WO2010042800A1 - Methods of reprogramming somatic cells and methods of use for such cells - Google Patents

Abstract

The present invention provides a method of reprogramming primate somatic cells by exposing a primate somatic cell with SALL4 under conditions sufficient to reprogram the cells; and culturing the exposed cells to obtain reprogrammed cells. Cells obtained by invention methods can be used as pharmaceutical compositions in clinical settings, for example, the treatment of hemophilia A in a subject.

Description

METHODS OF REPROGRAMMING SOMATIC CELLS AND METHODS OF USE FOR SUCH CELLS

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The present invention relates generally to the field of reprogramming somatic cells and more specifically to induced pluripotent stem cells (iPS) and more specifically to methods of reprogramming somatic cells and clinical uses for such cells generated by such methods.

Background Information

[0002] Stem cells possess properties that make them potentially invaluable in treatment of disease. The pluripotent nature of such cells makes them an attractive potential source of any of several types of differentiated cells that could act to replace lost function in patients. Techniques have been available for generating stem cells from embryos (embryonic stem, or ES, cells) for several years, as well as for the inducing ES cells to differentiate in a controlled fashion to any of a number of different cell types. However, ethical issues as well as challenges in overcoming immune rejection have severely hampered progress with the clinical use of ES cells to treat disease. Ideally, a suitable stem cell for use in treating disease would not be of embryonic origin, but rather would be obtainable from the patient, so as to reduce or eliminate the likelihood of immune rejection (as well as to reduce ethical concerns). Yamanaka and colleagues have reported techniques for generating autologous stem cells from adult cells such as fibroblasts (Takahashi, K., and Yamanaka,Cell /25:663-676, 2006). Reprogramrning of fibroblasts to cells resembling ES cells, termed induced pluripotent stem (iPS) cells, is one of the most exciting findings in recent scientific history, and has tremendous therapeutic potential. To date, autologous iPS cells have proven to be nearly identical to ES cells, but without the ethical limitations of ES cells and with the important further advantage that they potentially avoid the immune barrier, iPS cells are an attractive source of differentiated cells for cell therapies. [0003] Yamanaka and colleagues reported the production of both human and murine iPS stem cells by introducing four genes, namely, Oct3/4, Sox2, c-Myc, and Klf4, into human or murine fibroblasts (Takahashi, K., and Yamanaka, S. Nature 448:313-317, 2007; Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S.. Cell 737:861-872, 2007; Lewitzky M, Yamanaka S. Curr Opin Biotechnol. 2007;18:467-473). Other investigators have subsequently reported the conversion of somatic cells to iPS cells, in each case by the introduction of multiple genes (typically four) into the progenitor somatic cells ( Park IH, Zhao R, West JA, et al. Nature. 2008;451:141-146; Yu J, Vodyanik MA, Smuga-Otto K, et al. Science. 2007;318:1917-1920). Yamanaka et al. noted in their studies that progeny mice transplanted with iPS cells tended to develop tumors and they attributed this problem to the use of c-Myc, an oncogene, as one of the pluripotency- inducing genes.

[0004] Pluripotency-inducing genes are typically introduced into somatic cells by the use of recombinant viruses that can intregrate into the host cell genome. Such integration contributes to the risk of generation of abnormal, including malignant, phenotpyes. One approach to reducing such risk is to use non-integrating adenoviruses to introduce the genes necessary to generate a pluripotent phenotype. A different strategy would be to reduce the number and/or type of genes used to induce pluripotency: the fewer genes used to promote pluripotency the smaller the amount of exogenous genetic material inserted into the cell and thus the lower the likelihood that the introduced exogenous genetic material causes adverse effects by altering cellular behavior. Similarly, the fewer the genes inserted into the genome, the lower the risk of abnormality by virtue of genetic insertion into chromosomal DNA. For example, Nakagawa et al attempted to generate iPS cells from mouse fibroblasts using just Oct3/4, Sox2, and Klf4 genes, omitting from the c-myc oncogene. They reported success using this approach, although they noted that efficiency of iPS production was reduced (Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Okita, K., Mochiduki, Y., Takizawa, N.,and Yamanaka, S. Nature Biotechnol 2d:101-106, 2008). Omission of the c-myc oncogene could reduce potential abnormalities both by elimination of the need to introduce an oncogene as well as by introducing less exogenous genetic material into progenitor cells. Clearly there would be an advantage to an approach to iPS production that involved a minimal amount of genetic destabilization of the cells, preferably by a reduction in the number of exogenous genes required for induction of the pluripotent phenotype, and ideally by the use of only a single gene to promote pluripotency.

[0005] With procedures in place for safely, efficiently and reproducibly producing iPS cells and controlling their further programming into cells that could be medically useful it will become possible to treat patients with certain clinical deficiencies. For example, Hanna et al recently deomstrated that iPS cells could reverse the sickle cell anemia phenotype in a suitable mouse model (Hanna J, Wernig M, Markoulaki S, et al. Science. 2007;318:1920- 1923). There are a number of other diseases that promise to be addressable by appropriate cell therapy and one of these diseases in Type A hemophilia.

[0006] Hemophilia is a congenital coagulation disorder. Hemophilia A occurs in 1-2 in 10,000 male individuals and represents roughly 90% of hemophilia cases. Hemophilia B affects 1 in 30,000 individuals and hemophilia C is even less common. Hemophilias are sex linked disorders characterized by deficiencies of Factor VIII in Hemophilia A and Factor IX in Hemophilia B. Factor XI in Hemophilia C is a recessive disorder. While the genes encoding Factor VIII and Factor IX are located on the X chromosome, the gene encoding Factor XI is located on chromosome 4 and Factor XI deficiency is much less frequent than other hemophilias.

[0007] Hemophilia A is caused by mutations within the genomic sequence of the Factor VIII gene. There are over 50 mutations that are known to cause deficiencies of Factor VIII protein. The majority (-65%) of the mutations are point mutations and deletions that occur throughout the gene. The other 35% of mutations are due to inversions occurring between introns of Factor VIII. Mutations of Factor VIII lead to depleted protein production, and inefficient clotting. As seen in Figure 1, activated Factor VIII participates in the activation of Factor X. Thus, individuals with hemophilia do not bleed more intensely than individuals with wild-type Factor VIII, but they are unable to clot efficiently following tissue injury.

[0008] Clinically, hemophilia is characterized by spontaneous and prolonged hemorrhage that can result in disability and death. The most common complication is chronic arthritis caused by bleeding within the joints. Traumatic injuries to individuals with hemophilia will cause rapid blood loss and, if not treated rapidly, will lead to death. Current therapies include fixed-dose prophylaxis, protein replacement therapies, and most recently, gene therapies. Current prophylaxis and protein replacements therapies are limited by incomplete efficacy, high cost, restricted availability and the possible development of neutralizing antibodies against Factor VIII in chronically treated individuals. However, much of the clinical presentation in hemophilia patients can be relieved by expression of only 3% of wild-type clotting protein, and in most cases an individual with 30% of wild-type clotting factor would be phenotypically normal.

[0009] There are several suitable methods for the expression of exogenous Factor VIII protein. Protein replacement therapies are suitable for short term treatments, such as surgeries and traumatic injuries. However, if individuals are continually treated with recombinant Factor VIII protein, they risk the development of neutralizing antibodies against Factor VIII. Thus, the allogenic nature of the treatment results in immune rejection of the foreign protein. Recently, gene therapy approaches have been implemented to generate an endogenous Factor VIII protein by using viral methods to introduce the cDNA of the Factor VIII gene. Although these approaches provided encouraging results in animal models, studies in larger animal models and phase I clinical trials have failed. Most often, the failure of these studies is due to immune rejection of capsid proteins necessary for the virus used to introduce Factor VIII cDNA.

[0010] Cell therapy has long been considered an option for hereditary diseases such as hemophilia. Recently, direct introduction of allogenic liver progenitor cells into the liver has resulted in successful treatment in a hemophilia B mouse model. The liver progenitor cells functionally integrated into the recipient liver expressed Factor IX and phenotypically corrected the mouse model. However, for this approach to be applicable in other models and in humans, we must successfully overcome the immune barrier. One option is to generate ES cell banks of cell lines carrying various HLA haplotypes. However, the extremely broad variety of immune types within the heterogeneous US population is a disincentive for this approach, as are ethical issues surrounding sourcing and use of ES cells. Both of these limitations promise to be overcome by therapy with cells derived from iPS cells rather than ES cells. [0011] One potential utility of iPS cells in a cellular therapy is bone marrow transplantation. Often in disease such as leukemia, bone marrow transplantation is indicated. However, lack of a suitable donor is often problematic.

[0012] Lung tissue is a very unique and specialized tissue within the body. It can become irreversibly damaged in a variety of ways, including by scarring or fibrosis. Unfortunately, the low availability of donor lungs prevents widespread use of lung transplantation. Thus, methods to generate lung tissue in vitro are highly desirable. The alveolar epithelium is composed of type I and type II pneumocytes, comprising 95% and 5% of the surface area of the alveolar lining, respectively. It has previously been demonstrated that ES cells can be differentiated into functional epithelial alveolar cells. Differentiated ES cells could efficiently produce both Type I and Type II pneumocytes. These pneumocytes effectively engrafted within various mouse models of lung injury suggesting functional integration of these differentiated ES cells. Further, since Type II pneumocytes are responsible for lung fluid clearance postnatally and are able to trans-differentiate into Type I pneumocytes in times of tissue injury, it suggests that Type II pneumocytes are the epithelial stem cell of the lung endothelium.

[0013] Induced pluripotent stem (iPS) cells have recently been derived by viral transduction of multiple factors (e.g., Oct4, Sox2, Klf4 and c-myc) into a variety of somatic cell types. iPS cells are indistinguishable from ES cells thus far and may present a therapeutic advantage in the use of future cellular therapies. It has already been demonstrated that mouse tail-tip fibroblasts can be established in culture, expanded, and infected with a cocktail of viruses that express multiple transcription factors necessary to retrodifferentiate the fibroblasts to an ES cell-like pluripotent state. The resultant iPS cells can then be differentiated, transplanted back to the original mouse, and functionally engrafted, providing therapeutic benefit.

[0014] More recent studies have demonstrated that the number of factors required to retrodifferentiate somatic cells to iPS cells can be reduced to one or two. Huangfu et al (ref) reported that the addition of a histone deacetylase inhibitor (valproic acid) to cultured human fibroblasts enabled the production of iPS cells using retrovirus vectors containing only Oct 4 and Sox2. [0015] The SALL gene family, SALLl, SALL2, SALL3, and SALL4, were originally cloned on the basis of their DNA sequence homology to Drosophila spalt (sal). In Drosophila, spalt is a homeotic gene essential for development of posterior head and anterior tail segments. It plays an important role in tracheal development, terminal differentiation of photoreceptors, and wing vein placement. In humans, the SALL gene family is associated with normal development, as well as tumorigenesis. SALL proteins belong to a group of C₂H₂ zinc finger transcription factors characterized by multiple finger domains distributed over the entire protein.

SUMMARY OF THE INVENTION

[0016] The present invention is based on the finding that somatic cells receiving an exogenous SALL4 gene alone can convert to iPS cells. iPS cells produced by receiving an exogenous S ALL4 gene or a functional fragment thereof, or more classically by receiving a mixture of three or four genes are able to differentiate into other cell types, such as endothelial cells or hepatic cells and thereby correct a genetic disorder, for example type A hemophilia. Such iPS cells can be induced to generate hematopoietic stem cells and populate bone marrow.

[0017] In one embodiment an iPS cell is disclosed, wherein the iPS cell contains an exogenously added SALL4 gene sequence and wherein the iPS cell self renews in culture.

[0018] Li one aspect the iPS cell contains, in addition to an exogenous SALL4 gene sequence, sequences of one or more of the following exogenous genes: Oct4, Sox2, KLF4.

[0019] In another aspect, the iPS cell is derived from a fibroblast cell.

[0020] In another aspect, the iPS cell is derived from a variety of cell types including hepatocytes, lung alveolar cells, bone marrow or blood cells, gastrointestinal epithelial cells, skin cells, nervous system cells and renal cells.

[0021] In another embodiment, an iPS cell of the invention is induced in culture by exposure to an appropriate combination of growth and induction factors to form a progenitors of a differentiated cell. [0022] In one aspect, the progenitor cell is introduced into an individual and populates that individual with functionally differentiated cells.

[0023] In another embodiment, an iPS cell of the invention produces Factor VIII.

[0024] In another aspect, the iPS cell contains an exogenously added SALL4 gene.

[0025] In another aspect, the iPS cell contains, in addition to an exogenous SALL4 gene, one or more of the following exogenous genes: Oct4, Sox2, KLF4.

[0026] In one aspect, a Factor Vlll-producing cell derived from iPS cells is introduced into a patient diagnosed with Hemophilia A.

[0027] In one aspect, cells derived from iPS cells are introduced into a patient with genetic disorders.

[0028] In one aspect, cells derived from iPS cells are introduced into a patient with tissue injuries and/or organ dysfunction.

[0029] In one aspect, SALL4 gene with or without one or more of following exogenous genes, Oct4, Sox2, KLF4 is used in situ for somatic cell reprogramming allowing treatment of tissue injuries.

[0030] In one aspect, small molecules useful for somatic cell reprogramming by stimulating SALL4 expression or enhancing SALL4 function are utilized in methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is an illustration of the coagulation cascade leading to a blood clot following injury. Boxed factors are known to cause hemophilias in humans. F-factor, a- activated.

[0032] Figure 2 shows fibroblast cells 7 days post-transduction of SALL4 retrovirus.

[0033] Figure 3 is a phase contrast comparison of newly induced ES-like cells cultured in the presence or absence of feeder layer and LIF (leukemia inhibitory factor). [0034] Figure 4 shows the growth and morphological status of induced ES-cells cultured without feeder layer and LIF.

[0035] Figure 5 shows embryoid bodies generated from newly-induced ES-like cells.

[0036] Figure 6 is a demonstration of reprogramming of iPS cells derived from mouse fibroblasts infection with a single transcriptional factor, mouse SALL4. SSEA-I, NANOG, and OCT4 are all well characterized embryonic stem cell makers.

[0037] Figure 7 shows enhancement of reprogramming via addition of SALL4.

[0038] Figure 8 shows the scoring of the number of ES-like colonies. O=Oct4; S=Sox2; N=Nanog. Human fibroblasts were infected with retroviruses each containing O, S and N or O and S or O and N with or without SALL4-containing retrovirus.

[0039] Figure 9 shows scoring of the number of ES-like colonies 7 days post-infection with retroviruses containing Oct4 (O), Sox2 (S) and Nanog (N) with and without SALL4- containing retrovirus. Ix, 2x, 3x and 4x represent fold-increases in titer of human retrovirus containing the SALL4 gene. The virus title was approximately 10⁹ cfu/ml. We used 1 ml per one well in a 6-well plate for infection at the Ix titer.

[0040] Figure 10 shows ES-like colonies induced by infection of human retrovirus expressing SALL4 in human skin fibroblasts. Arrow indicates an ES-like colony.

[0041] Figure 11 shows iPS cells derived from retroviral transduction of OCT4, SOX2, and KLF4.

[0042] Figure 12 demonstrates reprogramming of iPS cells derived by induction with three factors, Oct4, Sox2 and KLF4. SSEA-I, NANOG, and OCT4 are all well characterized stem cell makers, while SALL4 is a novel marker with emerging roles in stem cell pluripotency.

[0043] Figure 13 shows hematoxylin and Eosin staining of teratomas formed by the injection of iPS cells into the flanks of NOD-SCID mice. All three embryonic germ layers were clearly visible within the tumor. [0044] Figure 14 shows low magnification of EBs derived from iPS cells. EBs can give rise to embryonic (i.e., endoderm, ectoderm, and mesoderm) but not extra-embryonic (i.e., trophectroderm) tissues.

[0045] Figure 15 shows immunofluorescence staining of iPS cells differentiated into endothelial progenitor cells. The expression of endothelial progenitor markers peaked at day 7 and decreased as cells differentiated further into mature endothelial cells.

[0046] Figure 16 shows immunofluorescence staining of endothelial cells to demonstrate co-expression of Factor VIII and endothelial progenitor markers.

[0047] Figure 17 shows immunofluorescence staining of iPS cells differentiated into liver precursor cells.

[0048] Figure 18 shows demonstration of phenotype correction of hemophilia A bleeding disorder post tail-clip challenge. Uninjected Mice A continued to bleed while the injected mice stopped bleeding.

[0049] Figure 19 shows mRNA expression of FVIII in hemophilia A mice injected with endothelial progenitor cells derived from iPS cells 9 days post-transplantation. The graph shows relative quantitative comparison of FVIII expression in various tissues obtained from hemophilia A mice with and without treatment. 10⁶ endothelial cells derived from differentiation of iPS cells were delivered via injection into the liver. After treatment, the mice were subjected to tail-clip challenge and monitored every 4 hours. Tissues were extracted from treated and untreated mice.

[0050] Figure 20 shows the generation of iPS cells for the production of hematopoietic progenitor cells.

[0051] Figure 21 shows the fate of iPS-derived hematopoietic progenitor cells following injection into immunodeficient mice.

DETAILED DESCRIPTION OF THE INVENTION

[0052] Investigators have typically introduced into somatic cells, a combination of transcription factors, including but not limited to Oct4, Sox2 and KLF4, and optionally c- Myc, to generate iPS cells. While such methods are well established it presents several problems in potential clinical uses. One such problem is that introducing a number of different exogenous genes into the genome of a stable organism might be detrimental. Reactivation of exogenous genes could lead to malignant transformation (especially when the transcription factor c-Myc, which is an oncogene, is used) of iPS cells. Additionally, the likelihood of insertional mutagenesis increases when it is necessary to introduce several genes and such mutagenesis has the potential for triggering cellular dysfunction.

[0053] Recent research in many laboratories has been aimed at reducing the number of transcription factors necessary to generate iPS cells. Small molecule approaches have proven to be promising; however, they still require the introduction of multiple transcription factors. Other potential methods for techniques to generate iPS cells while reducing the risk of adverse side effects include introduction into somatic cells of the protein products of the pluripotency-inducing genes and reduction in the number of exogenous pluripotency- inducing genes used and introducing pluripotency-inducing genes without the use of retroviruses, which could carry additional risks per se.

[0054] The present invention provides methods that show reprogramming of somatic cells to iPS cells using fewer exogenous transcription factors than the number used by others. S ALL4 is a stem cell transcription factor with important regulatory roles in early development. SALL4 has been shown to play a key role in the maintenance of pluripotency in ES cells. Previously the inventors have also shown that SALL4 is able to bind and transcriptionally regulate the transcriptional factors Oct4, Nanog, Sox2 and Klf4, all of which have been used to induce the reprogramming of somatic cells.

[0055] Once a ready supply of stem cells is available, such cells can be treated in ways known in the art that promote further programming such that the cells are able to differentiate to various cell types characteristic of the three germ cell layers and/or express genes characteristic of differentiated cells. For example, stem cells obtained from hemangiomas when treated with vascular endothelial growth factor (VEGF) differentiate into endothelial- like tissue (Ma, Y., Fink, L.M., Ward, D.C. and Waner, M, US Patent 11/809871, 2007). Similar strategies may be implemented to induce stem cells to express differentiated gene products that might be useful for treating diseases, including, as a non-limiting example, type A hemophilia.

[0056] As used herein, "iPS cells" refer to cells that are substantially genetically identical to their respective differentiated somatic cell of origin and display characteristics similar to higher potency cells, such as ES cells, as described herein. The cells can be obtained from various differentiated (i.e., non-pluripotent and multipotent) somatic cells.

[0057] iPS cells exhibit morphological (i.e., round shape, large nucleoli and scant cytoplasm) and growth properties (i.e., doubling time; ES cells have a doubling time of about seventeen to eighteen hours) akin to ES cells. In addition, iPS cells express pluripotent cell- specific markers (e.g., Oct-4, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, and SSEA-I). iPS cells, however, are not immediately derived from embryos and can transiently or stably express one or more copies of selected potency-determining factors at least until they become pluripotent. As used herein, "not immediately derived from embryos" means that the starting cell type for producing iPS cells is a non-pluripotent cell, such as a multipotent cell or terminally differentiated cell, such as somatic cells obtained from a post-natal individual.

[0058] hi the methods described herein, at least one factor can be introduced into, and expressed in, differentiated somatic cells, whereupon the somatic cells convert in culture to cells having properties characteristic of pluripotent cells, such as human ES cells (i.e., express a combination of one or more of the following markers: OCT4, Nanog, SALL4, SSEA-I₃ SSEA-3, SSEA-4, TRA-1-60 or TRA-1-81 and appear as compact colonies having a high nucleus to cytoplasm ratio and prominent nucleolus), that can differentiate into cells characteristic of all three germ layers, and that contain the genetic complement of the somatic cells of a post-natal individual. Apart from genetic material introduced to encode the potency- determining factors, the reprogrammed (i.e., converted) cells are substantially genetically identical to the somatic cells from which they were derived. In a preferred aspect of the invention, SALL4 is sufficient for converting cells to iPS cells. It should be understood, that various isoforms of SALL4 are included in the invention. These include but are not limited to SALLl, SALL2, SALL3, and SALL4 as well as SALL4 mRNA spliced forms, SALL4A and SALL4B. [0059] Genetic material encoding SALL4 or other factors can be introduced by transfection or transduction into the somatic cells using a vector, such as an integrating- or non-integrating vector. Of particular interest in the present invention are retroviral vectors. Retroviral vectors, particularly lentiviral vectors, are transduced by packaging the vectors into virions prior to contact with a cell. After introduction, the DNA segment(s) encoding the potency-determining factor(s) can be located extra-chromosomally (e.g., on an episomal plasmid) or stably integrated into cellular chromosome(s). Approaches for achieving homologous recombination are among preferred embodiments.

[0060] Cell types pass through various levels of potency during differentiation, such as totipotency, pluripotency and multipotency. Of particular interest herein are pluripotent cells. As used herein, "pluripotent cells" refer to a population of cells that can differentiate into all three germ layers (e.g., endoderm, mesoderm and ectoderm). Pluripotent cells express a variety of pluripotent cell-specific markers, have a cell morphology characteristic of undifferentiated cells (i.e., compact colony, high nucleus to cytoplasm ratio and prominent nucleolus) and form teratomas when introduced into an immunocompromised animal, such as a SCID mouse. The teratomas typically contain cells or tissues characteristic of all three germ layers. One of ordinary skill in the art can assess these characteristics by using techniques commonly used in the art. See, e.g., Thomson et al., supra. Pluripotent cells are capable of both proliferation in cell culture and differentiation towards a variety of lineage-restricted cell populations that exhibit multipotent properties. Multipotent somatic cells are more differentiated relative to pluripotent cells, but are not terminally differentiated. Pluripotent cells therefore have a higher potency than multipotent cells. As used herein, "reprogrammed pluripotent primate stem cells" (and similar references) refer to the pluripotent products of somatic cell reprogramming methods. Such cells are suitable for use in research and therapeutic applications currently envisioned for human ES cells.

[0061] The present invention broadly relates to novel methods for reprogramming differentiated somatic cells into higher-potency cells, such as pluripotent cells, by administering at least two potency-determining factors into somatic cells to achieve a higher level of potency in the reprogrammed cells than in the somatic cells. Advantageously, the present invention allows the generation of pluripotent cells, such as iPS cells, from somatic cells without requiring an addition of cell surface receptors for introducing the potency- determining factors to the somatic cells. As used herein, "reprogramming" refers to a genetic process whereby differentiated somatic cells are converted into de-differentiated, pluripotent cells, and thus have a greater pluripotency potential than the cells from which they were derived. That is, the reprogrammed cells express at least one of the following pluripotent cell- specific markers: SSEA-3, SSEA-4, TRA-1-60 or TRA 1-81. Preferably, the reprogrammed cells express all these markers.

[0062] Suitable somatic cells can be any somatic cell, although higher reprogramming frequencies are observed when the starting somatic cells have a doubling time about twenty- four hours. Somatic cells useful in the invention are non-embryonic cells obtained from a fetal, newborn, juvenile or adult primate, including a human. Examples of somatic cells that can be used with the methods described herein include, but are not limited to, bone marrow cells, epithelial cells, fibroblast cells, hematopoietic cells, hepatic cells, intestinal cells, mesenchymal cells, myeloid precursor cells and spleen cells. Another type of somatic cell is a CD29.sup.+ CD44.sup.+ CD166.sup.+ CD105.sup.+ CD73.sup.+ and CD31.sup.- mesenchymal cell that attaches to a substrate. Alternatively, the somatic cells can be cells that can themselves proliferate and differentiate into other types of cells, including blood stem cells, muscle/bone stem cells, brain stem cells and liver stem cells. Multipotent hematopoietic cells, suitably myeloid precursor or mesenchymal cells, are specifically contemplated as suited for use in the methods of the invention.

[0063] A viral-based gene transfer and expression vector is a genetic construct that enables efficient and robust delivery of genetic material to most cell types, including non- dividing and hard-to-transfect cells (primary, blood, stem cells) in vitro or in vivo. Viral- based constructs integrated into genomic DNA result in high expression levels. In addition to a DNA segment that encodes a potency-determining factor of interest, the vectors include a transcription promoter and a polyadenylation signal operatively linked, upstream and downstream, respectively, to the DNA segment. The vector can include a single DNA segment encoding a single potency-determining factor or a plurality of potency-determining factor-encoding DNA segments. A plurality of vectors can be introduced into a single somatic cell. The vector can optionally encode a selectable marker to identify cells that have taken up and express the vector. As an example, when the vector confers antibiotic resistance on the cells, antibiotic can be added to the culture medium to identify successful introduction of the vector into the cells. Alternatively, the vector can optionally encode a marker protein such as green fluorescence protein (GFP). Integrating vectors can be employed, as in the examples, to demonstrate proof of concept. Retroviral (e.g., lentiviral) vectors are integrating vectors; however, non-integrating vectors can also be used. Such vectors can be lost from cells by dilution after reprogramming, as desired. A suitable non-integrating vector is an Epstein-Barr virus (EBV) vector. Ren C, et al., Acta. Biochim. Biophys. Sin. 37:68-73 (2005); and Ren C, et al., Stem Cells 24:1338-1347 (2006), each of which is incorporated herein by reference as if set forth in its entirety. The vectors described herein can be constructed and engineered using art-recognized techniques to increase their safety for use in therapy and to include suitable expression elements and therapeutic genes. Standard techniques for the construction of expression vectors suitable for use in the present invention are well-known to one of ordinary skill in the art and can be found in such publications such as Sambrook J, et al., "Molecular cloning: a laboratory manual," (3rd ed. Cold Spring Harbor Press, Cold Spring Harbor, N. Y. 2001), incorporated herein by reference as if set forth in its entirety.

[0064] In yet another aspect, the invention features a method for repairing or regenerating a tissue or differentiated cell lineage in a subject, the method involves obtaining the reprogrammed cell of a previous aspect and administering the cell to a subject (e.g., a subject having a myocardial infarction, congestive heart failure, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, cirrhosis, Parkinson's disease, Alzheimer's disease, diabetes, cancer, arthritis, wound healing, immunodeficiency, aplastic anemia, anemia, and genetic disorders) and similar diseases, where an increase or replacement of a particular cell type/ tissue or cellular de-differentiation is desirable. In one embodiment, the subject has damage to the tissue or organ, and the administering provides a dose of cells sufficient to increase a biological function of the tissue or organ or to increase the number of cell present in the tissue or organ. In another embodiment, the subject has a disease, disorder, or condition, and wherein the administering provides a dose of cells sufficient to ameliorate or stabilize the disease, disorder, or condition. In yet another embodiment, the subject has a deficiency of a particular cell type, such as a circulating blood cell type and wherein the administering restores such circulating blood cells. [0065] In yet another embodiment, the method increases the number of cells of the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ. In yet another embodiment, the method increases the biological activity of the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ. In yet another embodiment, the method increases blood vessel formation in the tissue or organ by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated control tissue or organ. In yet another embodiment, the cell is administered directly to a subject at a site where an increase in cell number is desired. In one embodiment, the site is a site of tissue damage or disease. In yet another embodiment, the site shows an increase in cell death relative to a corresponding control site.

[0066] In yet another embodiment, the method increases the number of cells of hematopoietic bone marrow cells in an individual by at least about 5%, 10%, 25%, 50%, 75% or more compared to the corresponding number of hematopoietic bone marrow cells in an untreated individual. In yet another embodiment, the method increases the number of circulating hematopoietic cells in an individual by at least about 5%, 10%, 25%, 50%, 75% or more compared to the corresponding number of circulating hematopoietic cells in an untreated individual. In yet another embodiment, the method increases the biological activity of hematopoietic cells in an individual by at least about 5%, 10%, 25%, 50%, 75% or more compared to a corresponding untreated individual.

[0067] The following examples are intended to illustrate but not to limit the invention in any manner, shape, or form, either explicitly or implicitly. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLE 1 REPROGRAMMING SOMATIC CELLS TO iPS CELLS

[0068] To determine if SALL4 could reprogram mouse somatic cells to iPS cells, a high titer retrovirus expressing the mouse SALL4 coding sequence was generated and used to infect fibroblast cells. After a period of approximately 3-7 days following retroviral infection, ES-like colonies emerged with a morphology consistent with iPS cells (Figure T). Cells were plated using ES medium containing Knockout DMEM (KDMEM, Invitrogen), 15% ES qualified FBS (PAA), 2 mM L-GIn (Invitrogen), IxIO^"4 M nonessential amino acids (Invitrogen), 1 ^χlθ^~^ M 2-mercaptoethanol (Sigma), IX pen strep (Invitrogen) and lOOOIU/ml LIF (Chemicon). The medium was changed every other day.

[0069] As one way to determine if these cells had properties expected of iPS cells, we withdrew LIF (leukemia inhibitory factor) from the culture media. LIF is an essential factor in the maintenance of pluripotency and self-renewal capabilities of stem cells in vitro. After only a few days, the ES-like colonies started to differentiate and exhibited morphology similar to fibroblasts in the absence of LIF. The cells appeared to have lost their ES-like morphology. As an additional test, cells exposed to exogenous SALL4 gene were plated in culture dishes in the absence of feeder cells (Figure 3).

[0070] Mitomycin Treated EmbryoMax Primary Mouse Embryo Fibroblasts Strain CFl (Chemicon) were used as feeder cells. To culture the feeder cells, plates or flasks were coated with ES Qualified 0.1% Gelatin (3 ml/25 cm²) for at least 30 minutes and inactivated feeder cells were plated and incubated in these containers at 37°C with 5% CO2. Plates or flasks containing such feeder layers may be used for 12-14 day after plating; feeders are typically passaged no more than passage 4-5 times. iPS or ES cells are plated on semi-confluent feeders.

[0071] Feeder cells have been well established in many laboratories to provide stability and support for stem cell growth in vitro. After about 13 days of culture without feeder cells, the cells died. The poor health and eventual death of the derived ES-like cells cultured in the absence of the feeder layer and LIF from the growth media (Figure 4) strongly suggested that SALL4-induced cells indeed had stem cell-like characteristics.

[0072] In order to further determine the nature of these ES-like colonies, we differentiated the cells into embryσid bodies (EBs). It is generally accepted that only cells possessing self- renewal capabilities can generate EBs. We were able to successfully derive EBs from SALL4-induced iPS cells, further suggesting that we have derived iPS cells from infection of a single transcription factor, SALL4 (Figure 5). [0073] In addition, the derived iPS cells exhibited ES-like features expressing the well- defined ES cell markers SSEA-I, Nanog and Oct-4 (Figure 6).

[0074] Further experiments showed that SALL4 could enhance somatic cell reprogramming by Oct4, Sox2 and KLF4 (Figure 7). The observed reprogramming efficiency in the presence of SALL4 was approximately 10-fold greater than that in the presence of Oct4, Sox2 and KLF4 but in the absence of SALL4.

[0075] To determine if SALL4 could be used for reprogramming of human somatic cells to iPS cells, a high titer retrovirus expressing the human SALL4 coding sequence was prepared and used to infect human fibroblast cells. Various combinations of the genes Oct4, Sox2 and KLF4 with or without the SALL4 gene were tested to determine the minimal number of factors that could induce reprogramming of fibroblast cells into an ES-like state. ES-like colonies were scored after 7 days post infection with SALL4-containing retrovirus. Our data indicated that SALL4 enhanced somatic cell reprogramming 4-fold when in combination with Oct4, Sox2 and KLF4 vs the results with Oct4, Sox2 and KLF4 alone (Figure 8). We noted further that by increasing the titer of SALL4-containing virus added to the pluripotency-inducing gene combination the number of colonies of ES-like cells markedly increased. For example, at the highest SALL4-containing retrovirus titer (4-fold increase vs the study described in Figure 8) we observed a 54-fold increase in the number of colonies formed (Figure 9).

[0076] Finally, human fibroblasts were infected with only SALL4-containing retrovirus. After 3-7 days post infection, the data showed that SALL4 alone could reprogram human somatic cells (Figure 10).

[0077] Therefore, the data show that the mouse S ALL4 gene sequence is capable of reprogramming mouse fibroblasts and human SALL4 gene sequence is able to reprogram human fibroblast cells. EXAMPLE 2 GENERATION OF INDUCED PLURIPOTENT STEM (iPSΪ CELLS

[0078] Because of the desirability of avoiding the use of an oncogene in the process of reprogramming somatic cells to iPS cells, iPS cells were generated without use of the c-myc gene. Although the efficiency of faithful reprogramming was much lower than the levels observed in the presence of c-myc, it was possible to derive iPS cells following retroviral infection with only 3 factors, namely OCT4, SOX2, and KLF4 (Figure 11). Further characterization of the resulting cells revealed that they expressed typical stem cell markers such as SSEA-I, NANOG, and OCT4 as well as novel markers such as SALL4 (Figure 12).

[0079] Our observations suggested that reprogrammed cells induced by OCT4, SOX2, and KLF4 were indeed true iPS cells. To ensure that iPS cells induced by OCT4, SOX2, and KLF4 could give rise to all germ layers of the developing embryo, we injected the cells into the flanks of NOD-SCID mice. Genuine stem cells under these conditions give rise to a teratoma composed of endodermal, ectodermal, and mesodermal cell lineages. Indeed, the injected cells gave rise to teratomas composed of all three germ layers within two weeks following injection (Figure 13). These data indicate that the cells we derived from fibroblasts are indeed iPS cells because they possess stem cell-like properties. By virtue of the omission of c-myc oncogene as an inducing gene, iPS cells induced by OCT4, SOX2, and KLF4 might have fewer abnormalities and thus be more effective as a source of cells for cell therapy.

[0080] Derivation of embryoid bodies from iPS cells

[0081] It has previously been demonstrated that direct injection of stem cells into mice is inappropriate for therapeutic use since, as shown above, injected stem cells form tumors of all three germ layers and thus could lead to uncontrolled differentiation into many different cell type unrelated to, and in some instances adverse to, the clinical need. Thus, it is advantageous to differentiate cells prior to therapeutic use. EBs are aggregates of ES cells or iPS cells that have passed through the first stage of embryonic development. That is, EBs have already made a lineage commitment to embryonic tissues and cannot generate extraembryonic trophectoderm. These experiments show that it is possible using the invention methods to generate EBs from fibroblast-derived iPS cells (Figure 14). Although EBs have made an early lineage commitment, this does not affect their therapeutic potential because they can still generate all cell lineages of the developing embryo and particularly for the purposes of this example, endodermal lineages.

[0082] Derivation of endothelial cells from iPS cells

[0083] Coagulation Factor VIII is secreted from liver sinusoidal cells and endothelial cells. Endothelial cells are thought to secrete the majority of Factor VIII protein. Thus, this example shows differentiation of EBs into endothelial cells using a previously established protocol of exposing the cells to culture media containing recombinant vascular VEGF (Nishikawa S. Embryonic stem cells as a source of hematopoietic and vascular endothelial cells in vitro. J Allergy Clin Immunol. 1997;100:S102-104; Vittet D, Prandini MH, Berthier R, et al. Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps. Blood. 1996;88:3424-3431; Festag M, Sehner C, Steinberg P, Viertel B. An in vitro embryotoxicity assay based on the disturbance of the differentiation of murine embryonic stem cells into endothelial cells. I: Establishment of the differentiation protocol. Toxicol In Vitro. 2007;21:1619-1630; Festag M, Viertel B, Steinberg P₅ Sehner C. An in vitro embryotoxicity assay based on the disturbance of the differentiation of murine embryonic stem cells into endothelial cells. II. Testing of compounds. Toxicol In Vitro. 2007;21:1631-1640). Following differentiation, the cells expressed FIk-I and CD31 (PECAM-I), markers for endothelial progenitor cells (Figure 15). By the use of an immunofluorescence assay with FVIII antibody, we demonstrated that the FIk-I and CD31 (PECAM-I) cells were producing FVIII (Figure 16).

[0084] Derivation of hepatic cells from iPS cells

[0085] Liver precursor and mature cells were also derived from iPS cells expressing Factor VIII and liver-specific markers including alpha fetoprotein, albumin, CYPlAl and HNF-4a (Figure 17).

[0086] Phenotypic correction of a preclinical mouse model of hemophilia A using iPS cells derived from fibroblasts

[0087] Following successful differentiation of iPS cells into endothelial progenitor cells and liver cells, we first focused on endothelial progenitor cells. 10⁶ cells were injected directly into the liver of a sublethally-irradiated male mouse model carrying mutations to the FVIII gene, the gene responsible for the coagulation disorder in hemophilia A. C57BL6 normal mice and uninjected hemophilia male mice were used as control groups. One week after transplantation of endothelial progenitor cells into the liver, all groups of mice were subjected to tail-clip challenge. The group of hemophilia mice not injected with the endothelial progenitor cells (negative control) died within 2-4 hours after tail clipping. Hemophilia A mice (n=4) treated with the iPS-derived endothelial cells surpassed the 2-4 hours challenge and survived as did the C57BL6 mice (N=3) (Figure 18).

[0088] As further evidence that the injected cells had protected the mice by secreting FVIII, levels of FVIII expression from blood plasma in injected mice were measured. We detected levels of FVIII in these mice that were comparable to non-injected controls. Since the endothelial cells were stained with FVIII via immunofluorescence, we assume that the increase of FVIII levels in treated hemophilia mouse tissues is due to the endothelial cells that were transplanted into the mice. mRNA expression profiling of FVIII shows 3-fold increases of FVIII levels in liver of treated vs untreated mice, approximately 7-fold increases in heart and kidney, and about 8-fold increases in spleen (Figure 19).

[0089] The data with the preclinical mouse model for type A hemophilia demonstrate that transplantation with endothelial progenitors derived in vitro from iPS cells can correct a potentially lethal phenotype in an animal model. This phenotypic correction provides proof of concept of the clinical application of converting somatic cells into iPS cells and subsequently programming those cells to correct genetic disorders, such as, in the example shown above, the generation of endothelial cells for treatment of hemophilia.

EXAMPLE 3 PRODUCTION QF HEMATOPOIETIC CELLS

[0090] Fibroblasts derived from the tail-tip of C57BL/6 mice (Figure 20A) were induced to pluripotency by introduction of the transcription factors OCT4, Nanog and Klf4, as described for Example 2. ES-like morphology was observed after 7 days post induction. The cells were positive for stem cell markers such as OCT4, Nanog, SALL4 and SSEA-I as determined by the use of immunoflurescence staining (not shown). The induced cells were able to form EBs in suspension. [0091] EBs were digested into single cells and cultured as described in Example 1 except that the medium was supplemented with a mixture of cytokines and induction factors (FLT3 ligand, SCF, TPO thrombopoieitin, interferon-gamma, and VEGF) for 7 days. H&E staining of the cells showed hematopoietic-like cells including erythroid cell precursors and neutrophils. Flow cytometry analysis of the hematopoietic-like cells were positive for hematopoietic cell surface markers B220, Terl 19, Gr-I and CD34.

[0092] Resulting hematopoietic-like progenitor cells were transplanted into immunodeficient mice. The cells were tagged with green fluorescent protin (GFP) to monitor their survival and localization in vivo. One month post-transplantation, bone marrow of the mice were analysed. We observed cells positive for both GFP as well as B220, Terl 19 and GR-I, indicating that the transplanted progenitor hematopoietic stem cells were able to survive in, and repopulate, bone marrow niche.

[0093] Materials and Methods

[0094] W4 ES and iPS cells were cultured on gelatin coated Mitomycin-C treated semi- confluent CF-I Passage 3 feeder layer (Chemicon) using ES growth medium containing Knockout DMEM (Invitrogen), 15% ES-qualified FBS₅ 2 mM L-Glutamine (Invitrogen), IxIO^-4 M nonessential amino acids (Invitrogen), IxIO^-4 M 2-mercaptoethanol (Sigma), IX pen strep (Invitrogen) and 1000IU/ml LIF (Chemicon). Growth medium was changed everyday. Tail-tip fibroblasts derived from C57BL/6 mice were cultured using growth medium containing High Glucose DMEM (Invitrogen), 10% certified FBS (Invitrogen) and IX pen strep (Invitrogen). Growth medium was changed every other day. Plat-E retroviral packaging cells (Clontech) were cultured using growth medium containing RPMI 1640 (Invitrogen), 10% certified FBS (Invitrogen) and IX pen strep (Invitrogen). Growth medium was changed every other day. OP9 stromal cells (ATCC) were cultured using growth medium containing alpha-MEM (Invitrogen), 20% certified FBS and IX pen strep. Growth medium was changed every other day. Phoenix- Ampho (Stanford University) retroviral packaging cell line was cultured using growth medium containing RPMI 1640 (Invitrogen), 10% certified FBS (Invitrogen) and IX pen strep. 293FT packaging cells were cultured using High Glucose DMEM (Invitrogen), 10% certified FBS (Invitrogen) and IX pen strep (Invitrogen). Growth medium was changed every other day. [0095] Viral Transfection and Infection

[0096] T75 flasks seeded with 90% confluent Plat-E retroviral packaging cell line (<Passage 5) were transfected with retroviral constructs pMXs-Sall4 (Addgene) using Lipofectamine 2000 transfection reagent (Invitrogen) according to manufacturer's instruction. Briefly, 75ul of Lipofectamine 2000 was incubated with 1.875ml of OptiMEM serum-free media (Invitrogen) at room temperature for 5 minutes. After incubation, 30ug of purified DNA in 1.875ml of OptiMEM was added to the lipidrOptiMEM mixture and incubated for 20 minutes at room temperature. DNA:Lipid:OptiMEM mixture was added to the flask with packaging cells in 18.75ml growth media. Transfection was performed overnight in 5% CO₂37⁰C humidified incubator. After overnight transfection, DNArlipid containing media was removed and replaced with recovery media containing prewarmed High Glucose DMEM supplemented with 10% FBS, ImM sodium pyruvate (Sigma) and IX pen strep. Virus production was allowed for 48 hours. Recovery media was replaced for virus collection the next day. Virus containing supernatant was harvested and filtered using 0.45um filter. 100mm culture dish containing 3X10^Λ5 tail-tip fibroblasts was instantly infected with fresh viruses encoding Sall4. 8ug/ml Polybrene was added to the virus cocktail to enhance infection efficiency. Infection was performed overnight. Virus containing media was removed and replaced with fresh prewarmed growth media. The fibroblasts were allowed to recover for 8 hours. The second batch of viruses were collected, filtered and used instantly for re-infection. Infection was performed as previously. Infected cells were allowed to recover for 7 days. Media was changed every other day.

[0097] pSSI13772 lentivirus (Sierra Sciences LLC) was generated using 293FT packaging cell line through co-transfection of the GFP plasmid with pMD2G/psPAX2 packaging/envelope plasmids (Sierra Sciences LLC). Lipofectamine 2000 was used as the transfection reagent. The rest of the protocol was performed in a similar manner as retroviral production.

[0098] iPS Generation and Embryoid Body Formation

[0099] After 7 days, infected cells were transferred to a gelatin coated Mitomycin-C treated semi-confluent CF-I Passage 3 feeder layer in ES medium. Cells were cultured for 14 days. After 14 days, vertical ES-like colonies were picked, digested into single cell suspension and replated into new feeder layer in ES medium for expansion. Embryoid body formation was performed using W4 ES and iPS cells as previously described (3). Briefly, confluent flasks of W4 ES and iPS cells were trypsinized and cells were pelleted. Cells were resuspended with 5ml of EB medium containing Knockout DMEM (Invitrogen), 15% ES- qualified FBS, 2 mM L-Glutamine (Invitrogen), 1x10 M nonessential amino acids (Invitrogen), I xIO^-4 M 2-mercaptoethanol (Sigma), IX pen strep (Invitrogen) and plated into T25 flasks. Cells were incubated for 45 minutes in 5% CO₂37⁰C humidified incubator. Feeder depleted cells were then harvested and pelleted. EB was generated using the hang- drop method. Inverted plates containing the drops were incubated in 5% CO₂37⁰C humidified incubator for 2 days. After 2 days, the EBs were collected gently and replated into 100mm petri dish in EB medium for an additional 4 days. EB medium was replenished on the second day.

[0100] Teratoma Formation

[0101] 1 Ox 10^-6 iPS cells from vertical iPS clones were resuspended into 200ul of ES medium. An equal volume of Geltrex Reduced Growth Factor Basement Membrane Matrix (Invitrogen) was added to the cell suspension. Cells were injected subcutaneously into each flanks of SCID/NOD mice. After 15 days, the teratoma was collected and embedded in paraffin. Sections of the tissue were then stained with H&E.

[0102] In vitro differentiation of W4 ES and iPS cells into hematopoietic progenitor cells

[0103] Four day old EB bodies from W4 ES and iPS cells were collected and digested into single cell suspension using 2mg/ml collagenase IV (Invitrogen). EB bodies were incubated for 20 minutes in collagenase IV at 37⁰C water bath. After incubation, cells were washed with enzyme-free dissociation medium (Invitrogen) and was triturated until suspension became homogenous. Cells were plated into gel coated MitomycinC-treated semi-confluent OP9 stromal cells using HoxB4 retrovirus and infection was performed overnight. Cells were then collected and virus containing medium was discarded. Cells were plated into new gel coated Mitomycin C-treated semi-confluent OP9 stromal cells using IMDM (Invitrogen) supplemented with 10% ES-qualified FBS, IX pen strep, 100 ng/mL Flt3L (Peprotech), 100 ng/mL SCF(Peρrotech), 40 ng/mL TPO (Peprotech), 40 ng/mL VEGF (Peprotech), 10 ng/ml IFNγ (Peprotech). Cells were allowed to differentiate into hematopoietic progenitor cells for 7 days. Growth medium with cytokines was replenished every other day. After 7 days, cells were collected by trysinization and used for FACS, H&E staining, methylcellulose assay, transplantation and cell expansion. Cells to use for FACS were infected with GFP virus overnight and allowed to recover for one day before sorting.

[0104] Fluorescence Activated Cell Sorting

[0105] After cells have been treated with cytokines to promote hematopoietic progenitor differentiation, cells were trypsinized, pelleted and resuspended into single cell suspension. Cells were filtered using a sterile 0.70um mesh cell strainer (BD Biosciences). Cells were stained with C-kit, B220, Terl 19, Gr-I, CDl Ib₅ CD34 all from BD Pharmingen. Cells were then sorted using iCyt Reflection Cell Sorter (iCyt). Results were analyzed using FlowJo software.

[0106] Hematoxin and Eosin (H&E) Staining

[0107] 1,000 sorted cells were concentrated using cytospin and stained with H&E using Varistain Gemini slide stainer.

[0108] Methylcellulose Assay

[0109] Colony forming unit assay was performed as previous described. Briefly, 2XlO^"4. CD34+/B220+/Terl 19+/Gr-l+/c-kit+ sorted cells were plated in low adherent 35mm culture dish (Stem Cell Technologies) with semi-solid methylcellulose medium M3434 (Stem Cell Technologies). Progenitor colonies were scored after 5 days.

[0110] Induction of Acute Anemia using Phenylhydrazine Hydrochloride

[0111] One day before anemia was induced, mice were injected with anti-asialo GMl antibody (Wako) according to manufacturer's instructions. Acute anemia was induced in 4-6 weeks old C57BL/6 mice using 50mg/ml Phenylhydrazine HCL. 3 days after injection, cells were injected intravenously with UlOT⁶ CD34+/B220+/Terl l9+/Gr-l+/c-kit+ sorted. Anti- asialo GMl antibody was continuously injected 1 day, 1 week and 2 weeks after transplantation. [0112] Transplantation

[0113] The day before irradiation, the mice were injected with anti-asialo GMl antibody (Wako) via IP to deplete the mice of NK cells. 4-6 weeks old SCID/NOD and C57BL/6 were subjected to one dose of 400rads of radiation. The mice were allowed to recover overnight. 1 xlO^"6 CD34+/B220+/Terl 19+/Gr-l+/c-kit+ sorted cells were then injected intravenously. Anti-asialo GMl antibody was continuously injected 1 day, 1 week, 2 weeks after transplantation. Experiments were carried out with Institutional Animal Care and Use Committee approval.

[0114] Alkaline Phosphatase Staining

[0115] Alkaline phosphatase staining was performed using Alkaline Phosphatase detection kit (Chemicon, SCR004) according to manufacturer's instruction. Briefly, W4 ES cells and iPS were plated in gelatin coated semi-confluent feeder layer at low cell density. After 5 days, cells were fixed with 4% paraformaldehyde for 2 minutes. Fixative solution was removed and the cells were washed once with TBST rinse buffer containing 2OmM Tris- HCl, pH 7.4, 0.15M NaCl₅ 0.05% Tween-20. Cells were stained with 2:1:1 FRV:Naphthol:water solution and incubated for 15 minutes at room temperature protected from light. Cells were then washed once with TBST rinse buffer. TBST buffer was added to the cells to prevent the cells from drying.

[0116] Immunofluorescence Staining

[0117] Cells were fixed with PBS containing 4% paraformaldehyde for lOmin at room temperature. After washing with PBS (Ca+, Mg+), the cells were blocked with 5% normal goat and bovine serum albumin in 0.1% Triton X-100 for 1 hour at room temperature. Primary antibodies included SSEA-I (Chemicon), Oct-4 (Chemicon), Sox2 (Santa Cruz Biotechnology), Nanog (Santa Cruz Biotechnology), SALL4 (developed 'in house'). Secondary antibody used was anti-mouse FITC (Sigma). Nucleus were counterstained with lmg/ml 4',6-diamidino-2-phenylindole (DAPI) for 10 minutes. Table 1: Quantitative Real Time Polymerase Chain Reaction Analysis

[0118] Characterization of mouse Sall4-iPS cells

[0119] Tail-tip fibroblasts (TTF) were derived from C57BL6 mice. TTF were retrodifferentiated using a retroviral introduction of a single transcription factor, Sall4. Well established mouse Sall4-iPS clones have similar morphology to that of mouse W4 ES cells. Phase contrast images shows very distinct clones 20 days after transfection (Ia). Clones were picked and expanded. After 5 days, the clones were stained using alkaline phosphatase. Clones bearing red color is a positive stain indicative of stem cell property. Clones were also tested with well defined pluripotent markers, Sall4, Nanog, Ssea-1 and Oct4. All markers stained positive as well. [0120] Generation of hematopoietic cells in vitro

[0121] Day 6 embryoid bodies derived from mouse Sall4-iPS cells were digested into single cells and plated into OP9 stromal cells. Cells were maintained in growth media containing a cocktail of cytokines, FLT3L, SCF, VEGF, IFN-gamma, and TPO, for 5 days. After 7 days, cells were cytospun and stained with hematoxylin and eosin. The following images show erythroid precursor-like and neutrophil-like morphology. This is an evidence of successful generation of hematopoietic cells from mouse Sall4-iPS cells in vitro.

Prereduction of fibroblast with a single transcription factor, Sall4

Transduced cells grown in mouse stem cell media containing recombinant LIF and mouse feeder cells. Replenish media daily Rat clones evident.

ES cell-like morphology in culture predominant Clones were picked for characterization and differentiation studies.

- ^■ differentiation studies, cells were plated using f- Day 25 - hang-drop method to produce emforyoϊd bodies (EB) iPS dϊfferen- I ^J -Collect EBs and differentiated further for4 days in suspension

Tiated into J Day 26- Culture . embryoid ] bodies (^ Day 28 - dissociate EBs into single cells and plate the cells

Day 30 ^""into Mitomycin-C treated OP9 stromal feeder cells.

Day 31 . <&. -, » jlnffir.t the cells with HOXB4 retrovirus overnight.

EBs differenDay 32 <ζ_______g Begin differentiation of celts into hematopoietic cells by treatment tiated into -<? of five cytokines FLT3L, SCF, TPO,¥E©F,FNγ hematopoietic Day 34 cells

Day 36 Day 37 Collect hematopoietic cells for characterization, clonogenic (CFU) assays and transplantation.

[0122] To further confirm the hematopoietic cells derive mouse Sall4-iPS cells, surface markers analyses was performed using flow cytometry on mouse Sall4-iPS cells derived hematopoietic cells. After 7 days of cytokines treatment to induce differentiation of EBs into hematopoietic cells, cells were collected and stained with surface markers: B220 (lymphoid), GrI and CDl Ib (myeloid), Terl 19 (erythroid) and c-kit (hematopoietic progenitor).

[0123] The expression of hematopoietic markers was also determined using quantitative real-time PCR (qRT-PCR) analysis. Mouse Sall4-iPS cells-derived hematopoietic cells express specific hematopoietic markers at various time points after treatment of cytokines by quantitative real time PCR analysis. Markers specific to hematopoiesis and lymhoid development, lymphoid development, pre-B and T-cell development were up-regulated indicative of successful differentiation of mouse Sall4-iPS cells into myeloid and lymphoid cells.

[0124] Distinct reddish coloration of hematopoietic cells derived from mouse Sall4-iPS cells after prolonged culture in media containing cocktail of 5 cytokines, FLT3L, SCF, VEGF, IFN-gamma and TPO. Therefore, we sought if fetal hemoglobin was expressed using quantitative real time PCR analysis. Mouse Sall4-iPS cells-derived hematopoietic cells express specific hematopoietic markers at various time points after treatment of cytokines by quantitative real time PCR analysis. Markers specific to erythroid lineage, fetal hemoglobin B-Hl and adult hemoglobin B-major markers were up-regulated indicative of successful differentiation of mouse Sall4-iPS cells into erythroid cells. Fetal hemoglobin mRNA expression decreased after prolonged culture while adult hemoglobin was unchanged. The loss of fetal hemoglobin expression might due to successive maturation of the cells into terminal stage.

[0125] To determine myeloid differentiation potential, colony forming unit (CFU) assays were performed. After 7 days of cytokines treatment, CD34+ cells were sorted by FACS and 2x10⁴ cells were plated in methylcellulose semi-solid media, M3434. colony-forming units- granulocyte-erythroid-makrophage-megakaryocyte (GEMM) and colony-forming units- granulocyte-macrophage were detected at day 7 in culture. This data further proves integrity of hematopoietic cells derived from mouse Sall4-iPS cells.

[0126] Long-term engraftment of hematopoietic progenitor cells derived from Sall4-iPS cells in the bone marrow.

[0127] After 7 days of cytokines treatment, the cells were stained with H&E to verify success of hematopoietic differentiation frommouse Sall4-iPS cells. Cells were then collected and tested for CD34 surface marker. CD34+ cells were sorted and reanalyzedfor purity. 2x10⁶ CD34+ cells were labeled with lenti-GFP prior to transplantation onto lethally irradiated irnmunodeficientSCID mice and C57BL6 mice (the same strain that the TTF used for retrodifferentiation was derived originally). After 30 days of post transplantation, the mice were sacrificed to analyze engraftment and repopulation potential of hematopoietic cells.

[0128] 30 days post transplantation, the SCID mice were sacrificed and the bone marrow cells were collected. Surface markers of myeloid (Gx- 1, CDl Ib), lymphoid (B220), erythroid (Terl 19) and early hematopoietic markers (CDl 17, CD34) were analyzed by flow cytometry. The cells successfully engrafted into the bone marrow and were able to repopulate for a period of three months. Control SCED mice did not survive the lethal irradiation and died within 2 weeks while transplanted mice survived. This is a clear indication of therapeutic potential of mouse Sall4-iPS cells differentiated into hematopoietic lineages. Similar results were observed when cells were transplanted into C57BL6 mice.

EXAMPLE 4 SINGLE GENE TRANSFER TO PRODUCE iPS

[0129] Because of the desirability to introduce the minimum amount of exogenous genetic material into a human somatic cell in the process of generating iPS cells that have utility in cell therapy, we seek to generate iPS cells with a single gene transfer. The following protocol is carried out.

[0130] SALL4 gene sequences are introduced into somatic cells obtained from a patient by the use of high titer retrovirus or any other process for gene transfer, a number of which are well known in the art. After a period of approximately 3-7 days following retroviral infection, ES-like colonies emerge with properties consistent with iPS cells.

[0131] The iPS cells so produced are optionally cultured under conditions well known in the art to generate EBs. Cultures of iPS cells or EBs are submitted to a protocol well established in the art of exposing the cells to culture media containing recombinant vascular VEGF.

[0132] Following differentiation to progenitor endothelial cells, the cells are tested for expression of markers for endothelial progenitor cells, including but not limited to the markers FIk-I and CD31 (PECAM-I). By the use of an immunofluorescence assay with FVIII antibody, the cells are also tested for production of FVIII. [0133] Following demonstration of differentiation of iPS cells into endothelial progenitor cells that produce Factor VIII₃ these cells are injected into the patient from whom the original somatic cells had been obtained. The site of injection is optionally into the liver of such patient. The patient is subsequently evaluated for FVIII production.

EXAMPLE S

INSULIN SECRETING BETA CELLS DERIVED FROM MOUSE AND HUMAN

SALL4-JPS CELLS

[0134] Strategies of generation of insulin secreting β cells derived from mouse Sall4-iPS cells and human SALL4-ΪPS cellsTail tip fibroblasts were derived from C57BL6 mice. Fibroblasts within passages less than 4 were infected with Sall4 retrovirus and complete reprogramrning took place for a total of 14 days post infection based upon positive staining of pluripotent markers, qRT-PCR analysis of pluripotent transcripts, embryoid bodies formation and teratoma mass formation. Embryoid bodies (stage 1) were plated into gelatin coated plates and treated with factors such as nicotinamide, insulin, transferrin, and selenic acid which promotes differentiation into beta cells (Stage 2). After 9 days in differentiation, multi- lineage progenitors were present at this stage. Pancreatic cells were selectively differentiated by plating the cells into laminin-poly ornithine coated plates (Stage 3). Further differentiation took place for 18 days.

[0135] Insulin secreting β cells derived from mouse and human Sall4-iPS cells

[0136] Mouse and human Sall4-iPS cells were cultured using a hanging drop method for 2 days and an additional 4 days in suspension culture to generate embryoid bodies containing the three germ layer cell types without the presence of leukemia inhibiting factor (LIF) and feeder layer. EBs were allowed to attach in gelatin coated plates and cultured for 9 days (Stage 1). Phase contrast images were taken to compare different types of iPS cells generated in our laboratory and differentiated into insulin-secreting beta cells. On day 2 of Stage 1, the EBs started to attach to the tissue culture plates, and cells start to migrate from the EB core and proliferate. Day 8 of Stage 1, cobblestone-like cells, containing multilineage cells were formed as seen at high magnification. After 9 days of differentiation, the cells were treated with factors such as nicotinamide, insulin, transferrin, and selenic acid to promote differentiation into beta cells. Islet-like clusters started to form 3 days after treatment of the factors. Distinct islet-like clusters continued to form 9 days after treatment of the factors. Cells were selectively differentiated into insulin-secreting beta cells by differentiation in laminin-polyornithine coated plates (Stage 3). This type of coating further promoted robust differentiation of multilineage progenitor cells into insulin-secreting beta cells. Islet-like clusters persisted even after 18 days of differentiation.

[0137] Immunofluorescence staining of insulin-secreting beta cells using pancreatic markers showed slightly positive stain using insulin antibody. This is an indication that cells are starting to mature from a multilineage progenitor stage into more mature pancreatic beta cells. Glucagon, a marker for pancreatic alpha cells shows very minimal expression. C- peptide or proinsulin, is a marker for beta cells at progenitor stage showing very minimal expression as well. This is an indication that the cells starting to mature into beta cells.

[0138] Islet-like clusters derived from mouse Sall4-iPS cells and human SALL4-iPS cells secreting insulin in response to glucose stimulation

[0139] Various iPS-derived islet-like clusters (both human and mouse derived) response to glucose-stimulated insulin secretion. The islet-like clusters were sequentially treated with low (5.5mM Glucose with 5OuM Tolbutamide) and high (27.7 Glucose mM) concentrations of glucose. At low glucose concentration, insulin was detected in all iPS types at comparable level. At high glucose concentration, beta cells generated from human SALL4-ΪPS cells had a more robust response to glucose stimulation.

EXAMPLE 6 MYOCARDIOCYTES FROM SALL4-JPS CELLS

[0140] Prospects for cell based therapies of heart diseases are a burgeoning field. Their use in myocardial infarction and heart disease are among the potential uses of stem cell therapies to treat diseased and damaged tissues. These future treatments will involve the introduction of stem cells, or stem cell-like cells into the tissue and should mediate a functional recovery within the myocardial tissue.

[0141] Embryonic stem (ES) cells are well known for the ethical issues that they pose. Derived from the inner cell mass, ES cells can give rise to embryonic endoderm, ectoderm, and mesoderm while not contributing to the trophoblast lineage of the placenta. Because ES cells can give rise to all cells of the embryo proper in vivo and in vitro, their therapeutic potential is vast. Indeed potential clinical uses are still in the future there several potential problems that must be overcome. The most formidable challenge is the immune barrier.

[0142] Potential cell therapies for myocardial infarction and heart disease require a pure population of myocardiocytes for introduction into the heart. These cells have been derived from ES cells by several different groups, however, they still may be rejected by immune cells. Recent discoveries in stem cell biology have allowed the development of induced pluripotent stem (iPS) cells. These cells can be derived from fibroblast cell cultures by the viral transduction of as few as three transcription factors. iPS cells are indistinguishable from ES cells and have the ability to form teratomas, and contribute to the germ line in mice. Thus, iPS cells have tremendous potential for cell therapies. Recently, two separate groups described the derivation of myocardiocytes from iPS cells.

[0143] Immunofluorescence (IF) staining of human and mouse iPS cells derived cardiomyocytes with cardiac specific Markers

[0144] After being suspended 6 days for EB formation, cells were attached with special medium for cardiac differentiation. At day 14 of differentiation, cells were stained with cardiac specific markers toponin T, heavy chain cardiac myosin (MHC) and alpha sarcomeric actin as indicated. The expression of all markers (Figure 1) indicating mature cardiomyocytes that were successfully generated from both human and mouse sall4 iPS cells.

[0145] RT-PCR analysis of cardiac specific marker expression in cardiomyocytes differentiated from human and mouse iPS cells

[0146] At day 14 of differentiation, cells were used to analyze mRNA expressing for cardiac specific markers using RT-PCR. The cardiac specific markers included alpha heavy chain cardiac myosin (alpha-MHC), cardiac ventricle myosin light chain(MLV-2v), atrial natriuretic factor (ANF) and pluripotent stem cell marker OCT4. Mouse fetal heart cDNA was used as control. H9, W4 ES cells and both sall4 iPS cells express OCT4 highly indicating cells under pluripotent status. Both human and mouse Sall4-iPS cells derived cardiomyocytes strongly expressed all the cardiac markers while no OCT4 expression (Figure 2). This indicates that the iPS cells were successfully differentiated into mature cardiomycytes including ventricular and atrial cells.

[0147] Production and Characterization on murine iPS cells

[0148] Here we report the preparation of murine iPS cells from tail-lip fibroblasts using both 3-factor and 1 -factor retrovirus transduction protocols. iPS cells generated by either procedure can be differentiated into alveolar cells that can be efficiently engrafted into murine lung tissue.

[0149] The production and characterization of iPS cells generated from mouse tail-lip fibroblasts using either 3-factor or single-factor retrovirus transduction in our laboratory has been reported previously. Both three-factor derived iPS cells (3F-iPS) and single-factor derived iPS cells (Sall4-iPS) exhibit the expected ES cell-like characteristics. They express proteins known to be important in maintaining ES cell pluripotency (Sall4, Oct4, SOX2, Klf4, C-myc, and Nanog), they can be differentiated into cells representative of all three germ layers in vitro, they form teratomas in nude mice and, when injected into blastocysts, they yield viable mouse chimeras.

[0150] In vitro differentiation of 3F-iPS and Sall4-iPS cells into lung progenitor cells

[0151] Lung progenitor cells and mature lung cells were derived from 3F-iPS and Sall4- iPS cells by a three-stage differentiation process, following previously described method for mouse ES cells. This process, which involved 25-45 days of differential culture, is summarized in Figure IA. In brief, in stage 1, 5 x 10⁶ iPS cells were plated in a 100mm bacterial dish containing DMEM High glucose (Invitrogen), 10% certified FBS (Invitrogen), and IX pen-strep (Invitrogen). Cells were cultured for 2.5 days in this condition in a 5% CO2 humidified incubator in order to generate Embryiod Bodies (EBs). Medium containing free floating embryoid bodies (EBs) was collected and the EBs allowed to settle by gravity. EBs were collected and transferred back to the same Petri dish containing DMEM high glucose, 10% Knock-out serum replacement (KOSR-Invitrogen), IX pen strep and 100ng/ml Activin A (R&D Systems, San Diego, CA). Cells were cultured in this condition for 4.5 days. Media was changed every 2 days. EBs were collected and transferred into a new 100mm bacterial dish for an additional 2.5 days in media containing DMEM high glucose, 10% KOSR and IX pen strep. Media was changed every 2 days. It is of great importance to use only EBs that have not aggregated during the three conditions used in this stage for the succeeding steps due to extremely low efficiency of this method.

[0152] For stage 2 differentiation, EBs were collected and plated in several gelatin-coated culture dishes: 35mm culture dishes for qRT-PCR analysis (15-20 EBs)₃48 well plates for immunostaining (5-10 EBs) and two 100mm culture dishes for expansion (remaining EBs). EBs were allowed to attach and differentiate into definitive meso-endoderm progenitor cells for 11 days with just enough media (DMEM, 10% KOSR, Ix pen-strep) to cover the surface of the culture dishes to increase EB attachment. Only very few EBs will attach to the culture plates so extreme care must be taken during this culture stage. Media was changed and growth of the differentiated cells was monitored every 2 days.

[0153] In stage 3, lung progenitor cells were selected by growth in Small Airway Basal Medium (SABM) with bulletkit supplements containing BSA, Insulin, EGF for 5 days up to 45+ days. Media was changed and differentiation status monitored every 2 days.

[0154] Fluorescence Activated Cell Sorting

[0155] Cells from the final stage of lung differentiation method were analyzed and sorted by flow cytometry. I X lO⁶ cells were fixed using Fix Perm Kit (Invitrogen) according to manufacturer's instructions. Briefly, the cells were fixed with lOOul of Solution A and cells were incubated for 15 minutes. The cells were then washed once with IX PBS (Mediatech) and pelleted by centrifugation. lOOul of Solution B 9 was added and incubated with each with the following antibodies: primary mouse monoclonal antibodies to SPA, SPB, SPC and TTF-I (all from Santa Cruz Biotechnology) for 15 minutes. Cells were washed once with IX PBS and incubated with FITC anti-mouse secondary antibody (Southern Biotech) in the dark for 15 minutes. Cells were pelleted and 500ul of IX PBS was added. The cells were sorted using an iCyt Reflection Cell Sorter and the data analyzed using Flowjo software (XYZ). [0156] Transplantation of Lung progenitor cells into Irradiated C57BL/6 mice

[0157] The lung progenitor cells exposed to SAGM supplemented with bulletkit reagents for 14d days were infected with green fluorescent protein(GFP) lentivirus construct overnight. Double stained cells were then sorted using a cocktail of primary antibodies (SPA, SPB, SPC and TTF-I) with anti-mouse RPE secondary antibody and GFP positive cells. Following sorting, 2 X 10⁶ cells were transplanted into irradiated C7BL/6 mice via tail vein injection. The mice were subjected to a whole body irradiation. Treated mice (n=x) were sacrificed at various time points, 7 and 30 days, for engraftment and repopulation studies.

[0158] Quantitative Real Time Polymerase Chain Reaction Analysis

[0159] Total RNA from I X lO⁶ cells were extracted using Trizol reagent (Invitrogen) according to manufacturer's instruction, lug of RNA was then reversed transcribed using High Capacity cDNA Archive kit (Applied Biosystems).

[0160] Generation of induced Pluripotent Stem (iPS) Cells from Tail-tip Fibroblasts

[0161] The present experiments show that the inventors have produced iPS cells from murine and human origins using retrovirus transfection with vectors that express Oct4, SOX2 and Klf4 (PNAS) (Science). As noted previously by other investigators, iPS cells can be generated without the use of the known oncogene c-myc. While cell reprogramming without c-myc decreased efficiency by 3-4 folds, iPS cells are derived readily with only Oct4, Sox2, and Klf4 (designated 3F-iPS for their 3 factor derivation). We also used a different reprogramming protocol that employs a single retrovirus vector carrying the Sall4 gene. The Sall4 protein is a zinc-finger transcription factor expressed in human and murine ES cells that regulates the expression of Oct4, SOX2, Klf4, and C-myc (Sall4 PNAS). The introduction of exogenous Sall4 coding sequences into somatic cells produces iPS cells (termed Sall4-iPS cells) with an efficiency 10-100 fold better than 3 -factor reprogramming protocols (Science). C57BL/6 mice strain tail-tip fibroblasts were used to successfully derive 3F-iPS and Sall4- iPS cells. These 3F-iPS and Sall4-iPS cells expressed typical pluripotent stem cell markers, including ES-alkaline phosphatase, SALL4, OCT4, NANOG, and SSEA-I. Additionally, 3F- iPS and Sall4-iPS cells expressed Oct4, Sox2, and Klf4 similar to W4, a known ES cell line. They also formed embryoid bodies (EBs), composed of multiple tissue types, and they formed teratomas when injected into the flanks of SCID/NOD mice. These teratomas showed neural crest-like, muscle-like, and gut-like morphologies of the ectoderm, mesoderm, endoderm germ layers, respectively. Finally, when the iPS cells were injected into blastocysts and then implanted into pseudo pregnant mice, they produce viable chimeric mice. Collectively, the data suggest that both 3F-iPS and Sall4-iPS cells derived from mouse tail-tip fibroblasts have stem cell-like properties.

[0162] iPS Cells Differentiate into Lung Progenitor Cells

[0163] The following experiment shows differentiation of 3F-iPS and Sall4-iPS cells into lung alveolar cells following the three stages outlined in Figure . Retrodifferentiated cells using 3 factors and one factor (Sall4) were induced to differentiate into lung alveolar cells by treatment of Activin A, a known protein to enhance differentiation of mouse ES cells into definitive mesoendodermal lineages such as alveolar cells. The following panel shows the progress of differentiation after cells have been allowed to attach in gelatin coated plates. The removal of KOSR (in replacement for FBS) seem to have very little effect on attachment of the embryoid bodies in Stage 2 where EBs were attached for 9 days. In Stage 3 with the use of various growth factors and supplements, the EBS attached within 4 hours. The EBs were then monitored on a daily basis up to 55 days of differentiation. Day 22 shows stable attachment of EBs on the culture plate and cells on the edge of the plate continue to proliferate. More pronounced alveolar-like structures were observed at day 24th of differentiation. There were no significant differences between iPS cells derived from 3 factors or Sall4-only factor, as well as the mouse ES cells, W4.

[0164] mRNA expression of typical endoderm and mesoderm lung cell markers Brachyury, FoxA2, and Soxl7 were examined in the iPS-derived lung progenitor cells. Results of qRT-PCR confirmed that iPS-derived cells expressed upregulated Brachyury, FoxA2, and Soxl 7 in the differentiation stage. In addition, early and mature lung markers SPC, TTF-I and SPA, SPB, CClO₅ respectively, were analyzed to characterized the population of iPS-alveolar cells. Results of qRT-PCR confirmed upregulation of early and mature lung makers, indicating a heterogeneous population. This data suggests an abundance of lung epithelial alveolar cells even at the very late stage of differentiation and that during prolonged culture, iPS-derived progenitor cells mature slowly which is consistent with ES- derived progenitor cells. Together, the data document that endothelial early and mature lung alveolar cells can be derived from iPS cells using the EB differentiation method. Interestingly, TTF-I mRNA expression was markedly upregulated in this study which is in contrast to previously reported data.

[0165] iPS-alveolar cells from the final stage of lung differentiation was analyzed for early and mature lung markers and sorted by flow cytometry. Flow cytometry analysis (FCA) indicated upregulation of early markers SPC and TTF-I . Only mature lung marker SPB was stained while SPA could not be detected.

[0166] To further establish the expression of early and mature lung markers, we performed an immunofluorescence (IF) staining analysis at days 38. At day 38, early lung marker SPC is highly positive but TTF-I, also an early lung marker, stained negative (Figure F), consistent with earlier data (Bishop et al). immunostaining with lung specific markers were positive, including TTF-I, an early lung marker. This result confirmed a heterogeneous mixture of early and mature lung cells. The panel shows similar immunostaining results for 3F and sall4-only derived iPS cells differentiated into alveolar cells. At Day 38 of differentiation, alveolar progenitor markers expressed SPC and TTFl indicating slow progression of alveolar progenitors to mature alveolar cells.

[0167] Engraftment and Long Term Repopulation of Lung Epithelial alveolar cells in vivo

[0168] We next implanted the sorted GFP labeled iPS-alveolar cells into lateral tail vein of C57BL/6 mice and sacrificed at 7 days. This immunostaining revealed that the transplanted GFP positive cells appeared to structurally reconstitute the alveolar spaces in the lung. Finally, to further characterize the localization of GFP labeled cells, major organs, including heart, liver, spleen, kidney, and duodenum were embedded in paraffin and stained with anti- GFP and anti-SPC. Results indicate that no extrapulmonary migration of cells was detected and that only lung was positive for GFP and SPC markers. This confirms that the lung alveolar cells, injected via lateral vein, specifically home to the lung.

[0169] Lung disease is the 3^r leading killer in America and is responsible for 1 in 6 deaths. Lung tissue is a uniquely specialized tissue in the body that can become irreversibly damaged by scarring and fibrosis that occur in cystic fibrosis (CF), emphysema, and sarcoidosis. There is no cure for CF, emphysema, or sarcoidosis and treatment focuses on alleviating symptoms and/or removing damaged lung tissue. Therefore, methods to generate new lung tissue and replace damaged lung tissue are much sought after.

[0170] The use of ES (embryonic stem) cells to generate functional epithelial alveolar cells (here designated Lung-ES for the purpose of discussion) have been previously demonstrated. These Lung-ES were able to derive into both Type 1 (respiratory cells) and Type 2 (surfactant producing) pneumocytes and were successfully engrafted into mouse models with lung injury, suggesting functional integration. However, immune associated rejections are a concern when using ES derived cells.

[0171] iPS cells are indistinguishable from ES cells thus far and may present a major therapeutic advantage in the use of future cellular therapies. Previously, mouse tail-tip fibroblasts were induced to pluripotency, differentiated, and transplanted back to the original mouse; with the functional engraft providing therapeutic benefit. Thus, iPS cells can completely avoid the immune barrier associated with the use of ES cells in cellular therapies. By generating Lung-iPS cells and transplanting them into the lung parenchyma, we demonstrate a potential tissue generator. This proof-of-principle study shows that lung progenitor cells derived from iPS cells can engraft within the lung parenchyma and functionally integrate into the lung structure. It is clear that iPS cells will have tremendous therapeutic benefit in the future for the treatment of tissue replacement once several technical challenges are overcome.

[0172] Stage 1 conditions differentiate iPS cells into definitive endoderm and mesoderm layers in suspension, Stage 2 condition attaches the embryo-like (EB) structures to tissue culture dishes coated with gelatin, and Stage 3 conditions further differentiate the EBs into Type II progenitor pneumocytes. Well established early lung markers, Surfactant Protein C (SPC) and TTF-I were upregulated at all the time points tested. Mature lung markers such SPA, SPB and CClO were upregulated as well demonstating heterogenous population of early and mature lung cells. TTF-I mRNA expression was markedly upregulated. This data suggests an abundance of lung epithelial progenitor cells even at the very late stage of differentiation. Wild type Tail-tip fibroblasts were used as the calibrator during analysis and cell suspension from lung from wild type mice were used as positive control. Day 24 lung epithelial progenitor cells derived from 3F-iPS cells were tested: SPC and TTF-I early lung markers stained positive. Mature lung markers SPA and SPB were also tested and only SPB stained positive comparable to the early marker counterparts. Mature lung markers, SPA and SPB were slightly positive. These results indicate a heterogeneous population of early and mature lung cells present.

[0173] Production and Characterization of Induced Pluripotent Stem (iPS) Cells

[0174] Following the seminal work of Yamanaka and colleagues, we have been able to successfully derive iPS cells from fibroblasts of both mouse and human origin. The mouse fibroblasts used for this study were derived from the tail-tip of a C57BL/6 mouse strain, a close relative of the hemophilia A mouse model used, in order to reduce potential immune rejection. Like other investigators, we note that iPS cells can be generated without the introduction of c-myc, a known oncogene. Without c-myc the efficiency of faithful reprogramming was approximately 3 to 4-fold lower, however iPS cells were derived readily using only Oct4, Sox2 and Klf4. These iPS cells, (designated 3F-iPS for their 3 factor derivation), expressed typical pluripotent stem cell markers such as ES-alkaline phosphatase, SSEA-I, Nanog, Oct4 and SALL4. To ensure undifferentiated 3F-iPS cells could give rise to all three germ layers of the developing embryo, we injected the cells into the flanks of SCID mice. 3F-iPS cells gave rise to teratomas composed of all germ layers within one month following injection. These data clearly suggest that iPS cells derived from tail-tip fibroblasts have stem cell-like properties. To further ensure the pluripotent characteristics of 3F-iPS cells, , we generated Embryoid bodies (EBs) that were successfully differentiated to multiple tissue types with appropriate growth factor simulation.

[0175] 3F-ΪPS Cells Derived from Tail-tip Fibroblasts Differentiate into Endothelial Progenitor Cells and Mature Endothelial Cells in vitro

[0176] Endothelial cells are thought to secrete the majority of FVIII protein in vivo. To test the differentiation potential of 3F-iPS into endothelial cells in vitro, we applied a series of differentiation protocols that had been previously developed for ES cells by various laboratories. 3F-iPS cells were cultured using a hanging drop method for 2 days in LIF-free media where they readily formed spheroid EBs. By this method, the generation of EBs devoid of extraembryonic characteristics was ensured. After 2 day culture in a hanging drop, the EBs were collected and transferred to a nonadherent Petri dish and allowed to differentiate for an additional 2 days. Upon plating and culture of EBs in vascular endothelial growth factor (VEGF)-containing media on collagenase-coated dishes, the resultant differentiated cells showed typical endothelial cell morphology after 6, 12 and 18 days of growth. The endothelial cells derived from 3F-iPS were analyzed by immunofluorescence using various markers: FIk-I, an early endothelial progenitor marker, CD31 and FVIII, markers commonly used for cells or tissues of vascular endothelial origin. Early stages of endothelial cells differentiation (day 6) showed expression of FIk-I but not CD31 which indicate the presence of endothelial progenitor cells. Mature endothelial differentiation stages at day 12 and 18 showed weak to no expression of FIk-I and strong expression of CD31. The mature differentiated cells also expressed FVIII.

[0177] We next examined mRNA expression of liver and endothelial cell markers including FVIII by real-time PCR (RT-PCR). Results of RT-PCR confirmed that iPS- derived endothelial progenitor cells and differentiated cells expressed endothelial progenitor cell marker, FIk-I and mature endothelial cells markers, CD31 and FVIII. hi addition, other endothelial markers including ESGl, CD33, CD34, GATA3, VE-Cadherin, HPRT, TIEl and REXl were present in the iPS-derived population. Taken together, the data document that endothelial progenitor cells and mature endothelial cells can be derived from iPS cells using the EB differentiation method.

[0178] Phenotypic correction of a mouse model of hemophilia A using differentiated iPS cells derived from fibroblasts

[0179] Following successful differentiation of iPS cells into FVIII positive endothelial/ endothelial progenitor cells, we injected Ix 10⁶ of these cells directly into the livers of sublethally irradiated male mice carrying the deletion of exon 16 of the FVIII gene. C57BL/6 normal mice and non-injected hemophilia male mice were used as control groups. One week after endothelial progenitor cells were transplanted to the liver, all groups of mice were subjected to a tail-clip challenge. The group of hemophilia mice (N=6) not injected with the endothelial progenitor cells died within 2-8 hours after the challenge, hi contrast, hemophilia A mice (N=6) treated with the iPS-derived endothelial cells surpassed the 2-8 hours challenge and survived as did the C57BL/6 control mice (N=6). Furthermore, the levels of plasma FVIII in treated mice was measured over three months and revealed FVIII levels as high as 8-12% of controls. RT-PCR for FVIII mRNA also revealed a 3 fold increase of FVIII level in the liver compared to untreated mice, about a 7 fold increase in heart and kidney, and about an 8 fold increase in the spleen. Our data indicate that the preclinical mouse model can be rescued after transplantation with endothelial progenitors derived in vitro from iPS cells.

[0180] Long-term engraftment and structural integrity of transplanted iPS-derived cells

[0181] We monitored the plasma levels of FVIII in transplanted hemophilia mice for three months and observed that plasma FVIII levels increased progressively in three hemophilia mice after transplantation of the FVIII expressing cells. The increase in plasma FVIII in the transplanted hemophilia A mice is indicative of enduring function of transplanted cells.

[0182] Finally, we examined the liver distribution of GFP-labeled transplanted cells by immunofluroscent staining of liver tissue with antibodies against GFP and CD31. This immunostaining revealed that the transplanted GFP positive cells co-expressed CD31, a characteristic endothelial membrane marker and that these cells appeared to structurally reconstitute the sinusoidal endothelial compartment in the liver.

[0183] Hemophilia A is a sex-linked bleeding disorder characterized by the deficiency of coagulation Factor VIII causing prolonged bleeding due to the inability to efficiently clot. Treatment for hemophilia generally includes either fixed-dose prophylaxis or factor replacement therapy on an as needed basis. Regardless, neutralizing antibodies to the replacement protein have been reported and present a unique problem when treating hemophiliacs.

[0184] The monogenic nature of hemophilia makes it an ideal target for gene therapy. However, attempts at gene therapy so far have failed due to the lack of an adequate delivery system, unstained FVIII expression or immune rejection. Cellular therapies for hemophilia present a unique delivery method for FVIII protein in this disease. By generating iPS cells that express wild-type endogenous FVIII protein and transplanting them into the hepatic parenchyma, we demonstrated an effective phenotypic correction of hemophilia. This proof- of-principle study shows that endothelial progenitor cells derived from iPS cells can effectively express the FVIII protein, engraft within the hepatic parenchyma, and functionally integrate to provide the therapeutic benefit necessary for phenotypic correction of hemophilia. It is interesting to note that while the iPS derived endothelial cells were injected into the liver; higher levels of FVIII mRNA were detected in spleen, heart and kidney tissues of injected animals. Additional studies of GFP-tagged epithelial cells will be required to establish the complete whole-body distribution. Our study provides additional proof that correction of a single gene disorder using iPS cell technology is feasible and expands on the work of Hanna et al on correcting the sickle cell phenotype. It is clear that iPS cells will have tremendous therapeutic benefit in the future for the treatment of monogenic disorders once several technical challenges are overcome.

[0185] The major obstacles that must be overcome are the challenges surrounding the generation of iPS cells using multiple factors and retroviral transduction. In this study, we have produced iPS cells using the retroviral transduction of Oct4, Sox2, and Klf4 into mouse tail tip fibroblasts. While we have observed no tumor formation or other induced pathological problems in our study to date, long term follow-up throughout their life time will be need to determine if down-stream adverse effects will emerge. Ideally, one would like to remove the integration of transcription factors via randomly integrating viral vectors all together. Toward this end, recent studies have documented the ability to generate murine iPS cells using non-integrating adenovirus vectors expressing Oct4, Sox2, Klf4 and c-MYc. Adequate expression of 3-4 transcription factors using non- viral means may be difficult. Small molecules are another alternative to viral introduction of transcription factors and recent studies have suggested that this may be a viable option, although theoretically a small molecule may be necessary for each transcription factor in the reprogramming cocktail. There is little doubt that reducing the number of reprogramming factors would ease these problems and this may be possible in the near future.

[0186] Retroviral transfection and Fibroblast Infection

[0187] Three pMXs-based retroviral vectors encoding either Oct-4, Sox2 or Klf-4 were purchased from Addgene (Cambridge, MA). Vectors were transfected into a Plat-E packaging cell line (Clontech, Mountainview, CA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer's directions. Twelve hours after transfection, the medium was replaced with medium containing DMEM high glucose (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Invitrogen, Carlsbad, CA), IX pen/strep (Invitrogen, Carlsbad, CA) and ImM sodium pyruvate (Sigma, St Louis, MO). After 48 hr, virus- containing supernatant was collected for each transcription factor and combined with supplemental 8ul/mL polybrene (Chemicon, Billerica, MA). Medium was replenished and the transfected packaging cells were allowed to generate virus for an additional 24 hours. Filtered virus-containing supernatant was used to infect 2x10⁵ tail-tip fibroblasts (TTF) from C57BL/6 mice at passage 3-4.

[0188] iPS cell generation and embryonic body (EB) formation

[0189] Seven days after infection, fibroblasts were harvested by trypsinization and replated at 5^χlO⁴ cells per 100mm dish with Mitomycin-C treated feeder layer (Chemicon, Billerica, MA). Cells were plated using mouse ES medium containing Knockout DMEM (KDMEM₅ Invitrogen, Carlsbad, CA), 15% ES qualified FBS (PAA, Ontario, Canada), 2 mM L-Glutamine (Invitrogen, Carlsbad, CA), IxIO^""4 M nonessential amino acids (Invitrogen, Carlsbad, CA), IxIO^-4 M 2-mercaptoethanol (Sigma, St Louis, MO), IX pen/strep (Invitrogen, Carlsbad, CA) and 12.5 ng/ml LIF (Chemicon, Billerica, MA). The medium was changed every other day. Fifteen days after infection, single colonies were picked and transferred into 50ul 0.25% trypsin in IXPBS. After 3 minutes of incubation at 37°C, lOOμl mouse ES medium was added and the colony was dissociated by pipeting up and down. The cell suspension was transferred to a feeder layer in 24-well plates, and then passaged to 6 well plates and then 100mm tissue culture dishes. The iPS cells were designated 3F-iPS, denoting their derivation from a 3 factor retrodifferentation.

[0190] EBs from 3F-iPS were formed by a hanging drop method (25) using EB differentiation medium containing KDMEM, 15% ES qualified FBS, 2 mM L-Glutamine, IxIO^-4 M nonessential amino acids, IxIO^"4 M 2-mercaptoethanol, and IX pen/strep. After two days in hanging drop culture, EBs were collected and transferred to an ultra low binding dish (Corning, Corning, NY) for another two days in EB differentiation medium. [0191] Characterization of iPS Cells

[0192] Alkaline phosphatase staining was performed using an ES-alkaline phosphatase detection kit (Cat# SCR004, Chemicon, Billerica, MA) according to manufacturer's instruction. For immunofluorescence staining, cells were fixed with 4% paraformaldehyde (EMD, Gibbstown, NJ) diluted in IX PBS (Mediatech, Manassas, VA) for 10 min at room temperature. After washing with PBS₅ the cells were blocked with 5% normal goat and 5% bovine serum albumin (Sigma St Louis, MO), in 0.1% Triton X-100 (Fisher Scientific, Pittsburgh, PA) for 1 h at room temperature. Primary antibodies included mouse anti-SSEA-1 (1:25 Chemicon, Billerica, MA), mouse anti-Oct-4 (1:25 Chemicon, Billerica, MA), mouse anti-Sox2 (1:50 Santa Biotechnology), mouse anti-Nanog (1:50 Santa Biotechnology), and rabbit anti-SALL4 (1 :400 developed 'in house'). The cells were then stained with fluorescent-labeled secondary antibodies which include FITC labeled anti-mouse IgG (Sigma, St Louis, MO) and FITC labeled anti-rabbit-IgG (Southern Biotech, Birmingham, Alabama). Nuclei were counterstained with lmg/ml 4',6-diamidino-2-phenylindole DAPI (Sigma, St Louis, MO) for 10 minutes.

[0193] Teratoma Formation

[0194] 3F-iPS cells were harvested by 0.25% trypsin treatment, collected into tubes, centrifuged, and the pellets were resuspended in EB differentiation medium. A lO⁶ cell suspension in lOOul was mixed with an equal volume of Geltrex (Invitrogen, Carlsbad, CA) and injected subcutaneously into SCID mice (Jackson Laboratory, Bar Harbor, Maine). A total of 5 mice were injected. One month after injection, teratoma were dissected and fixed with formalin (Fisher Scientific, Pittsburgh, PA). Paraffin-embedded tissue was sectioned and stained with hematoxylin and eosin.

[0195] Endothelial cell differentiation

[0196] The EBs were transferred to 35mm tissue culture dishes after 4 days of growth in EB differentiation medium and allowed to differentiate into endothelial progenitor cells using medium containing KDMEM, 15% fetal bovine serum (FBS), 2 mM L-Glutamine, IxIO^"4 M nonessential amino acids, IxIO^-4 M 2-mercaptoethanol, Ix pen/strep, 20ng/ml bFGF (Invitrogen, Carlsbad, CA), 20ng/ml EGF (Invitrogen, Carlsbad, CA), 50ng/ml VEGF (R&D Systems, Minneapolis, MN), 20ng/ml IGF (Sigma, St Louis, MO), 50ug/ml Ascorbic acid (Sigma, St Louis, MO) and lug/ml Hydrocortisone (Sigma, St Louis, MO). For endothelial progenitor cell differentiation, cells were collected after 10 days of differentiation using Collagenase IV (Invitrogen, Carlsbad, CA) for identification and characterization. For mature endothelial cell differentiation, cells were passaged using 0.25% trypsin (Invitrogen, Carlsbad, CA) and replated on collagen IV coated dishes using endothelial cell culture medium containing EGM-2 (Lonza, Portsmouth, NH), 15% FBS, 2 mM L-Glutamine, IxIO^"4 M nonessential amino acids, IxIO^-4 M 2-mercaptoethanol, Ix pen/strep, 20 ng/ml bFGF, 20ng/ml EGF, 50ng/ml VEGF, 20ng/ml IGF, 50ug/ml Ascorbic acid and lug/ml Hydrocortisone. Cells were passed when dishes were 90% confluent.

[0197] Preparation of GFP and Labeling of endothelial cells

[0198] The GFP lentivirus (pSSI 13772, a kind gift from Dr Bill Andrews, Sierra Sciences LLC) was prepared as described (26). Prior to transplantation, endothelial progenitor cells were infected with GFP lentivirus overnight with supplemental 8 ug/ml Polybrene. 48 postinfection, the endothelial cells were collected by trypsinization, suspended into single cells and injected into the hemophilia A mice.

[0199] Characterization of endothelial cells by immunofluorescence and immunocytochemistry

[0200] Immunofluorescence methods used to characterize endothelial cells derived from 3F-iPS cells were similar to those used initially to characterize the 3F-iPS. Primary antibodies used included mouse anti-FLKl (1 :50 Alpha Diagnostic International), mouse anti-CD31 (1:25 BD Pharmingen), rabbit anti-CD31 (Biocare, Concord, CA, for histology), mouse anti-GFP (Chemicon, Billerica, MA) and mouse anti-Factor VIII (1 :50 Diagnostic Biosystems). Cells or tissue sections were then incubated with fluorescent-labeled secondary antibodies which include Rhodamine labeled anti-mouse IgG, Phycoerythrin labeled anti- rabbit-IgG (Southern Biotech, Birmingham, Al), FITC labeled anti-mouse-IgG (Sigma, St Louis, MO) and FITC labeled anti-rabbit-IgG (Southern Biotech, Birmingham, Al). Nuclei were counterstained with lmg/ml 4',6-diamidino-2-phenylindole DAPI (Sigma, St Louis, MO) for 10 minutes. [0201] RT-PCR

[0202] RNA was extracted from samples using Trizol Reagent (Invitrogen, Carlsbad, CA) according to manufacturer's instructions, lug of RNA from each sample was reversed transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Real-time PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) according to manufacturer's instructions. GAPDH (Eurogentec, San Diego, CA) was used as an endogenous control. Primer sequences used were as in the following table. Analysis was done using the Applied Biosystems SDS 7500 FAST software vl.4.

Table 2: Primer sequences used for real-time PCR

[0203] Plasma FVIII activity assay

[0204] Factor VIII activity was measured by chromogenic methodology using Chromogenix Coamatic® Factor VIII kit (Instrumentation Laboratory, Lexington MA). Assays were performed in duplicate according to the manufacturer instructions using the microplate method and end-point detection as described for measuring extremely low levels of factor VIII activity. A high (0-150% FVIII activity) and low (0-5% FVIII activity) standard curve was constructed using serial dilutions of normal human reference plasma (Precision BioLogic, Dartmouth, Nova Scotia) which was calibrated against the 5^th International WHO standard factor VIII and von Willebrand factor in plasma (NIBSC code 02/150). Murine FVIII protein shows 74% amino acid sequence homology with human FVIII and the murine protein demonstrates conservation of functionally important domains. Wild- type C57BL/6 mice have FVIII levels ~100% that of a normal human plasma standard.

[0205] Mouse model

[0206] All animal studies were performed following National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee at Nevada Cancer Institute. Six hemophilia A mice (Jackson Laboratory, Strain Name: B6; 129S4- F8tmlKaz/J, Stock Number: 004424) 6-8 weeks old were used for this study(28). Hemophilia A mice were originally produced by disruption of the FVIII gene through insertion of a targeting vector containing a neomycin gene cassette in the 3' end of exon 16. The targeting vector was introduced into 129S4/SvJae-derived Jl embryonic stem (ES) cells and was injected into C57BL/6 blastocysts. Genotype of each hemophilia A mice was verified by PCR as described. Hemophilia A mice used in this study were irradiated with 20OcGy radiation one day prior to transplantation. We administered 4 U of FVIII recombinant protein by intraperitoneal injection (Baxter Healthh Care, Deerfield, IL) prior to transplantation to ensure survival of the hemophilia A mice. All mice tested were also anesthetized with 0.5 mg/g Avertin via i.p. injection prior to transplantation. Each mouse was injected with 2X10⁶ endothelial progenitor cells derived from 3F-iPS cells via liver injection using 27-gauge needles. Brief pressure was applied to injection sites to ensure proper hemostasis. At various times (7, 30, 60 and 90 days) post transplantation, a tail clip bleeding challenge was performed by cutting lcm from the end of the mouse tail and was used to assess correction of the hemophilia phenotype as previously described (29). C57BL/6J control mice (n=9), the closest relative of the hemophilia A mice model, and irradiated hemophilia A mice (n=9) were injected with PBS as controls. Concurrently, plasma from each mice tested was collected for FVIII level measurement at the indicated times of tail-clip challenge.

EXAMPLE 7

[0207] Somatic cell reprogramming to create induced pluripotent stem (iPS) cells has been accomplished by numerous laboratories using retrovirus transduction, expression plasmids and non-integrating adenovirus to introduce multiple transcription factors, notably Oct4, Sox2, Klf4, and c-Myc. Here we describe a new reprogramming system using only a single protein, Sall4. Sall4 is expressed in human and murine ES cells. Sall4 regulates the expression of Oct4, Sox2, Klf4, and c-Myc. Viral transduction of Sall4 into the genomes of mouse or human somatic cells produces iPS cells with high efficiency that are pluripotent, karyotypically normal, form teratomas in nude mice, and differentiate into all three germ layers in vitro. Murine Sall4-iPS cells introduced into blastocysts yield viable mouse chimeras. These observations should facilitate future clinical applications of iPS technology.

[0208] In the discovery by Yamanaka and colleagues, they introduced Oct4, Sox2, Klf4, and c-Myc into the genome of mouse somatic cells via retroviral transduction and achieved reprogramming to an ES cell-like state. This observation was quickly confirmed by others and extended to the production of human iPS cells. The potential detrimental effects of the oncogenic protein c-Myc were quickly realized and it was successfully removed from the reprogramming cocktail. While this was a significant step toward potential clinical uses of iPS cells, elimination of integrative viral vectors and further lowering the number of exogenous transcription factors needed for reprogramming would be highly beneficial.

[0209] iPS cells also been derived using plasmid transfection and non-integrating adenoviral vectors. However, the efficiency of reprogramming was much lower in these studies than when viral integration was used, an observation that can likely be attributed to low transgene expression from non-integrating expression systems.

[0210] Huangfu and colleagues recently reported that the addition of a histone deacetylase inhibitor to cultured primary human fibroblasts enabled the production of iPS cells using only Oct4 and Sox2. This report not only documents that somatic cell reprogramming can be done with fewer factors but also suggests that appropriate chemical modulators may facilitate iPS cell production.

[0211] Here we demonstrate that both human and murine somatic cells (fibroblasts) can be converted to iPS cells with high efficiency using only a single transcription factor, Sall4. Sall4 is encoded by a gene with important roles in early embryonic development and pluripotency maintenance. We have recently shown that Sall4 can regulate the transcriptional levels of Oct4, Sox2, Klf4 and c-Myc(13). This suggested that Sall4 may have potential utility in somatic cell reprogramming. By introducing exogenous Sall4 coding sequences into somatic cells, we were able to derive iPS cells( termed Sall4-iPS cells) that expressed stem cell specific markers, generated teratomas in SCID/NOD mice, and contributed to chimeric animals following blastocyst injection. This finding should enable easier derivation of iPS cells using viral vectors or transfected plasmid constructs simply by reducing the number of required exogenous factors to unity. Our finding should also ease the transition of iPS cell technology into clinical practice.

EXAMPLE 8 GENERATION OF MOUSE iPS CELLS USING SALL4

[0212] iPS cells have been generated by different laboratories using various production protocols. However, the majority of groups have introduced multiple transcription factors, most notable Oct4, Sox2, Klf4, and c-Myc via retrovirus transduction. We sought to generate iPS cells using as few reprogramming factors as possible in order to enhance the potential progression of iPS technology into the clinic. We had previously shown that Sall4 regulates the expression of the Oct4, Sox2, Klf4, and c-Myc, and plays an important role in embryonic stem cell pluripotency. Thus, we sought to determine the utility of Sall4 in reprogramming somatic cells to a pluripotent state by introducing a retroviral construct encoding Sall4 (pMXs-Sall4 from Addgene) into tail-tip fibroblasts (TTF) derived from the wild-type C57BL/6 mouse.

[0213] After only 3-5 days, colonies began to show and they exhibited morphology closely resembling ES colonies and distinguishable from the parental TTF. Seven days following infection, the TTF derived cells were transferred to a feeder plates and cultured in ES media containing leukemia inhibiting factor (LIF) to ensure maintenance of pluripotency. Cells deprived of LIF and feeder layer eventually died. Growth on a feeder cell layer in the presence of LIF is well known to support self-renewal and pluripotency of ES cells. The TTF derived cells were able to maintain stem cell-like morphology, and self-renew similar to other iPS cells and the mouse ES cell line W4.

[0214] Although these ES -like cells were derived in the absence of a selectable marker, the cells exhibited both morphological and biochemical features similar to iPS cells previously derived in our laboratory using transduction with Oct4, Sox2 and Klf4 vectors. This finding suggests that retroviral introduction of Sall4 into mouse fibroblasts is sufficient to reprogram somatic cells to iPS cells, independent of other transcription factors or small molecules. These iPS cell lines were termed Sall4-iPS cells to denote their origin by transfection with a single vector containing only the Sall4 transcription factor.

[0215] Mouse SalI4-iPS Cells Express Markers of Pluripotency

[0216] W4 ES cells and Sall4-iPS cells were positively stained for ES- alkaline phosphatase, an enzymatic marker of self renewal. To verify the expression of stem cell specific markers in Sall4-iPS cells, the cells were subjected to immunostaining with antibodies to Oct4, Sox2 and Nanog, all of which are known markers of pluripotent stem cells. Expression for each stem cell marker was positive, hi contrast, the surrounding fibroblasts did not stain positive for either of these proteins. The levels of protein expression in Sall4-iPS cells were qualitatively similar to those expressed in the murine ES cell line W4. Surrounding feeder layer was used as an internal negative control and showed no detectable signals. The expression of pluripotency markers was also determined using quantitative realtime PCR (qRT-PCR) analysis. Three individual Sall4-iPS clones tested revealed an up- regulation in the endogenous expression of Oct4, Sox2, and Klf4. While Oct4 and Klf4 were consistently up-regulated, Sox2 expression was more varied. qRT-PCR analysis was also performed on the three Sall4-iPS cells clones to verify upregulation of endogenous Sall4 and minimal expression of viral Sall4 transgene. Primers derived from the 3' untranslated region were used for determination of endogenous Sall4 expression while for total Sall4 expression, we used primers from the Sall4 coding region. Total Sall4 gene expression in each of the three Sall4-iPS clones examined revealed only minimal expression of the Sall4 retroviral construct. This is an indication that once the TTF cells are reprogrammed (marked by upregulation of endogenous Sall4 expression), the expression of the exogenous Sall4 is silenced. This is likely through the same mechanism of silencing previously described.

[0217] We next sought to ensure a normal genetic makeup of the Sall4-iPS cells prior to further in vivo experimentation. Flow cytometry analysis of the Sall4-iPS cells using propidium iodide suggested that the cells have normal DNA content. Further, metaphase chromosome analysis revealed that the Sall4-iPS cells exhibit a normal diploid karyotype with no gross chromosome abnormalities detected. [0218] Sall4-iPS Cells Have Active ES-specific Promoter

[0219] To evaluate the DNA methylation status of ES cell specific promoter, we used bisulfate sequencing. As shown in Figure In, the mouse Sall4-iPS cells are highly unmethylated in the Nanog gene promoter whereas the CpG dinucleotides of this region were highly methylated in the parent TTF cells. These findings indicate that the Nanog promoter is active in Sall4-iPS cells.

[0220] Formation of Embryoid Bodies from Sall4-iPS cells

[0221] To determine the differentiation ability of newly derived Sall4-iPS cells in vitro, we derived embryoid bodies (EBs) using the hanging drop method. EBs are complex three dimensional spheroid structures which are composed of all three germ layers (endoderm, mesoderm and ectoderm). EBs can be differentiated spontaneously into all three germ layers after 15 day culture using an EB differentiation medium. To document differentiation into the various germ cell types we used immunofluorescent staining of various germ layer markers after spontaneous differentiation of EBs in culture plates. The markers included β- III-tubulin and nestin (markers of ectoderm), desmin (marker for mesoderm), α-fetoprotein (AFP, endoderm marker), and vimentin (mesoderm and parietal endoderm markers). The EB differentiated cells expressed all these markers.

[0222] qRT-PCR analysis was performed to confirm that these differentiated cells expressed mRNA transcripts corresponding to the proteins detected by immunostaining. Analysis showed upregulation of gene expression for all of the markers tested, confirming differentiation of EBs into the three germ layer cells. In contrast, expression of Oct4, Sox2, Klf4 and Sall4 was markedly decreased, consistent with lineage specific differentiation and a loss of pluripotency (data not shown). These data demonstrated that Sall4-iPS cells could differentiate into the three germ layers in vitro and is strongly indicative of an ES cell-like state.

[0223] Mouse Sall4-iPS Cells Are Able To Form Teratoma

[0224] For in vivo testing of pluripotency, we injected Sall4-iPS cells into the flanks of SCID/NOD mice to determine their pluripotent potential. When 5X10⁶ mouse Sall4-iPS cells were introduced into adult immunodeficient SCID/NOD mice through subcutaneous injection, the cells spontaneously formed teratoma-like masses containing ectodermal, mesodermal, and endodermal tissue types. The presence of three germ layers in teratomas derived from the injection of mouse Sall4-iPS cells indicates the pluripotent characteristics of the cells.

[0225] Sall4-iPS CeUs Are Able To Form Chimeric Mice

[0226] We next examined the ability of Sall4-iPS cells to contribute to chimeric mice. Sall4-iPS cells were derived from the C57BL/6 mouse, which has a black coat color. When these cells were injected into enucleated blastocysts of ICR mice (with a white coat) and implanted into the pseudo-pregnant uterus, chimeric mice were born. Chimeric mice were determined by the agouti coat color of the progeny. These data suggest that Sall4-iPS cells are competent iPS cells that can contribute to the full-term and adult development of chimeras. This data clearly demonstrates that we have derived iPS cells from the retroviral transduction of a single factor, Sall4, into somatic cells of mice.

[0227] High efficiency of somatic cell reprogramming by Sall4

[0228] Previous studies in nuclear transfer demonstrated that over-expression of Sall4 can enhance reprogramming by 15-590 fold. In this study, we investigated if somatic cell reprogramming by three defined factors, Oct4, Sox2 and Klf4 as established by Dr. Yamanaka's group, can be enhanced by the addition of Sall4. The number of ES-like colonies markedly increased by ~10 fold when Sall4 was added to the three factor reprogramming cocktail. Interestingly, when Sall4 was used as the only reprogramming factor, the number of ES-like colonies remarkably increased by —10-100 fold compared to that of three factors, depending on the amount of Sall4 virus used to initiate the reprogramming process.

[0229] In using human normal foreskin fibroblasts, the enhancement of the reprogramming was not as robust as the mouse counterpart with an increase of approximately 5 fold (data not shown) and this may be due to a species difference. Regardless of the lower number of ES-like colonies generated, the data demonstrated that SALL4 alone is sufficient to reprogramming for human somatic cells as well. [0230] Human iPS generation and characterization

[0231] We next sought to reprogram human somatic cells to iPS cells using viral transduction of SALL4. Using a lentivirus construct, we introduced the human SALL4 coding sequence into normal human foreskin fibroblasts. Six days after infection, the cells were harvested by collagenase IV and plated onto mitomycin C-treated mouse feeder cells and cultured in human ES medium. Lentiviral integration was verified by PCR and protein expression by Western blot in separate experiments (data not shown).

[0232] Approximately two weeks later (day 20), colonies of human ES-like cells appeared. The newly derived human SALL4-iPS cells demonstrated morphology similar to H9 ES cells and distinguishable from the parent foreskin fibroblasts. ES-alkaline phosphatase staining of human SALL4-iPS cells was also positive. The SALL4-iPS cells expressed well characterized pluripotency markers, OCT4, TRA-1-81, NANOG, SSEA-3 and TRA- 1-60 (Figure 4e). Consistent with the positive immunoflourescent staining of SALL4- iPS cells, the endogenous mRNA transcripts of NANOG and OCT4 were upregulated as shown by qRT-PCR. In each experiment the expression levels of the pluripotency markers and alkaline phosphatase was similar to the levels observed in the human ES cell line H9 but strikingly different than the parental FF cells from which the SALL4-iPS cells were derived.

[0233] To ensure that complete reprogramming had ensued following over-expression of endogenous SALL4, qRT-PCR analysis was performed on three individual human S ALL4- iPS clones using primers flanking the 3' untranslated region. Endogenous SALL4 expression was upregulated compared to the parent FF. Lentiviral SALL4 transcript was also analyzed and showed very minimal expression indicating silencing of exogenous SALL4 once the FF were completely reprogrammed. Similar pattern was observed as with mouse SALL4-iPS. Furthermore, chromosomal and flow cytometric analyses show normal diploid karyotype. These findings are very similar to that in mouse Sall4-iPS cells.

[0234] We next sought to determine if human S ALL4-iPS cells were pluripotent. To determine the differentiation ability of human iPS cells in vitro, we used a hanging drop method of cultivation to form EBs. Once the EBs were generated, the EBs were collected and transferred to gel-coated culture dishes for attachment. Following 16 days of spontaneous differentiation, the cells were tested by immunostaining for β-III-TUBULIN (ectoderm), DESMIN(mesoderm), and AFP (endoderm). Human SALL4-iPS cells all stained positive for the markers that define specific cell lineages of the germ layer origin.

[0235] We also injected 10⁶ human SALL4-iPS cells into the flanks of SCID/NOD mice to determine if the cells were able to form teratoma containing tissues of all three germ layers in vivo. Indeed, as shown in Figure 41, human SALL4-iPS cells generated teratoma consisting of all three embryonic germ layers. These data clearly suggest that viral introduction of SALL4 into the genomic sequence of human somatic cells is sufficient to reprogram somatic cells to an ES cell-like state.

[0236] Here we have shown that viral introduction of Sall4 into murine or human somatic cells is capable of reprogramming them to a pluripotent state. To ensure the accuracy of this finding, these experiments have been repeated at least 3 times independently by three different laboratory personnel. One of the most striking observations is the high efficiency of the retrodifferentiation process relative to that achieved using Oct4, Sox2 and Klf4. Addition of the Sall4 virus to the three factor reprogramming cocktail increases the efficiency approximately 10 fold, however, reprogramming with Sall4 virus alone is 10-100 fold more efficient than the combination of Oct4, Sox2 and Klf4 dependent on amount of Sall4 retrovirus used. Although the reason for this enhanced efficiency is unclear, Sall4 may modulate the activities of additional factors required for reprogramming more efficiently than the other transcription factors. Oct4, Sox2 or Klf4 also may interfere with the stimulatory effect of Sall4 by promoter competition at the viral transfection level or by other transcriptional regulatory processes.

[0237] Using a single transcription factor, Sall4, to reprogram somatic cells to pluripotent cells may change a fundamental paradigm in iPS cell biology. It has previously been thought that Oct4 was the dominant protein necessary for the reprogramming and subsequent stem cell pluripotency. However, we have shown that Sall4 regulates the transcriptional levels of Oct4, Sox2, Klf4, and c-Myc and this regulation likely contributes to the reprogramming mechanism. The use of a single reprogramming factor will also facilitate future uses of iPS cells. Small molecules, expression plasmids, non-integrating viral vectors, and homologous recombination techniques, can all bypass potential adverse genomic alterations associated with retro- and lentivirus vectors. Once genetically stable, iPS cell clones can be achieved robustly and reproducibly, future clinical applications of the technology may proceed quickly.

EXAMPLE 9 SUPPLEMENTAL MATERIAL AND METHODS

[0238] CeU Culture

[0239] Primary cultures of tail-tip fibroblasts (TTF) derived from C57BL/6 mice were maintained in growth medium containing High Glucose DMEM (Invitrogen, CA ), 10% certified FBS (Invitrogen, CA) and IX pen strep (Invitrogen, CA). 90% confluent flasks were passaged 1 :3. Cells within passage 3-4 were used for reprogramming. Plat-E retroviral packaging cells (Clontech, CA) were cultured in growth medium containing RPMI 1640 (Invitrogen, CA), 10% certified FBS (Invitrogen, CA) and IX pen strep (Invitrogen, CA). Cells within passage 5-10 were used for retrovirus production. W4 ES cells and mouse Sall4- iPS cells were cultured on gelatin-coated dishes bearing a mitomycin-C treated semi- confluent feeder layer within passage 3-4 and ES growth medium containing Knockout DMEM (Invitrogen, CA)₅ 15% ES-certified FBS, 2 mM L-Glutamine (Invitrogen, CA)₃ IxIO^"4 M nonessential amino acids (Invitrogen, CA), IxIO^"4 M 2-mercaptoethanol (Sigma, MO), IX pen strep (Invitrogen, CA) and 12.5 ng/ml LIF (Chemicon, CA). Growth medium was changed every day. Cells were passaged using 0.25% trypsin in PBS. 293FT cells were maintained in High Glucose DMEM, 10% certified FBS and IX pen strep. Cells less than 25 passages were used for lentiviral production. H9 ES cells and human SALL4-iPS cells were maintained in DMEM/F12 20% ES-qualified FBS, 2 mM L-Glutamine, IxIO^"4 M nonessential amino acids, 1 x 1O-* M 2-mercaptoethanol, IX pen strep and 4 ng/ml recombinant human basic fibroblast growth factor (Invitrogen, CA). For passaging, H9 and human SALL4-iPS cells were washed once with PBS and then incubated with 2 mg/ml collagenase IV (Invitrogen, CA) at 37⁰C for 5 minutes. An equal volume of IX PBS was added and the cells were transferred to a 15ml conical tube and pelleted by centrifugation. The solution was discarded and cells were resuspended in ES media for human SALL4-iPS and H9 ES cells. Cells were then transferred to a new gelatin-coated dish containing mitomycin-C treated semi-confluent feeder layer. The feeder cells were derived from a pool of day 13.5 embryos of CF-I mice were used for both mouse and human iPS cells as well as ES cells. These cells were maintained in High Glucose DMEM, 10% certified FBS and IX pen strep and were expanded up to passage 3. Confluent flasks were treated with 10 ug/ml mitomycin-C for 2.5 hrs. The cells were then washed 5 times with PBS and were collected by dissociation with 0.25% trypsin in PBS. Cells were then stored in freezing media containing 50% certified FBS, 40% High Glucose DMEM and 10% DMSO at -80^c until use.

[0240] Virus Production and iPS Generation

[0241] T75 flasks seeded with 90% confluent Plat-E retroviral packaging cell line (<Passage 5) were transfected with the retroviral construct pMXs-Sall4 (Addgene plasmid bank) using Lipofectamine 2000 transfection reagent (Invitrogen, CA) according to manufacturer's instructions. Briefly, 75ul of Lipofectamine 2000 reagent was incubated with 1.875ml of OptiMEM serum-free media (Invitrogen, CA) at room temperature for 5 minutes. After incubation, 30ug of purified pMXS-Sall4 DNA in 1.875ml of OptiMEM was added to the lipidrOptiMEM mixture and incubated for 20 minutes at room temperature. DNA:Lipid: OptiMEM mixture was added to the flask with packaging cells in 18.75ml growth media. Transfection was performed overnight in a 37⁰C humidified incubator with 5% CO₂. The media was removed and replaced with media containing pre-warmed High Glucose DMEM supplemented with 10% certified FBS, ImM sodium pyruvate and IX pen strep. An initial virus harvest was made 48 hr later (virus stock (VS) 1) and a second harvest was made 24 hr after the first (VS-2). Virus containing supernatants then were filtered through a 0.45um filter.

[0242] A 100mm culture dish containing 3X10⁵ tail-tip fibroblasts was infected with freshly prepared VS-I with virus encoding Sall4. 8ug/ml Polybrene (Chemicon, CA) was added to the medium to enhance infection efficiency. Infection was performed in a humidified incubator with 5% CO2 overnight at 37°C. Virus containing medium then was removed and replaced with fresh pre-warmed growth media. The fibroblasts were allowed to grow for an additional 8 hours then subjected to a second cycle of infection by the addition of fresh VS-2 medium. This infection was performed as described above for VS-I . Infected cells were allowed to grow for 7 days with medium being changed every other day. The pSSI-13772 GFP construct (Sierra Sciences LLC) was used both to test the efficiency of transfection on the Plat-E cell line and to prepare Sall4-iPS cells tagged with the cellular marker, GFP. Lipofectamine 2000 was used as the transfection reagent as previously described.

[0243] A lentivirus-SALL4 construct for human iPS production was prepared using Invitrogen's Gateway technology. Briefly, the SALL4 transcript cDNA was cloned into pENTR-3C, followed by LR clonase recombination (Invitrogen, CA) and cloned into the pDEST FG12 CMV cassette. Successful clones were confirmed by PCR and restriction enzyme digestion. 12ug of purified lenti virus SALL4 DNA, 4ug of pMD2G and lOug of psPAX2 (the two latter plasmids were from Addgene plasmid bank) were used to co-transfect the 293FT packaging cell line.

[0244] After 7 days, infected cells were transferred to a gelatin-coated dish bearing mitomycin-C treated semi-confluent feeder layer in ES medium. Cells were cultured for 14 additional days after which ES-like colonies (designated Sall4-iPS cells for its one factor derivation) were picked, and re-plated onto a new feeder layer in ES medium for expansion.

[0245] Characterization of Embryoid Bodies Formed by ES and iPS cells

[0246] Embryoid bodies were generated by a hanging drop method as previously described using ES cells (W4 and H9) and iPS cells of human and mouse origin. Briefly, confluent flasks of ES and iPS cells were trypsinized and the cells pelleted by centrifugation. Cells were re-suspended in 5ml of EB medium containing Knockout DMEM (Invitrogen), 15% ES-qualified FBS, 2 mM L-Glutamine, IxIO^-4 M nonessential amino acids, 1 xlO^"^ M 2- mercaptoethanol, IX pen strep and plated into a T25 flask. Cells were incubated for 45 minutes in a 37⁰C humidified incubator with 5% CO₂ in order to deplete feeder cells. Feeder depleted cells were then harvested and collected by centrifugation. A cell suspension with a cell density of 3.3xlO⁵ cells/50 ml was plated on 140 mm culture plates using an 8 channel automated pipettor to deposit 18 to 22 rows of drops of 15μl/drop onto each plate. The plates were inverted and cultured in a humidified incubator with 5% CO₂ for 2 days. After 2 days, 10 mis of PBS was added to each plate and the EBs were collected gently by swirling and re-plated into 100mm Petri dish in EB medium for an additional 4 days. EB medium was changed every other day. [0247] Differentiation of Embryoid Bodies (EBs) derived from Sall4-iPS Cells

[0248] To determine the differentiation ability of the EBs derived from Sall4-iPS cells, the day 6 EBs (2 day culture in hanging drops and 4 days in suspension culture maintained in EB medium) were transferred to gelatin-coated plates and allowed to attach for 10 days. The cells were maintained using EB medium and medium was changed every 2 days. For immunocytochemistry staining, 2-5 EBs on day 2 were plated in a gelatin coated 48-well plate and differentiated into the cells of the three germ layers for an additional 10 days. Markers used were the following: βlll-tubulin (a marker of ectoderm), desmin (a marker for mesoderm), a-fetoprotein (AFP, a marker for endoderm), nestin (a marker for ectoderm), and vimentin (markers of mesoderm and parietal endoderm). Secondary antibody controls were used. For qRT-PCR analysis, 10-15 EBs on day 2 were plated in gelatin coated 35mm culture dishes and differentiated into the cells of the three germ layers for 10 days in order to confirm that the differentiated EBs express the markers used for immunostaining.

[0249] Alkaline Phosphatase Staining of iPS cells

[0250] Alkaline phosphatase staining was performed using the ES-Alkaline Phosphatase detection kit (SCR004, Chemicon) according to manufacturer's instruction. Briefly, ES cells (W4 and H9) and iPS cells (both human and mouse origin) were plated on gelatin-coated dishes contianig mitomycin-C treated semi-confluent feeder layer at low cell density. After 5 days, cells were fixed with 4% paraformaldehyde for 2 minutes. Fixative solution was removed and the cells were washed once with TPBS (PBS with 0.1% Triton X-100) rinse buffer. Cells were incubated with 2:1:1 FRV:Naphthol:water solution for 15 minutes at room temperature protected from light. Cells were then washed once with TPBS rinse buffer, overlaid with TPBS buffer to prevent the cells from drying, and then visualized for AP staining by phase microscopy.

[0251] Immunofluorescence staining of iPS cells

[0252] Cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were then washed with PBS and blocked with 5% bovine serum albumin (BSA) in TPBS for 2 hour at room temperature. The cells were incubated with primary antibodies including mouse anti-Oct4 (1:150, Millipore, MA), mouse anti-Sox2 (1:100, Santa Cruz Biotechnology, CA), rabbit anti-Nanog (1:180 Santa Cruz Biotechnology, CA), rabbit anti- Sall4 (1 :400 developed 'in house')(75), rat anti- Stage-Specific Embryonic Antigen-3 (1 :25 Millipore, MA), mouse anti-TRA-1-60 (1:50, Millipore, MA), mouse anti- TRA-1-81 (1:50, Millipore, MA), rabbit anti-Desmin (1:20, Millipore, MA), rabbit anti-GFAP (1:500, Millipore, MA), mouse anti-β tubulin III (1:250 Millipore), mouse anti-Vimentin (1:250, Millipore, MA), mouse anti-Nestin (1:80 Millipore, MA), and goat anti-α fetoprotein (1:60 Santa Cruz Biotech, CA) overnight at 4⁰C in TPBS with 1%BSA. The cells were then washed 3 times with TPBS and incubated with appropriate fluorophore-labeled secondary antibodies: FITC labeled anti-mouse IgG (1:1000 Sigma, MO), FITC labeled anti-rabbit IgG (1:1000 Southern Biotech, Alabama), FITC labeled anti-goat IgG (1:1000 Southern Biotech, Alabama) and FITC labeled anti-rat IgG (1 : 1000 Southern Biotech, Alabama) for 1 hr in room temperature. The cells were then washed three times with TPBS and the nuclei were counterstained with lmg/ml 4',6-diamidino-2-phenylindole (DAPI) (Sigma, MO) for 10 minutes. Cells were washed three times with TPBS rinse buffer to reduce background fluorescence. Samples were analyzed on a Motic fluorescence microscope and images were acquired with a Q Imaging digital camera.

[0253] qRT-PCR

[0254] Total RNA from I X lO⁶ cells was extracted using Trizol reagent (Invitrogen) according to manufacturer's instructions. 1 ug of RNA was reversed transcribed using the High Capacity cDNA Archive kit (Applied Biosystems Inc.). The relative expression of various genes (listed in Tables 1 and 2) were quantified using a Power Sybr Green Master Mix (Applied Biosystems Inc.) according to manufacturer's instructions. All samples were normalized using GAPDH as the endogenous control. Human GAPDH control was purchased from Eurogentec. Data was acquired using a 7500 FAST Real Time PCR system (Applied Biosystems Inc, CA) and analyzed using the comparative C_T method via SDS FAST System software version 1.4.0. Table 3: qRT-PCR primers for mouse genes

Table 4: qRT-PCR primers for human genes

[0255] Bisulfite sequencing

[0256] Bisulfite treatment of DNA was performed with the EpiTect Bisulfite Kit (Qiagen, CA) according to manufacturer's instructions. Nanog promoter regions were amplified as previously described(iθ). Amplified PCR products were purified using gel filtration columns, cloned into the pGEM®-T Easy Vector (Promega, CA), and sequenced with Ml 3 forward and reverse primers.

[0257] Teratoma Formation

[0258] 5x10 iPS cells from day 20 clones (human and mouse origins) were re-suspended into 200ul of ES medium. An equal volume of Geltrex Reduced Growth Factor Basement Membrane Matrix (Invitrogen, CA) was added to the cell suspension and mixed by pipetting up and down. Cells were injected subcutaneously into each flank of 10 SCID/NOD mice. After 30 days, the teratomas were collected and embedded in paraffin. Sections of the tissue were then stained with hematoxylin and eosin.

[0259] Generation of Chimeric Mice

[0260] 10 clones from mouse Sall4-iPS cells cultured for 20 days in ES cell medium were picked up and made into a single cell suspension. Sall4-iPS cells were injected into blastocysts derived from ICR mice , which were then transplanted into the uteri of pseudo- pregnant ICR mice. Adult chimeras were obtained as determined by their coat color.

[0261] Karyotyping

[0262] For karyotyping analysis of iPS cells (human and mouse origins), 1 ug/ml colchicine (Sigma, MO) was added to each of the culture flasks and incubated for 2.5 hr. Treated cells were centrifuged and washed once with IX PBS. 10 ml of 75 mM KCl was added dropwise to 10ml tubes with gently mixing and incubated for 6 minutes at room temperature. Cells were then centrifuged and the supernatant was discarded. The hypotonically swollen cells were then fixed by dropwise addition of 10 ml of fixative solution containing a 1 :3 methanol: glacial acetic acid mixture and incubated for 20 minutes at room temperature. The cells were centrifuged and washed three times with fresh 10 ml fixative solution. The cells were centrifuged and a small residual solution was retained. Re-suspended cells were then deposited dropwise onto dry ice-chilled glass slides (optimum dropping height, 12 inches). The slides were removed from the dry ice bed and allowed to warm to room temperature. Slides were incubated in 7O⁰C water bath overnight and stained with DAPI for 15 min. Chromosome spreads were analyzed on a Olympus BX51 fluorescence microscope and images were acquired using Olympus DP70 camera.

[0263] DNA Ploidy

[0264] To evaluate DNA content of the iPS cells (human and mouse origins), 5 X 10⁵ cells were fixed and permeabilized using Fix & Perm Cell Permeabilization kit (Invitrogen, CA) following manufacturer's instructions. Briefly, about 5 X 10⁵ cells were fixed with lOOul of Reagent A for 15 minutes. The cells were washed once with IX PBS. lOOul Reagent B was added to permeabilized the cells and incubated with mouse anti-Oct4 (1:50 Millipore, CA) for 15 minutes. Cells were washed once with IX PBS. A small amount of residual PBS was retained and FITC-labeled secondary antibody was added (1:1000 Santa Cruz Biotechnology, CA). Cells were incubated for 15 minutes at room temperature in the dark, washed twice with IX PBS and propidium iodide (Invitrogen, CA) was then added according the manufacturer's instructions. The cell suspension was subjected to flow cytometry using an FC500 cytometer, gating on cells positive for Oct4 in order to eliminate any contaminating residual fibroblasts, and DNA content measurements, as determined by propidium iodide staining, were obtained. Data was analyzed using FlowJo software (Tree Star Inc., Ashland Oregon).

[0265] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

We claim:

1. An isolated pluripotent stem cell characterized as having an exogenously added SALL4 gene and being able to self renew in culture.

2. The cell of claim 1, further comprising at least one or more exogenously added genes selected from the group consisting of Oct4, Sox2, and KLF4.

3. The cell in claim 1, wherein the cell is generated from a fibroblast cell.

4. The cell in claim 1, wherein the cell is generated from a hepatic cell.

5. An isolated pluripotent stem cell that produces Factor VIII.

6. The cell of claim 5, wherein the cell is generated from a fibroblast cell.

7. The cell of claim 5, wherein the cell is generated from a hepatic cell.

8. The cell of claim 5, wherein the cell contains an exogenously added SALL4 gene.

9. The cell of claim 5, further comprising at least one or more exogenous genes selected from the group consisting of Oct4, Sox2, and KLF4.

10. A method for treating a subject diagnosed with type A hemophilia, comprising administering to the subject a cell of claim 5.

11. A cell of claim 1 , wherein the cell can be differentiated to a cell type selected from the group consisting of an osteoblast, chondrocyte, adipocyte, fibroblast, marrow stroma, skeletal muscle, smooth muscle, cardiac muscle, occular, endothelial, epithelial, hepatic, pancreatic, hematopoietic, glial, neuronal and an oligodendrocyte cell type.

12. The cell of claim 11 , wherein the hepatic cell expresses liver-specific markers selected from alpha fetoprotein, albumin, CYPlAl and HNF-4a or a combination thereof.

13. A method of treating a blood coagulation factor deficiency in a subject comprising: (a) providing a cell of claim 1 comprising a nucleic acid encoding a blood coagulation factor operably linked to an expression control element; and

(b) administering an amount of the cell wherein the blood coagulation factor is expressed at levels having a therapeutic effect on the subject such that the therapeutic effect is an increase in coagulation of blood.

14. The method of claim 13, wherein the blood coagulation factor is Factor VII, Factor VIII, Factor X, Factor XI, Factor XIII, or Protein C.

15. The method of claim 1 , wherein the cell is a human cell.

16. A method of reprogramming somatic cells comprising:

exposing a somatic cell of an individual with SALL4 under conditions sufficient to reprogram the cells; and

culturing the exposed cells to obtain reprogrammed cells.

17. The method of claim 16, wherein the somatic cells are obtained from a postnatal individual.

18. The method of claim 17, wherein the reprogrammed cells are substantially genetically identical to the post-natal individual.

19. The method of claim 16, wherein the somatic cells are obtained by in vitro differentiation of a stem cell.

20. The method of claim 16, wherein the exposing step includes introducing a vector encoding SALL4 into the somatic cells.

21. The method of claim 20, wherein the vector is a viral-based vector.

22. The method of claim 21 , wherein the viral-based vector is a retroviral vector.

23. The method of claim 22, wherein the retroviral vector is a lentiviral vector.

24. The method of claim 16, wherein SALL4 is introduced to the somatic cells as a reprogramming sequence in which a nucleic acid sequence encoding SALL4 is operably linked to a heterologous promoter.

25. The method of claim 16, further comprising exposing the cell to a nucleic acid sequence encoding a transcription factor selected from the group consisting of Oct-4, Sox2, Nanog and Lin28.

26. The method of claim 16, wherein the reprogrammed cells are pluripotent.

27. The method of claim 16, wherein the reprogrammed cells (i) express a cell marker selected from the group consisting of two ore more of OCT4, Nanog, SALL4 and SSEA-I; (ii) exhibit morphology characteristic of pluripotent cells; and (iii) form teratomas when introduced into an immunocompromised animal.

28. An enriched population of pluripotent cells produced according to the method of claim 16, wherein the population contains more than one cell.

29. The enriched population of cells as claimed in claim 28, wherein the pluripotent cells account for at least 60% of the population.

30. The enriched population of cells as claimed in claim 28, wherein the pluripotent cells account for at least 80% of the population.

31. The enriched population of cells as claimed in claim 28, wherein the pluripotent cells account for at least 95% of the population.

32. A cell culture comprising euploid pluripotent cells having a genome of a preexisting differentiated cell of an individual.

33. The cell culture of claim 32, wherein the indiviudal is a human.

34. The method of claim 16, wherein the method further comprises providing the cell to a subject for the repair or regeneration of a tissue or organ.

35. The method of claim 34, wherein the method increases function of the tissue or organ.

36. A cell of claim 1, wherein the reprogrammed cell expresses a cardiomyocyte specific gene selected from the group consisting of connexin43, Mef2C, Nkx2.5, GAT A4, cardiac troponin I, cardiac troponin T, and Tbx5.

37. A cell of claim 1, wherein the reprogrammed cell expresses an endothelial cell marker that is CD31 or FIk-I .

38. A cell of claim 1, wherein the cell expresses a neuronal marker selected from the group consisting of nestin and beta-tubulin.

39. A cell of claim 1, wherein the cell is an adipocyte positive for Oil red O or acetylated LDL uptake.

40. A cell of claim 1, wherein the cell has the morphology of a hematopoietic cell when stained with hematoxlin and eosin.

41. A method for repairing or regenerating a tissue in a subject, the method comprising

(a) obtaining a reprogrammed cell of claim 1 , and

(b) administering the cell to the subject.

42. The method of claim 40, wherein the administering provides a dose of cells sufficient to increase a biological function of the tissue or organ or circulating cell population of the subject.

43. The method of claim 40, wherein the administering provides a dose of cells sufficient to ameliorate or stabilize a disease, disorder, or condition of the subject.

44. The method of claim 40, wherein the subject has a disease selected from the group consisting of myocardial infarction, congestive heart failure, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, cirrhosis, Parkinson's disease, Alzheimer's disease, diabetes, cancer, arthritis, immunodeficiency and wound healing.

45. A tissue comprising a reprogrammed cell of claim 1.

46. A pharmaceutical composition comprising an effective amount of a cell of claim 1 in a pharmaceutically acceptable excipient for administration to a subject.

47. A kit for tissue repair or regeneration comprising a reprogrammed cell obtained by the method of claim 1, and instructions for use of the cell in methods of tissue repair or regeneration.