A Laflamme et al. 2007. Culture sequentially supplemented with Activin A for 24 h, and BMP 4 for 4 days. Nat Biotechnol. 2007 Sep;25(9): 1015-24. Epub 2007 Aug 26), L Yang et al. (L Yang et al. 2008. Defined medium supplemented with BMP4 (1 day), BMP4, Activin A and bFGF (days 1 -4), Activin A and bFGF (days 4-8), and DKK1 and VEGF. Nature. 2008 May 22;453(7194):524-8. Epub 2008 Apr 23), and X Q Xu et al. (X Q Xu et al. 2008. SB203580 (p38 MAP kinase inhibitor) PGI2 (prostaglandin member accumulated in END2-CM). Differentiation. 2008 Nov;76(9):958-70. Epub 2008 Jun 13).

Stem cells can be induced to differentiate to the neural lineage by culture in media containing appropriate differentiation factors. Such factors may include one or more of activin A, retinoic acid, basic fibroblast growth factor (bFGF), and antagonists of bone morphogenetic protein (BMP), such as noggin (Niknejad et al. European Cells and Materials Vol.19 2010 pages 22-29). Cells differentiating towards the neural lineage may be identified by expression of neural markers, such as Pax6, Nestin, Map2, β-tubulin III and GFAP. Cells of the neural lineage may cluster to form neurospheres (which may be nestin-positive cell aggregates), and these may be expanded by application of selected growth factors such as EGF and/or FGF1 and/or FGF2.

Differentiation may be into a specific cell type selected from one of a gland cell, a hormone secreting cell, a neural cell, a metabolic cell, a blood cell, a germ cell, an immune cell, a contractile cell, or a secretion cell.

The cultured stem cells may also be used for the formation of embryoid bodies. Embryoid bodies, and methods for making them, are known in the art. The term "embryoid body" refers to spheroid colonies seen in culture produced by the growth of embryonic stem cells in suspension. Embryoid bodies are of mixed cell types, and the distribution and timing of the appearance of specific cell types corresponds to that observed within the embryo. Embryoid bodies may be generated by plating out embryonic stem cells onto media such as semi-solid media. Methylcellulose media may be used as described in Lim et al, Blood. 1997;90:1291-1299.

Embryonic stem cells may be induced to form embryoid bodies, for example using the methods described in Itskovitz-Eldor (2000, supra). The embryoid bodies contain cells of all three embryonic germ layers.

The embryoid bodies may be further induced to differentiate into different lineages for example by exposure to the appropriate induction factor or an environmental change. Graichen et al (2007, supra) describes the formation of cardiomyocytes from human embryonic stem cells by manipulation of the p38MAP kinase pathway. Graichen demonstrates induction of cardiomyocyte formation from stem cells by exposure to a specific inhibitor of p38 MAP kinase such as SB203580 at less than 0 micromolar. Differentiated cells may be employed for any suitable purpose, such as regenerative therapy, as known in the art.

Stem cells obtained through culture methods and techniques according to this invention may be used to differentiate into another cell type for use in a method of medical treatment. Thus, the differentiated cell type may be derived from a stem cell obtained by the culture methods and techniques described herein which has subsequently been permitted to differentiate. The differentiated cell type may be considered as a product of a stem cell obtained by the culture methods and techniques described herein which has subsequently been permitted to differentiate. Pharmaceutical compositions may be provided comprising such differentiated cells, optionally together with a pharmaceutically acceptable carrier, adjuvant or diluent. Such pharmaceutical composition may be useful in a method of medical treatment.

Uses

Cells produced, obtained or propagated by the methods described herein may be used for a variety of commercially important research, diagnostic, and therapeutic purposes. The cells may be used directly for these purposes, or may be differentiated into any chosen cell type using methods known in the art. Progenitor cells may also be derived from the cells. The differentiated cells or progenitor cells, or both, may be used in place of, or in combination with, the cells for the same purposes. Thus, any use described in this document for nuclear reprogrammed cells, pluripotent cells or stem cells applies equally to progenitor cells and differentiated cells derived from those cells. Similarly, any uses of differentiated cells will equally apply to those cells for which they are progenitors, or progenitor cells.

The uses for nuclear reprogrammed cells, pluripotent cells or stem cells are generally well known in the art, but will be described briefly here. Therapeutic Uses

The methods and compositions described here may be used to propagate nuclear reprogrammed cells, pluripotent cells or stem cells for regenerative therapy. The cells may be expanded and directly administered into a patient. They may be used for the re- population of damaged tissue following trauma.

Pluripotent stem cells may be used directly, or used to generate ectodermal, mesodermal or endodermal progenitor cell populations, for regenerative therapy. Progenitor cells may be made by ex vivo (e.g. in vitro) expansion or directly administered into a patient. They may also be used for the re-population of damaged tissue following trauma.

Thus, hematopoietic progenitor cells may be used for bone marrow replacement, while cardiac progenitor cells may be used for cardiac failure patients. Skin progenitor cells may be employed for growing skin grafts for patients and endothelial progenitor cells for endothelization of artificial prosthetics such as stents or artificial hearts.

Pluripotent cells may be used as sources of ectodermal, mesodermal or endodermal progenitor cells for the treatment of degenerative diseases such as diabetes, Alzheimer's disease, Parkinson's disease. Pluripotent cells may be used as sources of mesodermal or endodermal progenitors for NK or dendritic cells for immunotherapy for cancer. The methods and compositions described here enable the production of ectodermal, mesodermal or endodermal progenitor cells, which may of course be made to further differentiate using methods known in the art to terminally differentiated cell types.

Thus, any uses of terminally differentiated cells will equally apply to those ectodermal, mesodermal or endodermal progenitor cells (or stem cells) for which they are sources. Nuclear reprogrammed cells, pluripotent cells, stem cells, ectodermal, mesodermal or endodermal progenitor cells and differentiated cells produced by the methods and compositions described herein may be used for, or for the preparation of a

pharmaceutical composition for, the treatment of a disease. Such disease may comprise a disease treatable by regenerative therapy, including cardiac failure, bone marrow disease, skin disease, burns, degenerative disease such as diabetes, Alzheimer's disease, Parkinson's disease and cancer.

The subject to be treated may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient. Therapeutic uses may be in human or animals (veterinary use). Libraries

For example, populations of undifferentiated and differentiated cells may be used to prepare antibodies and cDNA libraries that are specific for the differentiated phenotype. General techniques used in raising, purifying and modifying antibodies, and their use in immunoassays and immunoisolation methods are described in Handbook of Experimental Immunology (Weir & Blackwell, eds.); Current Protocols in Immunology (Coligan et al., eds.); and Methods of Immunological Analysis (Masseyeff et al., eds., Weinheim: VCH Verlags GmbH). General techniques involved in preparation of mRNA and cDNA libraries are described in RNA Methodologies: A Laboratory Guide for Isolation and

Characterization (R. E. Farrell, Academic Press, 1998); cDNA Library Protocols (Coweli & Austin, eds., Humana Press); and Functional Genomics (Hunt & Livesey, eds., 2000). Relatively homogeneous cell populations are particularly suited for use in drug screening and therapeutic applications.

Drug Screening Nuclear reprogrammed cells, pluripotent cells, stem cells and differentiated cells may also be used to screen for agents or factors (such as solvents, small molecule drugs, peptides, polynucleotides, and the like) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of the respective cells.

Nuclear reprogrammed cells, pluripotent cells, stem cells and differentiated cells may be used to screen for factors that promote pluripotency, or differentiation. In some

applications, differentiated cells are used to screen factors that promote maturation, or promote proliferation and maintenance of such cells in long-term culture. For example, candidate maturation factors or growth factors are tested by adding them to cells in different wells, and then determining any phenotypic change that results, according to desirable criteria for further culture and use of the cells.

Particular screening applications relate to the testing of pharmaceutical compounds in drug research. The reader is referred generally to the standard textbook "In vitro Methods in Pharmaceutical Research", Academic Press, 1997, and U.S. Pat. No. 5,030,015),as well as the general description of drug screens elsewhere in this document. Assessment of the activity of candidate pharmaceutical compounds generally involves combining the respective cells with the candidate compound, determining any change in the

morphology, marker phenotype, or metabolic activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed change.

The screening may be done, for example, either because the compound is designed to have a pharmacological effect on certain cell types, or because a compound designed to have effects elsewhere may have unintended side effects. Two or more drugs can be tested in combination (by combining with the cells either simultaneously or sequentially), to detect possible drug-drug interaction effects. In some applications, compounds are screened initially for potential toxicity (Castell et al., pp. 375-410 in "In vitro Methods in Pharmaceutical Research," Academic Press, 1997). Cytotoxicity can be determined in the first instance by the effect on cell viability, survival, morphology, and expression or release of certain markers, receptors or enzymes. Effects of a drug on chromosomal DNA can be determined by measuring DNA synthesis or repair. [3H]thymidine or BrdU incorporation, especially at unscheduled times in the cell cycle, or above the level required for cell replication, is consistent with a drug effect. Unwanted effects can also include unusual rates of sister chromatid exchange, determined by metaphase spread. The reader is referred to A. Vickers (PP 375-410 in "In vitro Methods in Pharmaceutical Research," Academic Press, 1997) for further elaboration.

Tissue Regeneration

Cells propagated according to the methods and compositions described here (and differentiated cells derived therefrom) may be used for therapy, for example tissue reconstitution or regeneration in an individual patient in need thereof. The cells may be administered in a manner that permits them to graft to the intended tissue site and reconstitute or regenerate the functionally deficient area.

Nuclear reprogrammed cells, pluripotent cells, stem cells or differentiated cells derived therefrom may be used for tissue engineering, such as for the growing of skin grafts.

They may be used for the bioengineering of artificial organs or tissues, or for prosthetics, such as stents.

Differentiated cells may also be used for tissue reconstitution or regeneration in a human patient in need thereof. The cells are administered in a manner that permits them to graft to the intended tissue site and reconstitute or regenerate the functionally deficient area.

For example, the methods and compositions described here may be used to modulate the differentiation of stem cells. Differentiated cells may be used for tissue engineering, such as for the growing of skin grafts. Modulation of stem cell differentiation may be used for the bioengineering of artificial organs or tissues, or for prosthetics, such as stents.

In another example, neural stem cells are transplanted directly into parenchymal or intrathecal sites of the central nervous system, according to the disease being treated. Grafts are done using single cell suspension or small aggregates at a density of 25,000- 500,000 cells per .mu.L (U.S. Pat. No. 5,968,829). The efficacy of neural cell transplants can be assessed in a rat model for acutely injured spinal cord as described by McDonald et al. (Nat. Med. 5:1410, 1999). A successful transplant will show transplant-derived cells present in the lesion 2-5 weeks later, differentiated into astrocytes, oligodendrocytes, and/or neurons, and migrating along the cord from the lesioned end, and an improvement in gate, coordination, and weight-bearing.

Certain neural progenitor cells are designed for treatment of acute or chronic damage to the nervous system. For example, excitotoxicity has been implicated in a variety of conditions including epilepsy, stroke, ischemia, Huntington's disease, Parkinson's disease and Alzheimer's disease. Certain differentiated cells as made according to the methods described here may also be appropriate for treating dysmyelinating disorders, such as Pelizaeus-Merzbacher disease, multiple sclerosis, leukodystrophies, neuritis and neuropathies. Appropriate for these purposes are cell cultures enriched in

oligodendrocytes or oligodendrocyte precursors to promote remyelination. Hepatocytes and hepatocyte precursors prepared using our methods can be assessed in animal models for ability to repair liver damage. One such example is damage caused by intraperitoneal injection of D-galactosamine (Dabeva et al., Am. J. Pathol. 143:1606, 1993). Efficacy of treatment can be determined by immunohistochemical staining for liver cell markers, microscopic determination of whether canalicular structures form in growing tissue, and the ability of the treatment to restore synthesis of liver-specific proteins. Liver cells can be used in therapy by direct administration, or as part of a bioassist device that provides temporary liver function while the subject's liver tissue regenerates itself following fulminant hepatic failure.

Cardiomyocytes may be prepared by inducing differentiation of stem cells by modulation of the MAP kinase pathway for example with SB203580, a specific p38 MAP kinase inhibitor, as described in Graichen et al (2007, supra). The efficacy of such

cardiomyocytes may be assessed in animal models for cardiac cryoinjury, which causes 55% of the left ventricular wall tissue to become scar tissue without treatment (Li et al., Ann. Thorac. Surg. 62:654, 1996; Sakai et al., Ann. Thorac. Surg. 8:2074, 1999, Sakai et al., J. Thorac. Cardiovasc. Surg. 118:715, 1999). Successful treatment will reduce the area of the scar, limit scar expansion, and improve heart function as determined by systolic, diastolic, and developed pressure. Cardiac injury can also be modelled using an embolization coil in the distal portion of the left anterior descending artery (Watanabe et al., Cell Transplant. 7:239, 1998), and efficacy of treatment can be evaluated by histology and cardiac function. Cardiomyocyte preparations can be used in therapy to regenerate cardiac muscle and treat insufficient cardiac function (U.S. Pat. No. 5,919,449 and WO 99/03973).

Cancer Stem cells propagated according to the methods and compositions described here and differentiated cells derived therefrom may be used for the treatment of cancer.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer. Further examples are solid tumor cancer including colon cancer, breast cancer, lung cancer and prostate cancer, hematopoietic malignancies including leukemias and lymphomas, Hodgkin's disease, aplastic anemia, skin cancer and familiar adenomatous polyposis. Further examples include brain neoplasms, colorectal neoplasms, breast neoplasms, cervix neoplasms, eye neoplasms, liver neoplasms, lung neoplasms, pancreatic neoplasms, ovarian neoplasms, prostatic neoplasms, skin neoplasms, testicular neoplasms, neoplasms, bone neoplasms, trophoblastic neoplasms, fallopian tube neoplasms, rectal neoplasms, colonic neoplasms, kidney neoplasms, stomach neoplasms, and parathyroid neoplasms. Breast cancer, prostate cancer, pancreatic cancer, colorectal cancer, lung cancer, malignant melanoma, leukaemia, lympyhoma, ovarian cancer, cervical cancer and biliary tract carcinoma are also included.

Nuclear reprogrammed cells, pluripotent cells, stem cells and differentiated cells according to the methods and compositions described here may also be used in combination with anticancer agents such as endostatin and angiostatin or cytotoxic agents or chemotherapeutic agent. For example, drugs such as adriamycin, daunomycin, cis-platinum, etoposide, taxol, taxotere and alkaloids, such as vincristine, and

antimetabolites such as methotrexate. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. I, Y, Pr),

chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

Also, the term includes oncogene product/tyrosine kinase inhibitors, such as the bicyclic ansamycins disclosed in WO 94/22867; 1 ,2-bis(arylamino) benzoic acid derivatives disclosed in EP 600832; 6,7-diamino-phthalazin-1-one derivatives disclosed in EP 600831 ; 4,5-bis(arylamino)-phthalimide derivatives as disclosed in EP 516598; or peptides which inhibit binding of a tyrosine kinase to a SH2-containing substrate protein (see WO 94/07913, for example). A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include

Adriamycin, Doxorubicin, 5-Fluorouracil (5-FU), Cytosine arabinoside (Ara-C),

Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin,

Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincristine, VP-16, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Nicotinamide, Esperamicins (see U.S. Pat. No. 4,675,187), Melphalan and other related nitrogen mustards, and endocrine therapies (such as diethylstilbestrol (DES), Tamoxifen, LHRH antagonizing drugs, progestins, anti- progestins etc).

Non-pluripotent cells

Non-pluripotent cells may be cells of any kind. Preferably, they are eukaryotic. For example, the cells may be:non-human cells, e.g. Xenopus, rabbit, guinea pig, rat, mouse or other rodent (including cells from any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle, horse, non-human primate or other non-human vertebrate organism; and/or non-human mammalian cells; and/or human cells.

In some preferred embodiments the non-pluripotent cells are somatic cells, preferably human somatic cells. A somatic cell is generally a cell that forms the body of an organism, and is typically not a gamete, germ cell, egg cell, gametocyte or undifferentiated stem cell. As such, the non-pluripotent cells / somatic cells may be terminally differentiated.

In some embodiments the non-pluripotent cells may be selected as having PARP activity (e.g. PARP1 and/or PARP2 activity). Cells may be obtained from cell lines or from humans or animals. For therapeutic application, non-pluripotent cells may be obtained from subjects requiring treatment in order to minimise the risk of rejection of the induced pluripotent cells when re- transplanted in the subject.

Structure Specific Recognition Protein 1 (SSRP1 )

Structure specific recognition protein 1 , also known as SSRP1 , is a human protein.

The NCBI accession number for human SSRP1 is NM 003146.2 (NP_003137.1

Gl:4507241 ). The amino acid sequence for human SSRP1 is shown in Figure 10.

The SSRP1 protein is a subunit of a heterodimer that, along with SUPT16H, forms chromatin transcriptional elongation factor FACT. FACT interacts specifically with histones H2A H2B to effect nucleosome disassembly and transcription elongation.

In this specification, reference to "SSRP1" includes homologues, mutants, derivatives or fragments of the full-length polypeptide represented in Figure 10 [SEQ ID NO: 16].

Homologues may have a defined level of sequence identity with the amino acid sequence shown in Figure 10. Homologues may be non-human equivalents. A homologue may also function as a heterodimer with SUPT16H (or homologue thereof) to form a homologue of chromatin transcriptional elongation factor.

For example, an SSRP1 homologue may have one of at least 60% sequence identity, or one of 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in Figure 10.

Mutants may comprise at least one modification (e.g. addition, substitution, inversion and/or deletion) compared to the corresponding wild-type polypeptide. The mutant may display an altered activity or property, e.g. binding.

Derivatives include variants of a given full length protein sequence and include naturally occurring allelic variants and synthetic variants which have substantial amino acid sequence identity to the full length protein. Protein fragments may be up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or 650 amino acid residues long. Minimum fragment length may be 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 5, 16, 17, 18, 19, 20 or 30 amino acids or a number of amino acids between 3 and 30.

Derivatives may also comprise natural variations or polymorphisms which may exist between individuals or between members of a family. All such derivatives are included within the scope of the invention. Purely as examples, conservative replacements which may be found in such polymorphisms may be between amino acids within the following groups:

alanine, serine, threonine;

glutamic acid and aspartic acid;

arginine and leucine;

asparagine and glutamine;

isoleucine, leucine and valine;

phenylalanine, tyrosine and tryptoph,

Derivatives may also be in the form of a fusion protein where the protein, fragment, homologue or mutant is fused to another polypeptide, by standard cloning techniques, which may contain a DNA-binding domain, transcriptional activation domain or a ligand suitable for affinity purification (e.g. glutathione-S-transferase or six consecutive histidine residues).

In some embodiments, cells are contacted with SSRP1 such that the amount of SSRP1 present in the cell is increased. SSRP1 may be administered in the form of FACT.

SSRP1 provided for this purpose may be recombinant SSRP1. Recombinant SSRP1 may be generated by expression of a nucleic acid encoding SSRP1 from a suitable vector in a suitable host cell. Techniques for recombinant expression of mammalian proteins are well known in the art. Recombinant SSRP1 may be purchased, e.g. from Active Motif, Carlsbad, CA, USA.

Nucleic acid encoding SSRP1

A nucleic acid encoding SSRP1 may be any nucleic acid, e.g. polynucleotide, containing a nucleotide sequence encoding SSRP1. The nucleic acid may be a gene, protein coding sequence or an expression cassette, e.g. SSRP1 gene operably linked to a regulatory sequence (e.g. promoter, enhancer).

In this specification the term "operably linked" may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence are covalently linked in such a way as to place the expression of a nucleotide coding sequence under the influence or control of the regulatory sequence. Thus a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of a nucleotide coding sequence which forms part or all of the selected nucleotide sequence. Where appropriate, the resulting transcript may then be translated into a desired protein or polypeptide.

Some methods of the present invention involve the introduction to cell(s) of nucleic acid encoding SSRP1 such that an effective amount of SSRP1 is expressed in the cell(s). In these methods the nucleic acid encoding SSRP1 is normally exogenous, i.e. not naturally being present in the cell. Some methods of the present invention involve expressing in the cell an effective amount of SSRP1 or a nucleic acid encoding SSRP1. These methods may involve addition of exogenous nucleic acid encoding SSRP1 or may involve induction of increased levels of expression of SSRP1 , compared to those normally present in the cell, from the cell's endogenous nucleic acid encoding SSRP1 (e.g. from the naturally occuring copy of SSRP1 encoding nucleic acid present in the genome of the cell).

Both approaches may involve overexpressing SSRP1 or a nucleic acid encoding SSRP1.

Suitable molecular biology techniques for expression of SSRP1 in cells in accordance with the present invention are well known in the art, such as those set out in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989. Exogenous nucleic acid encoding SSRP1 may be provided in the form of a vector. A vector is an oligonucleotide molecule (e.g. DNA or RNA) used as a vehicle to transfer genetic material into a cell. The vector may be an expression vector for expression of the genetic material in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the gene sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express plant aspartic proteases from a vector according to the invention. Suitable vectors include plasmids, binary vectors, viral vectors and artificial chromosomes (e.g. yeast artificial chromosomes).

Many plasmids are commercially available for use in genetic engineering. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location.

A viral vector is a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors, herpes simplex virus and the like.

In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene. Retroviral mediated gene transfer or retroviral transduction refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.

Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. Retroviral vectors, such as lentivirus based vectors, may be preferred because they allow for integration of the nucleic acid encoding SSRP1 into the genome of the host cell, and therefore provide a means of stable and long term expression of an effective amount of SSRP1.

Effective amount and Overexpression

In the present specification an effective amount refers to an amount of SSRP1 or nucleic acid encoding SSRP1 that is sufficient to initiate and/or produce a change in status of a cell from non-pluripotent to pluripotent. An effective amount of SSRP1 administered to ceils may be an amount that leads to a level of SSRP1 in a cell that is at least 1.1 times that in a comparator cell of the same type which has not been administered exogenous SSRP1. More preferably, the level may be selected from one of at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least .7, at least 1.8, at least 1.9, at least 2.0, at least 2.1 , at least 2.2, at least

2.3, at least 2.4 at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.5, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, or at least 10.0 times that in the comparator cell.

An effective amount of nucleic acid encoding SSRP1 administered to cells may be an amount that leads to a level of nucleic acid encoding SSRP1 in a cell that is at least 5% more over normal endogenous levels of the nucleic acid (e.g. RNA transcript) in the cell, or alternatively one of at least 10%, 15%, 20%, 25%, 30%, 35%, 38%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%.

Some methods of the present invention involve the introduction to cell(s) of nucleic acid encoding SSRP1 , or SSRP1 protein, such that an effective amount SSRP1 is expressed in the cell(s). Some methods of the present invention involve expressing in the cell an effective amount of SSRP1 or a nucleic acid encoding SSRP1.

In some embodiments an effective amount may be provided by overexpression of SSRP1 , Over-expression of SSRP1 or of a nucleic acid encoding SSRP1 comprises expression at a level that is greater than would normally be expected for a cell of a given type. This may involve an increase in transcription of a polynucleotide into mRNA and/or the process by which the transcribed mRNA is translated into peptides, polypeptides or proteins that is greater than a base line expression in a cell of an endogenous

polynucleotide or gene or one that is exogenous and expressed at base line levels.

As such, over-expression may be determined by comparing the level of expression of a marker between cells that have been transformed with exogenous SSRP1 or nucleic acid encoding SSRP1 , or have been induced to overexpress endogenous SSRP1 , with a cell of the same type that has not been so transformed or induced. Levels of expression may be quantitated for absolute comparison, or relative

comparisons may be made. In some embodiments over-expression may be considered to be present when the level of expression in a cell is at least 1.1 times that in a comparator cell of the same type. More preferably, the level of expression may be selected from one of at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1 , at least 2.2, at least 2.3, at least 2.4 at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.5, at least 4.0, at least 5.0, at least 6.0, at least 7.0, at least 8.0, at least 9.0, or at least 10.0 times that in the comparator cell.

Overexpression may comprise at least 5% more of a nucleic acid encoding SSRP1 , e.g. RNA transcript, over endogenous levels of RNA transcript, or alternatively one of at least 10%, 15%, 20%, 25%, 30%, 35%, 38%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%.

In some embodiments the effective amount may be provided by increasing the level of expression of the endogenous nucleic acid encoding SSRP1. This may also lead to the levels of over-expression described above. Expression of an effective amount of SSRP1 may be achieved from an endogenous nucleic acid encoding SSRP1 (e.g. genomic nucleic acid encoding SSRP1 ) induced to overexpression or from an exogenous SSRP1 polynucleotide or an equivalent thereof introduced into the cell and expressed in the cell.

Induction of overexpression of endogenous nucleic acid encoding SSRP1 may involve inserting a regulatory element, e.g. enhancer element, in the genome of the cell such that it is operably linked to the genomic nucleic acid encoding SSRP1 leading to upregulation of transcription of the SSRP1 gene and SSRP1 overexpression in the cell. Methods of this invention include the introduction of transgenes that are inducible by, for example, chemical agents or physical agents. In this instance, SSRP1 can be made to be overexpressed in the cell, thereby causing the reprogramming of a non- pluripotent cell to the pluripotent cell.

PARP

Poly (ADP-ribose) polymerase (PARP) refers to a family of proteins including DNA repair and programmed cell death. The catalytic domain is responsible for Poly (ADP-ribose) polymerisation, thereby forming a PAR polymer.

PARP activity is often mainly attributable to PARP1 (e.g. NCBI accession number NP 001609.2 Gl: 156523968). PARP activity can be readily measured, e.g. by using nicotinamidase to measure nicotinamide generated upon cleavage of NAD+ during PARP-mediated poly-ADP-ribosylation of a substrate (EMD Millipore PARP1 Enzyme Activity Assay), thereby providing a direct, positive signal assessment of the activity of PARP1.

Sequence identity

Percentage (%) sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with residues in the given listed sequence (e.g. referred to by the SEQ ID No.) after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity is preferably calculated over the entire length of the respective sequences.

Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalW 1.82. T-coffee or Megalign (DNASTAR) software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used. The default parameters of ClustalW 1.82 are: Protein Gap Open Penalty - 10.0, Protein Gap Extension Penalty = 0.2, Protein matrix = Gonnet, Protein/DNA ENDGAP = -1 , Protein/DNA GAPDIST = 4. Methods according to the present invention may be performed in vitro or in vivo. The term "in vitro" is intended to encompass experiments with materials, biological substances, cells and/or tissues in laboratory conditions or in culture whereas the term "in vivo" is intended to encompass experiments and procedures with intact multi-cellular organisms. The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference. Brief Description of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1. Xenopus SSRP1 protein addition to S-phase extract recapitulates mitotic remodelling. (A). Nuclei derived from erythrocyte cells were incubated in M-phase extracts for 45 minutes. Extracts were driven into interphase by addition of CaCh and relative efficiency of DNA replication was monitored by incorporation of ³²P-a-dATP as shown in the autoradiograph. Erythrocyte DNA was incubated directly in S-phase extract in the presence of 2 μΜ GST or 2 μΜ recombinant GST-SSRP1. (B). Chart showing absolute amount of DNA replicated as in (a). Samples were taken at 30, 60, 120, 180 minutes post nuclei addition to egg extract. (C). Erythrocytes nuclei were replicated following pre-incubation in M-phase extract or direct addition to S-phase extract in the presence of 100 μΜ Olaparib, 100 nM recombinant MBP-SSRP1 or Xenopus MBP-

SPT16 as indicated in the autoradiograph. Replication efficiency was evaluated at 2h and is shown in the chart. (D). Analysis of inter-origin distance of erythrocytes DNA in S- phase extract after addition of recombinant GST-SSRP1 or GST alone or after preincubation in mitotic extract (M-phase). Permeabilized erythrocytes nuclei were incubated for 75 minutes in S-phase extract supplemented with 5 mg/ml aphidicolin and Digoxigenin d-UTP (DigU). Fibers were combed on silanized coversiips. Lower panel, DigU; upper panel, merged DigU (green, dots)/DNA (red, fibers). The centre-to-centre distances between adjacent DigU tracks and their frequency were measured and plotted on the graphs. The centre-to-centre distance between DigU tracks is indicated in kb. (E).

Immunoblot (left panel) detecting the ORC1 , MCM7 and RPA levels on the erythrocytes chromatin in the presence of GST-SSRP1 or GST alone. Levels of histone H2B were used to normalize the amount of chromatin bound proteins loaded on the gel (right panel). Samples were taken at 60 minutes post nuclei addition. Figure 2. SSRP1 is required for efficient replication of sperm nuclei. (A). Immunoblot detecting chromatin binding of ORC1 , SSRP1 , MCM7 and RPA. Histone H2B was used as a loading control. Demembraneted sperm nuclei were incubated in S-phase extract at 16°C and chromatin was isolated at the indicated time points. (B). Immunoblot detecting chromatin binding of ORC1 , ORC2, MCM7, and RPA in mock (M) or SSRP1 depleted (D) extract. Histone H2A was used as a loading control. Chromatin was extracted 60 minutes after nuclei addition to egg extract. (C). The efficiency of DNA replication in mock (lane 1 ) and SSRP1 -depleted extract (lane 2) at 120 minutes post nuclei addition was tested. SSRP1 -depleted extract (lane 2) was supplemented with SSRP1 recombinant protein at 1 μΜ (lane 3) or 2 μΜ (lane 4) or GST alone (lane 5). (D). DNA combing of embryonic DNA either incubated in mock or SSRP1 -depleted extract. Lower panel, DigU; upper panel, merge DigU (green, dots)/DNA (red, fibers). Distribution of interorigin distance is reported in the panel below.

Figure 3. Human SSRP1 overexpression in human cells increases cell proliferation and DNA replication efficiency. (A). BrdU/DAPI double staining of HEK293 cells transfected with FLAG-empty vector and FLAG-SSRP1 ; magnification 40X. Cells were visualized by DAPI (blue) and BrdU incorporation (red). (B). Chart showing quantification of BrdU positive cells by FACS analysis. Experiment was repeated three times. Error bars represent standard deviation (SD) (C). Chart showing results of proliferation assay with HEK293 cells transfected with FLAG-empty vector and FLAG-SSRP1 and incubated for different times (24h, 48h and 72h). The number of viable cells was determined by the Resazurin dye test. (D). Charts showing analysis of inter-origin distance in HEK293 FLAG-empty vector (left) or FLAG-SSRP1 (right). Logarithmically growing cultures were incubated with 100 μΜ BrdU for 30 minutes. Centre-to-centre distances between adjacent BrdU tracks were measured. The centre-to-centre distance between BrdU tracks is indicated in kb. (E). Chromatin fractions from HEK293 FLAG-empty vector and two different clones overexpressing SSRP1 (FLAG-SSRP1-4 and FLAG-SSRP1-12) were separated on SDS page gel. The blots were incubated with anti Histone H4, anti Histone H4K20me1 , anti FLAG, anti SSRP1 and anti tubulin antibodies. Right panel, chart showing optical density (OD) quantification of signal from anti SSRP1 and H4K20me1 ; signal intensity normalized with anti total H4 signal. (F). IgG or a-SSRP1 conjugated beads were incubated with crude extract from HEK293 cells. Beads-bound proteins were separated on a SDS gel and revealed by immunoblot for the presence of SSRP1 and SET8. Input is a fraction of total extract. Figure 4. Excess of SSRP1 induces DNA damage-free cell reprogramming. (A). Nuclear Transfer (NT) experiment scheme (see text). (B). Effects of Xenopus MBP- SSRP1 or MBP protein injection (Control) on tadpole development following NT. (C). NT experiment (three independent experiments combined). Eggs were injected with MBP- SSRP1 or MBP protein (Control). St 31-33 endoderm cells were used as donor.

Swimming tadpoles were counted and muscular response was judged 4 days after NT

(D). iPS cells formation. Staining of SSRP1 or OKSM-induced colonies with SSEA4, TRA- 1-60, OCT4 and Nanog. (E). Chart showing SSRP1 and OKSM-mediated iPS colony formation efficiency. Colony formation was evaluated 20 days after transduction with SSRP1 lentivirus. OKSM (Oct4, Klf-2, Sox2, and c-Myc) encoding lentivirus was used as a control. (F). Chart showing transcript levels of SOX2 and OCT4 in clones expressing different levels of SSRP1 as shown in Fig (3E) obtained by qPCR and plotted as fold increase over GAPDH expression. (G) Immunofluorescence showing phosphorylation levels of histone H2AX in OKSM and SSRP1 iPS colonies. (H) Immunoblot showing levels of SSRP1 , phosphorylated p53Ser15 (P-p53) and γ-Η2ΑΧ in fibroblasts, OKSM and SSRP1 colonies (C). MEK2 was used as loading control. Right panel, chart showing quantification of H2AX phosphorylation (γ-Η2ΑΧ) and P-p53 in three different

experiments. Signal intensity for both γ-Η2ΑΧ and P-p53 was normalized to MEK2 signal. Error bars show SD.

Figure 5. Screening. (A). Mitotic egg extract was precipitated by centrifugation following incubation with increasing amount of Polyethylene Glycol (PEG). Either Pellet (P) or supernatant (S) fractions collected at different PEG concentrations were assayed for their ability to induce replication of somatic erythrocyte nuclei directly incubated in S- phase extracts. (B). Autoradiography showing somatic nuclei replication following incubation of the different PEG fractions (P, pellet; S, supernatant) at different PEG concentration in S-phase egg extract for 120 minutes. As positive control erythrocytes nuclei were pre-incubated in mitotic extract before releasing extract into interphase with 0.4 mM CaCI2. (C). Chart showing Replication efficiency of different fraction plotted on a graph. (D). Fractions 9% P and 15% P were separated on a SDS gel and protein were analysed by mass spectrometry. The table shows the most significant hits.

Figure 6. SSRP1 is required for mitotic remodelling in a Topoisomerase II independent fashion. (A). Schematic representation of experimental procedures.

Permeabilized erythrocytes nuclei were incubated in mitotic arrested mock (M) or SSRP1- depleted (D) extract for 45 minutes. Recovered chromatin was isolated and incubated either in mock (M) or SSRP -depleted (D) S-phase extract. (B). DNA replication efficiency was scored as a percentage of DNA replication in mock treated extracts (M-M). (C). The effect of Topoisomerase II inhibition on mitotic remodelling driven by SSRP1 was tested. Permeabilized erythrocyte nuclei were incubated in S-phase extract in the presence or in the absence of recombinant SSRP1 and 50 mg/ml ICRF193

Topoisomerase II inhibitor as indicated. As control erythrocytes nuclei were pre-incubated in mitotic extract in the presence or absence of ICRF193 before releasing extract into interphase with 0.4 mM CaC . (D). The effect of Topoisomerase II inhibition on inter- origin distance remodelling driven by SSRP1 was tested. Nuclei were incubated in the presence of digoxygenin-dUTP. DNA fibers were spread and analysed by

immunofluorescence. ssDNA in red (fibers) and digoxygenin-dUTP in green (dots). Inter- origin distance shown in kb is reported under each representative DNA fiber analysis.

Figure 7. Table summarizing results from 3 independent nuclear transfer experiments. (*) Embryos were graded depending on their integrity; G5: intact blastula, G4: quarter of blastula was abnormally cleaved, G3: half of blastula was abnormally cleaved, G2: 3/4 of blastula was abnormally cleaved. (1 ) Percentage (%) shows embryos at each stage per cleaved embryos. (2) Muscular response was judged 4 days after NT. (3) Swimming tadpole includes normally and abnormally swimming tadpoles.

Figure 8. Overexpression of SSRP1 promotes iPS cells colony formation. (A).

Morphology of iPS cells colonies induced by OKSM or SSRP1. (B). Alkaline phosphatase staining of iPS cells colonies. (C). Embryoid bodies formation from OKSM or SSRP1- derived colonies. Images were acquired one week after incubation of colonies in absence of MEFs at 10X magnification. (D). Embryoid bodies (EBs) from SSRPI or OKSM- induced colonies were stained for β-Tublll, a-SMA, and AFP (ectoderm, mesoderm and endoderm markers, respectively). The middle panel shows the DAPI staining and the left panel shows merged images. Images were acquired after two weeks of incubation without MEFs, at 10X magnification. (E). Staining of differentiated EBs with the same antibodies as in (D). Left panel shows the bright field and the middle panel shows the DAPI staining.

Figure 9. SSRP1 -induced colonies show transcriptional signature of bona-fide iPS cells. (A). RT PCR showing mRNA levels of the indicated genes. (B). The table shows the fold change of pluripotency and fibroblast specific genes in OKSM and SSRP1- induced colonies versus fibroblasts. RNA was extracted from SSRP1 and OKSM-induced colonies and fibroblasts and analysed with GeneChip Gene ST 2.0 arrays. (+) and (-) indicate a fold increase or decrease of at least 1.5, respectively; (++) and (--) indicate a fold increase or decrease of at least 3; (+++) and (— ) indicate a fold increase or decrease of at least 5. Figure 10. Amino acid sequence of human SSRP1. Figure 11. Photographs showing typical differentiated connective, neuronal, vascular and pigmented tissue structures present in teratomas obtained with 2 different SSRP1 induced iPS colonies (named SSRP1#1 and SSRP1#2). SSRP1 induced iPS cells were able to form teratoma when injected in mice. To evaluate the differentiation ability of human iPSC generated by SSRP1 the ability of the cells to form teratoma was tested. To perform this assay, human SSRP1-iPSC was injected into the kidney capsule and in the testis of SCID mice. 42 days post injection tumor formation was observed. 3 kidney tumors and 2 testis were collected from 3 SCID mice. Histological examination by hematoxylin and eosin staining showed that within the tumors, there was poor differentiated teratoma, which represent mesoderm and ectoderm. The low level of differentiation might be due to the high expression level of SSRP1.

Examples

Introduction

Replication origin density in embryonic cells of some vertebrate organisms such as Xenopus laevis is significantly higher than in somatic cells. This ensures rapid and accurate genome duplication during fast embryonic cell division. Somatic origin distribution reverts to the embryonic status when somatic nuclei are transferred to eggs and mitotic extracts in nuclear transfer experiments. Until now the molecular mechanisms regulating DNA replication origins distribution across the genome during nuclear transfer have been unknown.

Materials and Methods: Plasmids and recombinant proteins

Xenopus SSRP1 clone was obtained from Source Bioscience (IMAGE ID

5507010/BH74-H6). SSRP1 gene was cloned in pDonor221 (Invitrogen) using the oligos xSSRPIfw and xSSRPI rev (see Tablel ), and sub-cloned in pDEST15-GST and pDEST- MBP (kindly donated by Simon Boulton's lab). Xenopus SPT16 clone was obtained from Source Bioscience (IMAGE ID 5048405/AK 52-B 3). SPT16 was cloned in pDonor221 (Invitrogen) using the oligos xSPT16Hfw and xSPT16Hrev (see Tablel ), and subcloned in pDEST-MBP. Human SSRP1 was cloned in a pDEST15 vector from pENTR221 obtained from Imagenes (clone RZPDo839G05150D). Human SSRP1 was cloned into the Gateway destination vector pcDNA5FRT/TO (Invitrogen), modified with a FLAG-TAG (gift from Zuzana Horejesi, Clare Hall Laboratories). Protein purification

Expression of proteins tagged with Maltose binding protein MBP. Expression of MBP- SSRP1 , or MBP-SPT16 or MBP-TAG proteins in E.coli was induced for 3h at 30°C with 0.2 mM IPTG (SIGMA). The pellet was resuspended in lysis buffer (200 mM NaCI, 20 mM Tris pH7.5, 1 mM EDTA + protease inhibitors) and incubated 10 minutes on ice with 50 μΜ DNasel. Lysates were obtained through cell disruptor and clarified by centrifugation for 45 minutes at 21000 rpm at 4°C. Amylose resin suspension was used for purification on a column at 4°C. The column was washed three times with lysis buffer and proteins were eluted in 10 mM Maltose (Sigma) in lysis buffer. The protein was dyalized over night in 0 mM Tris pH8, 150 mM NaCI.

The expression of GST-SSRP1 or GST-TAG in E.coli was induced for 3h at 25°C with 0.2 mM IPTG. Pellet was resuspended in lysis buffer (300 mM NaCI, 200 mM HEPES pH7.5, 1 % Triton-X100, 1 mM DTT). Cell suspension was sonicated 3 times for 30 seconds and then incubated 10 minutes at 4°C with 25pg/ml DNasel. Lysate was clarified 45 minutes at 4°C at 2 000rpm. 2 ml of glutathione slurry were used to purify lysate from 1 litre culture. Lysate was incubated with glutathione beads (SIGMA) in rotation for 1 h at 4°C and then loaded on a column. After washing with lysis buffer, the protein was eluted with 150mM NaCI, 20mM HEPES pH7.5, 1 mM DTT and 20mM Glutathione pH8. SSRP1 and GFP control lentivirus (LV-EGFP) were obtained from Excellgen.

Antibodies

Rabbit Xenopus SSRP1 antiserum was generated against Xenopus GST-SSRP1 protein by Pettingill Technology Ltd, UK. Human SSRP1 was detected using anti-SSRP1 antibody from Abeam (ab26212). Anti histone H4 (monomethyl K20) was from Abeam (ab16974). Total histone H4 was detected with anti-histone H4 from Millipore (07-108). SET8 was detected with SET8 monoclonal antibody (B.540.4, Thermo Scientific).

Additional antibodies used in this study included antibodies against Mcm7 (Santa Cruz Biotechnology) and anti-histone H2B (Millipore). Monoclonal ORC1 antibody TK15 was previously generated in the Tim Hunt's laboratory (Tugal et al, 1998).

Extract fractionation

Egg extract for fractionation experiment shown in Fig 5 was prepared as previously described (Kubota and Takisawa, 1993) with some modifications. Briefly, 200 μΙ extract were diluted in 800 μΙ of LFB buffer 1/50 prepared from LFB1 (40 mM Hepes-KOH pH8, 20 mM K₂HP04-KH₂P04, 2mM MgCI2, 1 mM EGTA, 2 mM DTT, 10% Sucrose, protease inhibitors Leupeptine, pepstatine Aprotine 1 Mg each). LFB1/50 was prepared from

LFB1/100 diluted 1 :1 in LFB1. LFB1/100 was prepared from LFB1/ 000 diluted 1 :10 in LFB1. LFB1/100 was prepared from LFB1 plus 1 M KCI. The diluted extract was centrifugated 40 minutes at 80000xg in a swing out rotor at 4°C. The supernatant was decanted. 50% PEG 6000 (SIGMA) was added to LFB1 to obtain the starting

concentration (0.075 volumes for 3.5% PEG starting concentration) and the mix was incubated 30 minutes on ice. After 10 minutes centrifugation at 10.000 xg, the

supernatant was recovered and the pellet resuspended in 40 μΙ of LFB1/50. After collecting the aliquote, the supernatant was used for subsequent precipitations.

Erythrocyte nuclei were prepared as previously described (Lemaitre et al, 2005). Each fraction was then tested for its ability to induce replication of erythrocytes nuclei in S- phase extract. Each replication reaction was performed by adding 4 μΙ of each fraction to 16 μΙ of S-phase exctract and incubated for 120 minutes in the presence of 32P a-dATP.

Xenopus egg extract and chromatin isolation

Egg extracts and demembranated sperm nuclei were prepared as previously described (Lemaitre et al, 2005). To isolate chromatin fractions sperm nuclei (3000 nuclei/μΙ) were added to 40 μΙ of egg extracts for the indicated times. For immunoblotting, samples were diluted with 10 volumes of EB (100 mM KCI, 2.5 mM MgCI2, and 50 mM HEPES-KOH

(pH 7.5)) containing 0.25% NP-40 and centrifuged through a 30% sucrose layer at 10.000 g at 4°C for 5 minutes. Pellets were suspended in sample buffer loaded on a SDS-PAGE.

DNA replication assay

Sperm nuclei (3000 nuclei/μΙ) were added to 20 μΙ interphase egg extract. Samples were supplemented with 0.1 μΙ of ³²P-a-dATP (250 mCi; 3000Ci/mmol) and incubated at 23°C. Replication was stopped and analysed by agarose gel electrophoresis and

autoradiography. For quantitation, the intensity of the radioactive band was measured with the phosphoimager analysis programme (Amersham). Alternatively, to quantify absolute amount of replicated DNA samples were spotted on glass fiber filters and analysed through a β-counter as previously described (Lemaitre et al, 2005).

Immunodepletion and immunoprecipitation

For depleting 1 ml of Xenopus egg extract, 30 pg anti-SSRP1 antibodies were used. For immunoprecipitations, antibodies (10 pg) were conjugated to 30 μΙ protein A-Sepharose FF (Amersham) and added to 200 μΙ Xenopus egg extract. After 1 h of incubation, beads were washed and harvested. Samples were analysed by SDS-PAGE, transferred to a PVDF membrane and immunoblotted.

DNA combing

Sperm or erythrocytes nuclei were incubated in egg extract supplemented with digoxygenin-dUTP (Roche) directly added to egg. The reaction was stopped by adding the same volume of 1 % LMP agarose (Lonza) and transferred to a casting mould to prepare the plugs. Plugs were then treated with 2 mg/ml of proteinase-K (Roche) at 50°C overnight. The treatment was repeated, changing the proteinase-K solution, the day after, both over day and overnight. Subsequently, plugs were washed several times in TE buffer supplemented with 50 mM EDTA. The TE buffer was replaced with 50 mM MES buffer (pH 5.7) (3 ml/each plug) and plugs were incubated at 65°C for 15 minutes. Once melted, plugs were treated with β-agarase (3 units/plug; New England Biolab) at 42°C overnight. The resulting solution was used for stretching DNA fibres on silanized slides obtained from Genome Vision at a constant speed of 18 mm/minute. Slides were then dried at 65°C for 30 minutes and stained with fluorescent secondary antibodies against digoxygenin and DNA red staining dye according to manufacturer instructions (Genome Vision). Proliferation assay

HEK293 cells (2χ10³/100μΙ_) empty vector and SSRP1 expressing clones were plated into 96-well tissue culture microplates. For background control 100 μΙ medium without cells were used. After 24, 48, or 72 hours in culture 10 μΙ Resazurin solution (30025-1 , BIOTIUM) was added, and cells were incubated at 37° C for 3 hours. Fluorescence was measured on a PHERAstar FS (B G LABTECK) microplate reader using an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Fluorescence signal from each sample was deducted by background fluorescence from the background control. Each experiment was performed in quadruplicate. BrdU incorporation assay

HEK293 cells were incubated with 20 μΜ 5-bromo-2^' desoxyuridine (BrdU) (Sigma- Aldrich) at 37°C for 30 minutes, harvested and then fixed in 4% Paraformaldehyde (15710, Electron Microscopy Sciences) for 15 minutes at room temperature (RT) and permeabilised on ice cold phosphate-buffered saline (PBS) - 0,01 % Triton-x100 (PBST). After fixation, cells were washed with PBST and treated with 1 ,5 N HCI for 30 minutes at room temperature. Thereafter, cells were washed with PBS - 0.2% Triton-x100 for 10 min at RT and washed again. Cells were blocked in PBS 3% - bovine serum albumin (BSA) 0,01 % Triton-x100 (blocking solution), for 30 min at RT and incubated with 1 :250 anti BrdU antibody (OBT0030G, AbD SEROTEC) diluted in blocking solution for 1 h at room temperature. After washing with PBST cells were incubated with secondary antibody solution, Alexa Fluor^® 594 goat anti-rat IgG (Invitrogen) diluted in blocking solution, containing 1 pg/ml DAPI for 1 hour at RT in the dark. Samples were mounted with Vectashield mounting medium (H100, Vector Laboratories). Visualization of BrdU/DAPI double staining of HEK293 cells was obtained with fluorescent microscopy with magnification x 40. The total cell population was visualized by DAPI staining in blue, while the BrdU incorporation was visualized in red. Quantification of BrdU positive cells was performed using FACS analysis.

Somatic cell nuclear transfer

Somatic cell nuclear transfer to Xenopus eggs was done as described previously

(Gurdon, 2006). Briefly, endoderm cells from tail bud embryos (stage 31-33) were dissociated in calcium- and magnesium-free MBS. Endoderm cells were then transferred into the buffer containing SSRP1 protein. Single endoderm cell was picked up and injected into an egg enucleated by UV irradiation. This injection procedure allows injection of 5-10 ng of recombinant MBP-SSRP1 protein into an egg. As a control, MBP protein was injected with endoderm cells. The nuclear transplant embryos were cultured in MBS containing 0.1 % BSA at 16 °C overnight and then embryos at the blastula stage were moved to O. lxMBS.

Lentiviral mediated cell Reprogramming

Lentiviral reprogramming was performed following the Lentiviral Reprogramming System manual from Invitrogen. SSRP1 constitutive expressing lentivirus was obtained from Excellgene or constructed as described below OKSM single polycistronic mRNA expressing lentivirus (STEMMCA) was purchased from Millipore. Cell culture

Normal human fibroblast (GM00024C) were purchased from Coriell Cell Repositories (Camden, NJ) and grown in Eagle's Minimal Essential Medium plus 10% fetal bovine serum at 37°C plus 5% CO2. HEK293T cells were maintained in Dulbecco's Minimal Essential Medium containing 10% FBS and 1 % penicillin and streptomycin. iPS cells were generated and maintained in Human iPSC medium (DMEM/F-12 supplemented with 20% Knockout Serum Replacement (KSR), antibiotic-antimycotic solution, MEM non- essential amino acids solution, β-mercaptoethanol) supplemented with 12 ng/ml recombinant human basic fibroblast growth factor (bFGF) and valproic acid. For passaging, human iPS cells were washed once with PBS and then incubated with DMEM/F-12 containing 1 mg/ml collagenase IV at 37°C. When colonies at the edge of the dish started dissociating from the bottom, DMEM/F-12/collagenase was removed and washed with Human iPSC medium. Cells were scraped and collected into 15 ml conical tube. An appropriate volume of the medium was added, and the contents were transferred to a new dish on attachment factor (Invitrogen, cat. N. S-006-100)- coated mytomicin C-treated MEF feeder cells. The medium was changed daily.

Plasmid lentiviral expression vectors

SSRP1 pLentiviral expression vector was constructed using the Gateway LR Clonase II enzyme mixture (Invitrogen Cat. No. 1 1791-020) according to the manufacturer's protocol between Gateway-compatible entry clones and HiPerform pl_enti6.3/V5-DEST vector (Invitrogen, Cat. No. V533-06).

Lentivirus production and infection

HEK293T cells were plated at 70% confluence in 100 mm dish and incubated overnight. Cells were transfected with 3 pg pLenti6.3/V5-SSRP1 DNA plus 9 pg Virapower packaging mix using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Forty-eight hours after transfection the supernatant of transfected cells was collected and filtered through a 0.45 pm pore-size cellulose acetate filter (Whatman). Human fibroblasts were seeded at 8 x 10⁵ cells per 100 mm dish 1 day before

transduction. The medium was replaced with virus-containing supernatant, and incubated for 24 hr. Five days after transduction, fibroblasts were harvested by trypsinization and replated at 8 x 10⁴ per 100 mm dish on MEF feeder layer. Next day, the medium was replaced with human iPSC medium supplemented with 12 ng/ml bFGF and valproic acid (Sigma-Aldrich, Cat. No. P4543). The medium was changed every other day. Twenty days after transduction, colonies were picked up, mechanically dissociated to small clamps by pipetting up and down then transferred in 2-well plates.

RNA isolation and Reverse Transcription

Total RNA was purified with RNeasy Mini kit (Qiagen). Two micrograms of total RNA were used for reverse transcription reaction with Superscript II Reverse transcriptase (Invitrogen) and random primers, according to the manufacturer's instructions. PCR was performed with GoTaq (Promega) (for oligonucleotide description see Table 2). Antigen staining

Cells were fixed in 4% formaldehyde for 15 minutes at room temperature, permeabilized with 100% ice-cold methanol for 10 minutes at -20°C, and blocked with PBS containing 5% normal serum from the same species as the secondary antibody, and 0.1 % Triton X- 100 for 60 minutes. Cells were incubated with primary antibody overnight at 4°C, washed and incubated with Alexa Fluor (Invitrogen) secondary antibody for 2 hours. Oct4

(C30A3), Nanog antibody, SSEA4 (MC813), TRA-1 -60 (StemLight Pluripotency Antibody Kit, Cat. No. 9656), P-H2A.X (Ser139) (γ-Η2Α.Χ) and P-p53 (Ser15) primary antibodies were obtained from Cell Signaling. Secondary antibodies used were Alexa 488- conjugated donkey anti-Rabbit IgG (A21206, Invitrogen), Alexa594-conjugated goat anti- Mouse lgG2A (A21 135, Invitrogen). Alkaline phosphatase staining was performed using the Leukocyte Alkaline Phosphatase kit (Sigma). Western Blotting on cells

The cells were lysed with RIPA buffer (50 mM Tris-HCI pH7.5, 150 mM NaCI, 0.1 % SDS, 0.5% NaDeoxycholate, 1 % NP-40, 5 mM EDTA pH 8.0), supplemented with protease inhibitor cocktail (Calbiochem, Cat. No. 539 34). Cell lysates (20 μg) were separated by electrophoresis on 4% to 2% SDS-polyacrylamide gel and transferred to a polyvinylidine difluoride membrane (Whatman). The blot was blocked with TBST (20 mM Tris-HCI, pH 7.6, 136 mM NaCI, and 0.1 % Tween-20) containing 5% skim milk and then incubated with primary antibody solution at 4°C overnight. After washing with TBST, the membrane was incubated with secondary antibody for 1 hour at room temperature. Signals were detected with ECL Western Blotting Detection Reagents (GE Healthcare, Cat. No. RPN2106). Antibodies used for western blotting were anti-Ssrpl (1 :1000, 0D7, Abeam), anti-γ- H2A.X (S139) (1 :1000, Cell Signaling), anti-a-Tubulin (Clone B-5-1-2, T5168, Sigma- Aldrich).

In EB Vitro Differentiation and detection

For EBs formation, confluent undifferentiated human iPS cells were harvested by treating with collagenase IV and transferred in single cell suspension with a density of 10³ cells/ml into a 100 mm bacterial-grade dish. After 8 days as a floating culture, EBs were transferred to gelatin-coated plate and cultured in the same medium for another 8 days. Differentiated EBs were stained with anti-alpha smooth muscle Actin a-SMA (ab5694), anti-alpha 1 Fetoprotein (AFP) (ab94479) and anti-βΙΙΙ Tubulin mAb (promega G7121 ). Table 1. Oligonucleotides for cloning of SSRP1 and SPT16

Table 2. RT PCR oligonucleotides

Teratoma and Chimera formation assays

Teratoma and Chimera formation using SSRP1 derived iPS were performed by Applied Biosystems according to standard protocols. Briefly, five week old immune-deficient NOD/SCID mice were used for transplantation. They were maintained under non-specific pathogen-free (SPF) conditions. The cell-Matrigel mixture (100 μΙ) was injected into the kidney and testis of the mice. The transplanted animals were observed routinely once a week. They were sacrificed after development of palpable tumors or following an observation period of 30 weeks. The sacrificed animals were subjected to general inspection. When tumors were observed at the injection site, they were collected. In addition, the liver, lungs and spleen tissues of all the animals were inspected for tumor invasion into other organs. For the histological analysis the tumor containing tissues were fixed in 4% PFA, embedded in paraffin and serially sectioned into 6 micron sections. Every other section was mounted on slides (2-3 sections per slide, and 20 slides for each tumor). Slides, containing 8-12 sections from various parts of the tumor were stained with hematoxylin and eosin, and subjected to histological analysis by a certified pathologist. Chimera generation was obtained according to standard protocols by injecting blastula embryos obtained from albino mice with SSRP1 generated iPS cells. CD-1 pseudo- pregnant females were used to implant chimeric embryos. Pups were scored for agouti phenotype for the chimeric contribution, which was close to 90% for more than 50% of the pups.

Results:

SSRP1 protein addition to interphase extract recapitulates mitotic remodelling

Somatic nuclei replicate their DNA from fewer replication origins than their embryonic counterparts. This process can be reverted by different experimental procedures including incubation of somatic nuclei in embryonic egg extract and intact eggs (Ganier et al., 2011 ; Gurdon and Murdoch, 2008; Jaenisch and Young, 2008). However, the molecular mechanisms underlying this phenomenon are unknown. Transfer of somatic nuclei to Xenopus laevis eggs or mitotic egg extracts promotes the conversion of the somatic replication program to an embryonic one, following the removal of somatic chromatin-bound proteins such as transcription factors and histones (Ganier et al., 20 1 ; Kikyo et al., 2000; Lemaitre et al., 2005). Successful mitotic remodelling produced during incubation of erythrocyte somatic nuclei in mitotic (M-phase) egg extract allows their replication in interphase (S-phase) extract in which non-remodelled nuclei are unable to efficiently replicate (Fig. 1 A and (Lemaitre et al., 2005)). To identify factors responsible for such remodelling the inventor fractionated mitotic egg extracts with increasing amounts of polyethylene glycol (PEG) and incubated fractions in S-phase egg extract containing non- remodelled nuclei (Fig. 5A). They identified a fraction able to stimulate replication of somatic nuclei in S-phase egg extract without prior incubation in mitotic extract (Fig. 5B and 5C). Mass spectrometry analysis highlighted the presence in the most active fraction of histone chaperone and transcription factor FACT complex subunits SSRP1 and SPT16 (Singer and Johnston, 2004) (Fig 5D). To test the role of the FACT complex in nuclear remodelling they supplemented S-phase extract with recombinant FACT complex or its subunits SSRP1 and SPT16. SSRP1 alone was able to stimulate replication of somatic nuclei in S-phase extract. In contrast, somatic nuclei in S-phase extract alone did not replicate as previously reported (Fig. 1A and 1 B) (Lemaitre et al., 2005). The FACT complex was no better than SSRP1 in stimulating DNA replication suggesting that SSRP1 alone is sufficient to produce this effect (Fig. 1 C, lanes 8 and 4).

Recent evidence has shown that SSRP1 is posttranslational modified by the polyADP- ribosylation activity of PARP and that PARP activity is required for efficient nuclear reprogramming (Chiou et al., 2013). When olaparib was used to inhibit PARP activity (Menear et al., 2008) in egg extract the inventor found that both mitotic and SSRP1 - dependent stimulation of DNA replication of somatic nuclei was inhibited, suggesting that PARP activity is necessary for SSRP1 activity (Fig. 1 C). SSRP1 function might be facilitated by chromatin relaxation induced by PARP (Poirier et al., 1982). SSRP1 has been implicated in the elongation step of DNA replication (Abe et al., 2011 ) possibly through its interaction with the MCM complex (Tan et al., 2006). To determine whether the increased replication was due to an increased number of replication origins, as in the case of mitotic remodelling (Lemaitre et al., 2005) or to stimulation of the replication fork elongation rate, the inventor analysed the replication origin assembly and distribution on DNA fibers. Strikingly, they observed a significant increase in the number of replication origins on somatic nuclei added directly to S-phase extract in the presence of

recombinant SSRP1 , reaching levels comparable to embryonic nuclei and somatic nuclei pre-incubated in mitotic extracts (Fig. D). Mitotic remodelling is accompanied by an increase in the chromatin-bound ORC complex (Lemaitre et al., 2005). The inventor noticed that in the presence of recombinant SSRP1 , the amount of ORC1 bound to chromatin was also increased (Fig. 1 E). Increased ORC1 binding was paralleled by increased loading of the pre-replication complex (pre-RC) component MCM 7, member of the MCM helicase complex, and single strand DNA binding protein RPA, which is loaded when origins fire (Fig. 1 E). Thus, addition of recombinant SSRP1 to S-phase egg extract is sufficient to restore on somatic nuclei the density of active replication origins typically present during embryonic DNA replication. This process appears to be mediated by the stimulation of the pre-RC complex formation on somatic chromatin. Importantly, SSRP1 mediated increase of replication origin density is independent of its transcriptional role as transcription is absent in Xenopus egg extract.

The inventor then tested whether endogenous SSRP1 present in mitotic egg extract is required for mitotic remodelling of somatic nuclei. To this end they incubated somatic nuclei in M-phase extract depleted of SSRP1 and then transferred them to mock and SSRP1 depleted S-phase -extraets-te- irromtc>f-DNA-fep+icatiori. This experiment revealed that SSRP1 is required for mitotic remodelling as somatic nuclei pre-incubated in SSRP1- depleted M-phase extract were unable to replicate in mock-depleted S-phase extract (Fig 6A and 6B). As expected depletion of SSRP1 from S-phase inhibited DNA replication. The activity of Topoisomerase II has been implicated in mitotic remodelling of somatic

DNA (Lemaitre et al., 2005) and in repiicons resetting at the S-M phase transition (Cuvier et al., 2008). However, inhibition of Topoisomerase II with ICRF193 (Lemaitre et al., 2005) did not affect DNA replication efficiency or origin distribution in the presence of recombinant SSRP1 (Fig. 6C and 6D). These findings suggest that replication origins formation on somatic chromatin induced by recombinant SSRP1 is independent of Topoisomerase II activity and likely acts downstream this step.

SSRP1 is required for proper replication of embryonic DNA

To determine the molecular mechanisms underlying the effects of SSRP1 on DNA replication the inventor analysed the binding of endogenous SSRP1 to chromatin using sperm nuclei, which are efficiently replicated in S-phase extracts. Sperm nuclei were incubated in S-phase extract and chromatin was extracted at different times. SSRP1 was found to bind before the binding of the ORC complex (Fig. 2A). Furthermore, depletion of SSRP1 from egg extract greatly reduced ORC1 binding to sperm chromatin, increased the inter-origin distance and impaired replication efficiency of sperm nuclei (Fig. 2B, 2C and 2D). These findings indicate that SSRP1 facilitates pre-RC assembly and DNA replication also when sperm nuclei are used as template. The requirement for extra recombinant SSRP1 for the replication of somatic nuclei in S-phase extract suggests that endogenous SSRP1 in S-phase extract is limiting or less active towards somatic nuclei compared to mitotic and recombinant SSRP1 proteins.

SSRP1 over-expression in mammalian cells increases the efficiency of DNA replication and cell proliferation

To verify whether SSRP1 controls replication origin distribution in mammalian cells they produced stable 293 cell lines overexpressing FLAG-SSRP1. They analysed the BrdU incorporation of cell overexpressing FLAG-SSRP1 or FLAG-empty vector and found a significant higher number of BrdU positive cells in the presence of elevated levels of SSRP1 (Fig. 3A and 3B). The proliferation rate of SSRP1 expressing cells was also highly enhanced compared to control cells (Fig. 3C). They then monitored replication origin distribution by DNA combing. The analysis of DNA fibers recovered from

logarithmically growing cells stably overexpressing SSRP1 revealed a strong increase in the replication origin density, accompanied by a dramatic reduction from 118 kb to 33 kb of the average inter-origin distance (Fig 3D). Therefore, the ability of SSRP1 to induce the formation of de novo replication origins is conserved across different species. To understand the mechanisms underlying global increase in DNA replication origin density the inventor investigated the status of epigenetic modifications induced by SSRP1 overexpression in mammalian cells. To this end they analysed cellular clones expressing different levels of FLAG-SSRP1 protein and found increased genome wide methylation of histone H4 lysine 20 (H4K20) in cells with high levels of SSRP1 protein (Fig 3E). H4K20 methylation is recognized by the BAH domain of human ORC1 (Kuo et al., 2012; Noguchi et al., 2006), which is required for stable ORC1 chromatin binding. Mutations in this domain impair the formation of the pre-RC complex resulting in defective DNA replication in early stage of development (Bicknell et al., 2011 ). It is likely that increased genome wide H4K20 methylation contributes to increased ORC1 and therefore ORC complex binding to chromatin in cells overexpressing SSRP1. H4K20 methylation can be executed by Set8, which has been recently involved in the regulation of replication origins in mammalian cells (Tardat et al., 2010). Interestingly, they found that SSRP1 and Set8 interact in vivo (Fig. 3F). SSRP1 might facilitate chromatin binding of ORC1 by bringing Set8 to DNA and stimulating histone H4K20 methylation. Increased ORC complex binding and pre-RC assembly might also be favoured by known nucleosome remodelling activity of SSRP1 (Sharma et al., 2009).

SSRP1 mediates DNA damage free iPS cell formation

Reconfiguration of replication origin pattern in somatic cells is an essential step, which nuclei have to go through when transplanted into eggs or embryonic cells in nuclear transfer (NT) and animal cloning experiments (Gurdon and Wilmut, 201 1 ). Inefficient reconfiguration of replication origins in somatic cells that fail to adapt to the fast replication pace of embryonic division could contribute to inefficient reprogramming of somatic nuclei (Gurdon and Wilmut, 201 1 ). The inventor tested the hypothesis that SSRP1 improves the quality of the tadpoles obtained by NT experiments by promoting a more efficient reconfiguration of replication origin distribution of somatic nuclei injected into eggs. To this end they injected Xenopus laevis eggs with differentiated somatic nuclei isolated from stage 31 to 33 and assessed tadpoles development. They noticed that the presence of recombinant SSRP1 yielded a greater number of swimming tadpoles and improved their muscular responses (Fig 4A, 4B, 4C and 7). These results suggest that SSRP1 promotes the formation of healthier cells and tissues in NT experiments. Formation of induced pluripotent stem (iPS) cells has been shown to recapitulate many steps of embryonic development obtained through NT experiments (Abad et al., 2013; Yamanaka and Blau, 2010). Intriguingly, SSRP1 is one of the first genes activated in the process of iPS cells formation (Doege et al., 2012). The inventor tested whether SSRP1 has any direct role in the formation of iPS cells. To this purpose they infected human adult fibroblasts with a lentivirus expressing SSRP1 or OCT4, KLF4, SOX2 and c-MYC (OKSM) factors expressed from a single polycistronic mRNA. After few days, following the protocol set up by Yamanaka and colleagues (Takahashi et al., 2007), they transferred the infected cells onto mouse embryonic feeder (MEF) cells and monitored iPS cells colony formation. Strikingly, overexpression of SSRP1 alone was sufficient to induce the formation of iPS-like cells. These cells formed colonies that were

morphologically indistinguishable from the ones obtained with OKSM factors and were positive for early and late common pluripotency markers (Fig 4D, 8A and 8B). Consistent with this iPS-like cells obtained with SSRP1 overexpression were able to produce embryoid bodies (EBs), which were morphologically identical to EBs obtained from OKSM induced iPS cells (Fig 8C). SSRP1 induced EBs contained cells of ectoderm, endoderm and mesoderm derivation able to differentiate to more mature precursors (Fig. 8D and 8E). Importantly, SSRP1 -induced iPS cells, similar to OKSM induced iPS, were able to produce teratoma when injected in mice. These teratomas contained differentiated structures derived from embryonic germ layers (Figure 1 1). In addition, SSRP1 -derived cells were able to promote formation of chimeric mice with high efficiency, high chimeric grade and germline transmission (not shown). However, the number of iPS-like cells formation obtained with SSRP1 alone was lower when compared to the iPS cells obtained with OKSM factors (Fig. 4E).

To clarify the mechanisms underlying generation of iPS-like cells due to SSRP1 overexpression, the inventor monitored the expression profile of pluripotency-associated genes in primary fibroblasts infected with SSRP1 or OKSM lentivirus. They found that OCT4, KLF4, c-MYC and NANOG mRNAs were induced by SSRP1 alone to levels comparable to the ones obtained with OKSM factors (Fig. 9A). Intriguingly, SSRP1 mRNA was also induced by SSRP1 protein overexpression at levels significantly higher than the ones induced by OKSM factors. Increased levels of OCT4 and SOX2 mRNAs directly correlated to SSRP1 protein expression levels (Fig. 4F) as assessed in individual clone stably expressing different levels of SSRP1 (Fig 3E). To further validate the extent of nuclear reprogramming of SSRP1 induced iPS-like cells they monitored levels of mRNAs of genes normally associated to pluripotency or differentiation using genome-wide microarray analysis. Consistent with the other pluripotency features of SSRP1 induced iPS-like cells the inventor found that most of pluripotency-associated genes were upregulated whereas most of the mRNAs associated to fibroblast differentiation were downregulated to levels similar the ones obtained with OKSM (Fig. 9B). These data suggest that SSRP1 is able to promote iPS-like cells formation by triggering a

transcriptional program similar to the one induced by OKSM.

A key issue in the generation of iPS cells by the OKSM factors is the induction of replication stress, DNA damage and genome instability (Banito et al., 2009; Marion et al., 2009), thus potentially limiting their future use in clinical applications (Blasco et al., 2011 ). Reduced density of replication origins is a primary cause of DNA breakage at hard-to- replicate genomic DNA sequences (Letessier et al., 201 ). Therefore, the inventor hypothesized that due to its ability to increase replication origin density on somatic chromatin SSRP1 could promote nuclear reprogramming with reduced amount of replication stress and DNA damage. To test this hypothesis the inventor monitored induction of DNA damage by staining cells for histone H2AX phosphoryiated on serine 139 (Y-H2AX), a sensitive DNA damage marker (Ciccia and Elledge, 2010). They found that cells infected with a single lentivirus encoding for the OKSM factors produced significant higher levels of γ -H2AX compared to cells infected with SSRP1 encoding lentivirus (Fig 4G). To verify the extent of DNA damage accumulation during nuclear reprogramming they monitored genome- wide levels of γ -H2AX and nuclear

accumulation of p53 phosphoryiated on serine 15, an alternative marker of DNA damage response activation (Ciccia and Elledge, 2010). They found that SSRP1 induced iPS-like cells had significant lower levels of γ -H2AX and phosphoryiated p53 compared to iPS cells obtained with OKSM factors (Fig. 4H). Reduction of replication stress and DNA damage accumulation during nuclear reprogramming might be ascribed to a higher DNA replication efficiency produced by SSRP1 -mediated increase of DNA replication origin in somatic nuclei. Here the inventor describes the identification of SSRP1 as a key player in regulating replication origin assembly and pluripotency state. Since the establishment of the

Yamanaka protocol, which did not include SSRP1 , several methods have been reported to improve iPS cells formation efficiency. Recently Rais and colleagues showed that it is possible to obtain almost 100% reprogramming efficiency by depleting the Mbd3, a core component of the Mbd3/Nurd repressive complex (Rais et al., 2013). These findings, instead, show for the first time that nuclear reprogramming can be obtained with a single factor that induces expression of essential pluripotency genes typically present in iPS cells and at the same time controls the replication origins assembly. NT experiments performed by Gurdon indicated that egg cytoplasm carries as-of-yet undefined

determinants that accomplish somatic reprogramming within few cell divisions. Among these factors it is likely that SSRP1 plays a prominent role. The mechanism of action of SSRP1 is complex and includes increased replication origin formation through histone H4K20 methylation and direct stimulation of pluripotency genes transcription. SSRP1 - mediated stimulation of replication origin formation might help overcoming replication stress associated to high levels of transcription and metabolic requirements of embryonic cells. These findings might serve as paradigm to understand the relation between replication origin density and genome stability maintenance in developing vertebrate cells.

The inventor has shown that SSRP1 (Structure-specific recognition protein 1 ) triggers embryonic reconfiguration of somatic replication origins in Xenopus laevis egg extract by promoting the formation of the pre-replication complex. SSRP1 function requires PARP (Poly ADP-ribose polymerase) activity and increases the quality of embryos obtained by nuclear transfer experiments. Strikingly, SSRP1 overexpression in mammalian cells is sufficient to generate DNA damage free induced pluripotent stem (iPS) cells. These findings suggest replication origin regulation by SSRP1 is a key factor regulating pluripotency state and genome stability in developing vertebrate organisms.

References

Abad, M., Mosteiro, L, Pantoja, C, Canamero, M., Rayon, T., Ors, I., Grana, O., Megias, D., Dominguez, O., Martinez, D., et al. (2013). Reprogramming in vivo produces teratomas and iPS cells with totipotency features. Nature 502, 340-345.

Abe, T., Sugimura, K., Hosono, Y., Takami, Y., Akita, M., Yoshimura, A., Tada, S., Nakayama, T., Murofushi, H., Okumura, K., et al. (201 1 ). The histone chaperone facilitates chromatin transcription (FACT) protein maintains normal replication fork rates. J Biol Chem 286, 30504-30512.

Banito, A., Rashid, ST., Acosta, J.C., Li, S., Pereira, C.F., Geti, I., Pinho, S., Silva, J.C., Azuara, V., Walsh, M., et al. (2009). Senescence impairs successful reprogramming to pluripotent stem cells. Genes & development 23, 2134-2139.

Bicknell, L.S., Walker, S., Klingseisen, A., Stiff, T., Leitch, A., Kerzendorfer, C, Martin, C.A., Yeyati, P., Al Sanna, N., Bober, M., et al. (2011 ). Mutations in ORC1 , encoding the largest subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome. Nat Genet 43, 350-355.

Blasco, M.A., Serrano, M., and Fernandez-Capetillo, O. (201 1 ). Genomic instability in iPS: time for a break. The EMBO journal 30, 991 -993.

Chiou, S.H., Jiang, B.H., Yu, Y.L., Chou, S.J., Tsai, P.H., Chang, W.C., Chen, L.K., Chen, L.H., Chien, Y., and Chiou, G.Y. (2013). Poly(ADP-ribose) polymerase 1 regulates nuclear reprogramming and promotes iPSC generation without c-Myc. The Journal of experimental medicine 210, 85-98.

Ciccia, A., and Elledge, S.J. (2010). The DNA damage response: making it safe to play with knives. Molecular cell 40, 179-204.

Cuvier, O., Stanojcic, S., Lemaitre, J.M., and Mechali, M. (2008). A topoisomerase II- dependent mechanism for resetting replicons at the S-M-phase transition. Genes Dev 22, 860-865.

Doege, C.A., Inoue, K., Yamashita, T., Rhee, D.B., Travis, S., Fujita, R., Guarnieri, P., Bhagat, G., Vanti, W.B., Shih, A., et al. (2012). Early-stage epigenetic modification during somatic cell reprogramming by Parpl and Tet2. Nature 488, 652-655.

Ganier, O., Bocquet, S., Peiffer, I., Brochard, V., Arnaud, P., Puy, A., Jouneau, A., Feil, R., Renard, J. P., and Mechali, M. (201 1 ). Synergic reprogramming of mammalian cells by combined exposure to mitotic Xenopus egg extracts and transcription factors. Proc Natl Acad Sci U S A 108, 17331-17336.

Gore, A., Li, Z., Fung, H.L., Young, J.E., Agarwal, S., Antosiewicz-Bourget, J., Canto, I., Giorgetti, A., Israel, M.A., Kiskinis, E., et al. (2011 ). Somatic coding mutations in human induced pluripotent stem cells. Nature 471, 63-67.

Gurdon, J., and Murdoch, A. (2008). Nuclear transfer and iPS may work best together. Cell Stem Cell 2, 135-138.

Gurdon, J.B. (2006). Nuclear transplantation in Xenopus. Methods Mol Biol 325, 1-9.

Gurdon, J.B., and Wilmut, I. (201 1 ). Nuclear transfer to eggs and oocytes. Cold Spring Harbor perspectives in biology 3.

Hussein, S.M., Batada, N.N., Vuoristo, S., Ching, R.W., Autio, R., Narva, E., Ng, S., Sourour, M., Hamalainen, R., Olsson, C, et al. (2011 ). Copy number variation and selection during reprogramming to pluripotency. Nature 471, 58-62.

Jaenisch, R., and Young, R. (2008). Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132, 567-582.

Kikyo, N., Wade, P.A., Guschin, D., Ge, H., and Wolffe, A.P. (2000). Active remodeling of somatic nuclei in egg cytoplasm by the nucleosomal ATPase ISWI. Science 289, 2360- 2362.

Kuo, A.J., Song, J., Cheung, P., Ishibe-Murakami, S., Yamazoe, S., Chen, J.K., Patel, D.J., and Gozani, O. (2012). The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome. Nature 484, 1 15-1 19.

Lemaitre, J.M., Danis, E., Pasero, P., Vassetzky, Y., and Mechali, M. (2005). Mitotic remodeling of the replicon and chromosome structure. Cell 123, 787-801.

Letessier, A., Millot, G.A., Koundrioukoff, S., Lachages, A.M., Vogt, N., Hansen, R.S., Malfoy, B., Brison, O., and Debatisse, M. (201 1 ). Cell-type-specific replication initiation programs set fragility of the FRA3B fragile site. Nature 470, 120-123.

Marion, R.M., Strati, K., Li, H., Murga, M., Blanco, R., Ortega, S., Femandez-Capetillo, O., Serrano, M., and Blasco, M.A. (2009). A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 460, 1 149-1 153.

Mayshar, Y., Ben-David, U., Lavon, N., Biancotti, J.C., Yakir, B., Clark, AT., Plath, K., Lowry, W.E., and Benvenisty, N. (2010). Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell stem cell 7, 521-531. Menear, K.A., Adcock, C, Boulter, R., Cockcroft, X.L., Copsey, L., Cranston, A., Dillon, K.J., Drzewiecki, J., Garman, S., Gomez, S., et al. (2008). 4-[3-(4- cyclopropanecarbonylpiperazine-1 -carbonyl)-4-fluorobenzyl]-2H-phthalazin- 1 -one: a novel bioavailable inhibitor of poly(ADP-ribose) polymerase-1 . J Med Chem 51, 6581- 6591.

Noguchi, K., Vassilev, A., Ghosh, S., Yates, J.L., and DePamphilis, M.L. (2006). The BAH domain facilitates the ability of human Orel protein to activate replication origins in vivo. EMBO J 25, 5372-5382.

Poirier, G.G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C, and Mandel, P. (1982). Poly(ADP-ribosyl)ation of polynucleosomes causes relaxation of chromatin structure. Proc Natl Acad Sci U S A 79, 3423-3427.

Rais, Y., Zviran, A., Geula, S., Gafni, O., Chomsky, E., Viukov, S., Mansour, A.A., Caspi, I., Krupalnik, V., Zerbib, M., er a/. (2013). Deterministic direct reprogramming of somatic cells to pluripotency. Nature 502, 65-70.

Sharma, S., Thomas, D., Marlett, J., Manchester, M., and Young, J.A. (2009). Efficient neutralization of antibody-resistant forms of anthrax toxin by a soluble receptor decoy inhibitor. Antimicrob Agents Chemother 53, 1210-1212.

Singer, R.A., and Johnston, G.C. (2004). The FACT chromatin modulator: genetic and structure/function relationships. Biochemistry and cell biology = Biochimie et biologie cellulaire 82, 419-427.

Tada, M., Tada, T., Lefebvre, L., Barton, S.C, and Surani, M.A. (1997). Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells. The EMBO journal 16, 6510-6520.

Takahashi, K., Okita, K., Nakagawa, M., and Yamanaka, S. (2007). Induction of pluripotent stem ceils from fibroblast cultures. Nature protocols 2, 3081-3089.

Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676.

Tan, B.C., Chien, C.T., Hirose, S., and Lee, S.C. (2006). Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication. EMBO J 25, 3975-3985.

Tardat, M., Brustel, J., Kirsh, O., Lefevbre, C, Callanan, M., Sardet, C, and Julien, E. (20 0). The histone H4 Lys 20 methyltransferase PR-Set7 regulates replication origins in mammalian cells. Nat Cell Biol 12, 1086-1093.

Tuduri, S., Crabbe, L., Conti, C, Tourriere, H., Holtgreve-Grez, H., Jauch, A., Pantesco, V., De Vos, J., Thomas, A., Theillet, C, et al. (2009). Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription. Nature cell biology 11, 1315-1324.

Yamanaka, S., and Blau, H.M. (2010). Nuclear reprogramming to a pluripotent state by three approaches. Nature 465, 704-712.

Claims

Claims:

1. A method for generating a nuclear reprogrammed cell from a non-pluripotent cell, the method comprising (i) introducing into a non-pluripotent cell nucleic acid encoding SSRP1 or an effective amount of SSRP1 , or (ii) expressing in a non-pluripotent cell an effective amount of SSRP1 or nucleic acid encoding SSRP1.

2. The method of claim 1 , wherein the nuclear reprogrammed cell is a pluripotent cell.

3. A method of producing a pluripotent cell, the method comprising (i) contacting a non-pluripotent cell with nucleic acid encoding SSRP1 or an effective amount of SSRP1 , or (ii) expressing in a non-pluripotent cell an effective amount of SSRP1 or a nucleic acid encoding SSRP1.

4. A method of establishing an induced pluripotent stem cell, the method comprising the step of increasing the amount of SSRP1 in a non-pluripotent cell in a nuclear reprogramming step of said non-pluripotent cell.

5. The method of claim 4, wherein the method comprises expressing in a non- pluripotent cell an effective amount of SSRP1 or a nucleic acid encoding SSRP1.

6. The method of any one of claims 1 to 5, wherein the non-pluripotent cell is a somatic cell.

7. The method of any one of the preceding claims, further comprising culturing the nuclear reprogrammed or pluripotent cell(s) obtained under conditions to produce a population of cells.

8. The method of any one of the preceding claims, further comprising isolating the nuclear reprogrammed or pluripotent cells from non-pluripotent cells.

9. The method of any one of the preceding claims further comprising differentiating the nuclear reprogrammed or pluripotent cells into a somatic cell of a specific type.

10. Use, preferably in vitro, of a nuclear reprogrammed or pluripotent cell obtained by the method of any one of the preceding claims in producing a somatic cell.

1 1 . An isolated cell or isolated population of cells, preferably in vitro, produced by the method of any one of claims 1 to 9.

12. A somatic cell generated by differentiation of a nuclear reprogrammed cell or pluripotent cell obtained by the method of any one of claims 1 to 10.

13. A somatic cell or non-pluripotent cell, preferably isolated, into which nucleic acid encoding SSRP1 or a homologue thereof has been introduced so as to be expressed in the cell.

14. A somatic cell or non-pluripotent cell, preferably isolated, induced to overexpress endogenous SSRP1 or endogenous nucleic acid encoding SSRP1.

15. A somatic cell or non-pluripotent cell, preferably isolated, into which exogenous SSRP1 has been introduced.

16. Use, preferably in vitro, of nucleic acid encoding SSRP1 or SSRP1 for nuclear reprogramming of non-pluripotent cell(s), producing pluripotent cell(s) from non- pluripotent cell(s), or improving induced pluripotent stem cell establishment.

17. An inducer from a non-pluripotent cell to a nuclear reprogrammed cell, the inducer comprising nucleic acid encoding SSRP1 or SSRP1.

18. Use, in vitro, of SSRP1 protein in a method of nuclear transfer.

19. A method of in vitro nuclear transfer, the method comprising providing an enucleated egg cell, and in the presence of exogenous SSRP1 protein transferring the nucleus of a somatic cell into the enucleated egg cell.

20. Use, preferably in vitro, of a nuclear reprogrammed or pluripotent cell obtained by the method of any one of claims 1 to 9 in a method of determining the effect of a candidate agent or stimulus on the nuclear reprogrammed or pluripotent cell.