WO2010028019A2

WO2010028019A2 - Direct reprogramming of somatic cells using non-integrating vectors

Info

Publication number: WO2010028019A2
Application number: PCT/US2009/055698
Authority: WO
Inventors: Konrad Hochedlinger; Matthias Stadtfeld; Nimet Maherali
Original assignee: The General Hospital Corporation; President And Fellows Of Harvard College
Priority date: 2008-09-03
Filing date: 2009-09-02
Publication date: 2010-03-11
Also published as: WO2010028019A3

Abstract

Provided herein are methods for producing induced pluripotent stem cells with the use of a non-integrating vector. Reprogramming of cells using a non-integrating vector (e.g., adenoviral vector) permits the generation of induced pluripotent stem cells that do not contain leftover viral genes that can be reactivated in the cell. This type of transient reprogramming is further contemplated for production of induced pluripotent stem cells for delivery to an individual.

Description

DIRECT REPROGRAMMING OF SOMATIC CELLS USING NON-INTEGRATING VECTORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of the U.S. Provisional Application No. 61/093,850, filed September 3, 2008, the content of which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with Government support under Grant No. 1DP2OD003266-01 awarded by The National Institutes of Health (N1H). The Government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates to the production of induced pluripotent stem cells.

BACKGROUND

[0004] Reprogramming of cells by nuclear transfer (Wakayama et al., 1998; Wilmut et al., 1997) and cell fusion (Cowan et al., 2005; Tada et al., 2001) allows for the re-establishment of a pluripotent state in a somatic nucleus (Hochedlinger and Jaenisch, 2006). While the molecular mechanisms of nuclear reprogramming remain elusive, cell fusion experiments have implied that reprogramming factors can be identified in ES cells and be used to directly induce reprogramming in somatic cells. Indeed, a rational approach recently led to the identification of four transcription factors whose expression enabled the induction of a pluripotent state in adult fibroblasts (Takahashi and Yamanaka, 2006). Yamanaka and colleagues demonstrated that retroviral expression of the transcription factors Oct4, Sox2, c- Myc, and Klf4, combined with genetic selection for Fbxl5 expression, gives rise to induced pluripotent stem cells (iPS cells) directly from fibroblast cultures. Fbxl5-selected iPS cells contributed to diverse tissues in mid-gestation embryos, however, these embryos succumbed at mid-gestation, indicating a restricted developmental potential of iPS cells compared with ES cells.

[0005] Induced pluripotent stem cells have been generated from multiple cell types in both human and mouse cells by viral expression of Oct4 and Sox2, combined with either Klf4 and c-Myc (Takahashi, K., et al, (2007) Cell 131:861-872; Takahashi, K., et al (2003) Nature 423:5 41-5; Wernig, M., et al (2007) Nature 448:318-24; Okita, K., et al (2007) Nature 448:313-317; Maherali, N., et al., (2007) Cell Stem Cell 1:55-70; Park, I.H., et al (2008) Nature 451:141-146; Lowry, WE., et al., (2008) Proc Natl Acad Sci USA 105:2883-8) or LIN28 and Nanog (Yu, J., et al., (2007) Science 318:1917-1920). iPS cells are molecularly and functionally highly similar to ES cells, which makes in vitro reprogramming an attractive approach to produce patient- specific stem cells for studying and treating degenerative disease. Indeed, reprogrammed skin cells have recently been shown to alleviate the symptoms of Parkinson's disease (Wernig. M., et al., (2008) Proc Natl Acad Sci USA 105:5856-61) and sickle cell anemia (Hanna, J., et al., (2007) Science 318:1920-3) in mouse models. However, a major limitation of this technology is the use of integrating viruses, which permanently alter the genome and are associated with the risk of tumor formation due to the spontaneous reactivation of the viral transgenes (Okita, K., (2007) et al, supra). The low efficiency of reprogramming (0.01-0.1%) also raised the possibility that insertional mutagenesis may be a prerequisite for in vitro reprogramming (Hawley, R. G., (2008) MoI Ther 16:1354-5). Previous experiments showed that retroviral tagging of explanted hematopoietic stem cells selects for clones in which the retroviral construct had inserted proximal to self -renewal genes thus causing their activation (Kustikova, O., et al., (2005) Science 308:1171-4). While the sequencing of a limited number of insertion sites in iPS cells did not reveal common targets (Aoi, T., et al (2008) Science (express, 10.1126/science.1154884)), this possibility has not been unequivocally ruled out (Hawley, R.G. (2008), supra).

SUMMARY OF THE INVENTION

[0006] Induced pluripotent stem cells are a type of pluripotent stem cell artificially derived from a somatic cell by providing for the expression of stem cell-associated genes. iPS cells are generally derived by viral delivery of stem cell-associated genes into adult somatic cells (e.g., fibroblasts). Typically the production of iPS cells has been performed by introducing a nucleic acid sequence using a genome-integrating vector (e.g., retroviral vector or lentiviral vector). These vectors stably and randomly integrate into the genome and can induce DNA mutations. Furthermore, reactivation of viral transgenes at a later timepoint can be tumorigenic. For these reasons, iPS cells that were made with integrating vectors (or differentiated progeny of the iPS cells) cannot be administered safely to a subject. Described herein are methods for producing iPS cells that utilize non-integrating vectors, allowing the production of iPS cells that are devoid of viral integration remnants and are therefore suitable for administration to a subject. [0007] One aspect described herein is a method for producing an induced pluripotent stem cell from a somatic cell, the method comprising: (a) contacting a somatic cell with a non- integrating viral vector comprising a nucleic acid sequence encoding at least one reprogramming factor; and

(b) optionally isolating a reprogrammed cell of step (a).

[0008] In one embodiment of this aspect and all other aspects described herein, the somatic cell is a human cell.

[0009] In another embodiment of this aspect and all other aspects described herein, the somatic cell is a fibroblast.

[0010] In another embodiment of this aspect and all other aspects described herein, the somatic cell is an hepatocyte.

[0011] In another embodiment of this aspect and all other aspects described herein, the reprogramming factor is selected from the group consisting of Oct4, Sox2, c-Myc and Klf4. In an alternate embodiment, each of Oct4, Sox2, c-Myc and Klf4 are introduced to a cell using one or more non-integrating vectors.

[0012] In another embodiment of this aspect and all other aspects described herein, the non- integrating viral vector comprises an adenoviral vector.

[0013] In another embodiment of this aspect and all other aspects described herein, the method further comprises the step of passaging the cells.

[0014] In another embodiment of this aspect and all other aspects described herein, the induced pluripotent stem cell is substantially free from viral integration remnants. [0015] In another embodiment of this aspect and all other aspects described herein, production of the induced pluripotent stem cell is evidenced by detection of a stem cell marker and/or characteristic ES morphology and growth kinetics in cell culture (i.e., resemble ES cells).

[0016] In another embodiment of this aspect and all other aspects described herein, the stem cell marker is selected from the group consisting of SSEAl, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl,Oct4, SOX2, and Natl. [0017] Another aspect described herein relates to a cell composition produced by the method described above.

[0018] Also contemplated herein is the use of a cell composition produced by the methods described above for the treatment of a disease or a disorder.

Definitions [0019] The term "pluripotent" as used herein refers to a cell with the capacity, under different conditions, to differentiate to more than one differentiated cell type, and preferably to differentiate to cell types characteristic of all three germ cell layers. Pluripotent cells are characterized primarily by the ability to differentiate to more than one cell type, preferably to all three germ layers, as assayed using, for example, a nude mouse teratoma formation assay. Pluripotency is also evidenced by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is the demonstration of the capacity to differentiate into cells of each of the three germ layers.

[0020] The term "re-programming" as used herein refers to the process of altering the differentiated state of a terminally-differentiated somatic cell, multipotent cell or progenitor cell to a pluripotent phenotype. A "re-programming factor" as that term is used herein refers to any factor or combination of factors that promotes the re-programming of a somatic cell and can include, for example at least one nucleic acid sequence encoding a transcription factor (e.g., c-Myc, Oct4, Sox2 and/or KIf 4).

[0021] By "differentiated primary cell" or "somatic cell" is meant any primary cell that is not, in its native form, pluripotent as that term is defined herein. The term "somatic cell" also encompasses progenitor cells that are multipotent (e.g., produce more than one cell type) but not pluripotent (e.g., can produce cells from all three germ layers). It should be noted that placing many primary cells in culture can lead to some loss of fully differentiated characteristics. However, simply culturing such cells does not, on its own, render them pluripotent. The transition to pluripotency requires a re-programming stimulus beyond the stimuli that lead to partial loss of differentiated character in culture. Re-programmed pluripotent cells (also referred to herein as "induced pluripotent stem cells") are also characterized by the capacity for extended passaging without loss of growth potential, relative to primary cell parents, which generally have capacity for only a limited number of divisions in culture.

[0022] The term " vector " refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell. An "expression vector" is a specialized vector that contains the necessary regulatory regions needed for expression of a gene of interest in a host cell. In some embodiments the gene of interest is operably linked to another sequence in the vector. It is preferred that the viral vectors used herein are replication defective, which can be achieved for example by removing all viral nucleic acids that encode for replication. A replication defective viral vector will still retain its infective properties and enters the cells in a similar manner as a replicating adenoviral vector, however once admitted to the cell a replication defective viral vector does not reproduce or multiply. The term "operably linked" means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector.

[0023] As used herein, the term "non-integrating viral vector" refers to a viral vector that does not integrate into the host genome; the expression of the gene delivered by the viral vector is temporary. Since there is little to no integration into the host genome, non- integrating viral vectors have the advantage of not producing DNA mutations by inserting at a random point in the genome. For example, a non-integrating viral vector remains extra- chromosomal and does not insert its genes into the host genome, potentially disrupting the expression of endogenous genes. Non-integrating viral vectors can include, but are not limited to, the following: adenovirus, alphavirus, picornavirus, and vaccinia virus. These viral vectors are "non-integrating" viral vectors as the term is used herein, despite the possibility that any of them may, in some rare circumstances, integrate viral nucleic acid into a host cell's genome. What is critical is that the viral vectors used in the methods described herein do not, as a rule or as a primary part of their life cycle under the conditions employed, integrate their nucleic acid into a host cell's genome. It goes without saying that an iPS cell generated by a non-integrating viral vector will not be administered to a subject unless it and its progeny are free from viral remnants.

[0024] As used herein, the term "viral remnants" refers to any viral protein or nucleic acid sequence introduced using a viral vector. Generally, integrating viral vectors will incorporate their sequence into the genome; such sequences are referred to herein as a "viral integration remnant". However, the temporary nature of a non-integrating virus means that the expression, and presence of, the virus is temporary and is not passed to daughter cells. Thus, upon passaging of a re-programmed cell the viral remnants of the non-integrating virus are essentially removed.

[0025] As used herein, the term "free of viral integration remnants" and "substantially free of viral integration remnants" refers to iPS cells that do not have detectable levels of an integrated adenoviral genome or an adenoviral specific protein product (i.e., a product other than the gene of interest), as assayed by PCR or immunoassay. Thus, the iPS cells that are free (or substantially free) of viral remnants have been cultured for a sufficient period of time that transient expression of the adenoviral vector leaves the cells substantially free of viral remnants.

[0026] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0027] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0028] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the production of induced pluripotent stem cells using an adenoviral vector. Figure IA shows a primary ES-like colony that appeared in cells treated with adenoviruses expressing c-Myc, Klf4, Oct4, and Sox2; Figure IB shows a stable iPS colony after continuous passaging of the initial colony shown in Figure IA.

Figure 2 shows an analysis of pluripotency markers in Adeno-iPS cells. Figure 2A shows brightfield (upper panel) and fluorescence (lower panel) images of an Adeno-iPS cell clone established from Sox2-GFP fetal liver cells taken at passage 0 (PO) and passage 2 (P2). Figure 2B Expression of endogenous c-myc, Klf4, Oct4, Sox2 and nanog measured by qPCR in Adeno-iPS cells derived from fetal liver (FL), fibroblasts (TTF) and hepatocytes (HEP) as well as in V6.5 control ES cells. Figure 2C Bisulfite sequencing of the Oct4 and Nanog promotors in hepatocytes, ES cells and iPS cells derived from hepatocytes. Open circles represent unmethylated CpGs; closed circles denote methylated CpGs. Figure 2D Expression levels of endogenous GAPDH (G) as well as adenoviral c-myc (M), Klf4 (K), Oct4 (O) and Sox2 (S) in fibroblasts three days after infection with adenoviruses (TTF + 4 adenos), ES cells and Adeno-iPS cells derived from fetal liver, fibroblasts and hepatocytes.

Figure 3 shows absence of viral integration in Adeno-iPS cells. Figure3A shows a schematic drawing of the adenoviral vector indicating the position of the cDNA and the sizes of the respective DNA fragments after BamHI digestion. A pBluescript (pBS) sequence present in both the adenoviral vector and the Oct4IND transgene is highlighted. Figure 3B shows a PCR analysis for adenoviral integration in genomic DNA from the indicated AdenoiPS clones as well as from V6.5 ES cells (-). An arrowhead indicates the position of the positive control band amplified from vector DNA (+). Figure 3C shows a Southern blot analysis of BamHI- digested genomic DNA using DNA fragments constituting the entire adenoviral vector backbone as probes. Plasmid DNA of pAd-Sox2 diluted to the equivalent of 0.2, lor 5 integrations per genome and genomic DNA of HEK cells (which contains an adenoviral integration) were used as positive controls. The asterisk indicates the position of a 3kb band resulting from hybridization of the pBS sequence in the adenoviral probe to transgenic sequences in the Oct4IND allele. Solid arrowheads indicate the position of BamHI fragments of the adenoviral vector and open arrowheads highlight adenoviral sequences present in HEK cells.

Figure 4 shows an example of pluripotency of Adeno-iPS cells. Figure 4A-4C show images of teratomas produced from Adeno-iPS cells containing keratinized epithelium (Figure 4A), mucous epithelium (Figure 4B) and cartilage (Figure 4C). Figures 4D-4I depict fluorescence images showing the contribution of fluorescent protein-labeled Adeno-iPS cells to lung, brain and heart in a postnatal chimeric animal. Nuclei were counterstained with DAPI. The small insets in Figures 4D, 4F, and 4H highlight the fields magnified in Figures 4E, 4G, 41 while the insets in Figures 4E, 4G, 41 show the background fluorescent levels and DAPI staining of corresponding tissues in a non-chimeric littermate. Figures 4 J, and 4K show images of coat- color chimeras derived from fetal liver (Figure 4J) and hepatocytes (Figure 4K) Adeno-iPS cells. Figures 4L-4O show fluorescence and brightfield images of a wild type (Figures 4L, 4M) blastocyst and an Oct4-GFP (Figures 4N, 40) blastocyst obtained after mating a chimera mouse expressing GFP from the Oct4 promoter with a wild type female.

Figure 5 shows the structure of an exemplary adenoviral vector. Schematic of the basic features of the adenoviral vector showing the viral Inverted Terminal Repeats (FTR) as well as the E3 deletion. The viral El region was replaced with a transgenic expression cassette consisting of a cytomegalovirus (CMV) promoter, the intervening sequence (IVS) as well as the polyadenylation signal (polyA) of the human beta globin gene. The position were the cDNA of the individual reprogramming factors was integrated is shown. Small arrows indicate the location of PCR primers used to test for adenoviral integration into the genome. Figure 6 shows exemplary results of adenoviral infection efficiency in different cell types. Figures 6A, and 6B show images of fetal liver cells (Figure 6A) and tail-tip fibroblasts (Figures 6B) infected with adenoviruses expressing c-Myc, Klf4 and Sox2 either co-stained with antibodies against c-Myc and Sox2 or with an antibody against Klf4. Figures 6C, and 6D show immunofluorescence labeling of hepatocytes infected with four adenoviruses and stained for either c-Myc and Klf4 (Figure 6C) or Sox2 and Oct4 expression (Figure 6D). Nuclei of cells shown in Figures 6A-6D were counterstained with DAPI. Note that the majority of cells that got infected expressed more than one viral gene. Figure 6E shows a table summarizing the infection efficiency for the different cell types as well as the percentage of cells expressing all four transcription factors (TF), estimated based on the frequency of double- infected cells. In cells derived from Oct4IND mice, a high percentage (-90%) of cells has been shown to express the transgene (Maherali, N., et al., (2007), supra).

Figure 7 shows promoter methylation of exemplary Adeno-iPS cells. Bisulfite sequencing of the Oct4 and Nanog promotors in tail-tip fibroblasts, V6.5 ES cells and iPS cells derived from fetal liver (FL) or fibroblasts (TFF). Open circles represent unmethylated CpGs; closed circles denote methylated CpGs.

Figure 8 shows an exemplary Southern blot analysis using cDNA fragments of the four reprogramming factors as probes.

Plasmid DNA diluted to the equivalent of 0.2, 1 or 5 integrations per genome was used as positive controls (the position of the respective bands are indicated by closed arrowheads). Endogenous genes and pseudogenes also recognized by the cDNA probes are highlighted by open arrowheads. An asterisk in the Oct4 blot indicates the position of a band corresponding to the Oct4 cDNA integrated in the CoIlA locus in Oct4IND cells.

Figure 9 shows an exemplary timeline of adenoviral reprogramming experiments. Shown are experimental timelines for the derivation of Adeno-iPS cells from fetal liver (Figure 9A), postnatal fibroblasts (Figure 9B) and adult hepatocytes (Figure 9C). The images to the left show the respective starting populations at the time of adenoviral infection. To the right, an image of an established Adeno-iPS cell line is shown. Arrows highlight experimental manipulations of the cells at the indicated days (Figure 9D).

Figure 10 shows exemplary kinetics of adenoviral gene expression. Diagrams showing the decrease in expression of adenoviral myc, Klf4 and Oct4 in infected wildtype tail-tip fibroblasts as measured by qPCR. Expression 1.5 days after infection was set to 100%. The cells were kept sub-confluent to allow dilution of the adenoviral vectors by continuous cell divisions. Expression of adenoviral Sox2 was not measured for technical reasons.

Figure 11 shows the ploidy of exemplary Adeno-iPS cells. FACS diagrams (left panel) showing PI labeling of diploid (Figure HA) and tetraploid (Figure HB) Adeno-iPS cells. Note that tetraploid iPS cells also have increased forward (FSC) and side (SSC) scatter values (right panel).

DETAILED DESCRIPTION

[0029] Described herein are methods for producing induced pluripotent stem cells with the use of a non-integrating vector. The methods described herein are advantageous over previous reprogramming methods that utilize retroviral vectors for the delivery of stem cell genes, because retroviral vectors can disrupt the host genome and increase susceptibility of the cells to tumor formation. Reprogramming of cells using a non-integrating vector (e.g., adenoviral vector) permits the generation of induced pluripotent stem cells that do not contain leftover viral genes that can be reactivated in the cell. This type of transient reprogramming is contemplated for production of induced pluripotent stem cell for delivery to an individual.

Cells

[0030] While fibroblasts are preferred, essentially any somatic cell type can be used. Some non-limiting examples of cells include, but are not limited to, epithelial, endothelial, neuronal, adipose, cardiac, skeletal muscle, immune cells, hepatic, splenic, lung, circulating blood cells, gastrointestinal, renal, bone marrow, progenitor cells, and pancreatic cells. The cell can be isolated from any somatic tissue including, but not limited to brain, liver, lung, gut, stomach, intestine, fat, muscle, uterus, skin, spleen, endocrine organ, bone, etc. [0031] Where the cell is maintained under in vitro conditions, conventional tissue culture conditions and methods can be used, and are known to those of skill in the art. Isolation and culture methods for various cells are well within the abilities of one skilled in the art. [0032] Further, the parental cell can be from any mammalian species, with non-limiting examples including a murine, bovine, simian, porcine, equine, ovine, or human cell. For clarity and simplicity, the description of the methods herein refers to fibroblasts as the parental cells, but it should be understood that all of the methods described herein can be readily applied to other parent cell types. In one embodiment, the somatic cell is derived from a human individual.

[0033] Where a fibroblast is used, the fibroblast is flattened and irregularly shaped prior to the re-programming, and does not express Nanog mRNA. The starting fibroblast will preferably not express other embryonic stem cell markers. The expression of ES-cell markers can be measured, for example, by RT-PCR. Alternatively, measurement can be by, for example, immunofluorescence or other immunological detection approach that detects the presence of polypeptides that are characteristic of the ES phenotype.

Non-integrating viral vectors

[0034] While retroviral vectors incorporate into the host cell genome and can potentially disrupt normal gene function, non-integrating vectors have the advantage of controlling expression of a gene product by extra-chromosomal transcription. It follows that since non- integrating vectors do not become part of the host genome, non-integrating vectors tend to express a nucleic acid transiently in a cell population. This is due in part to the fact that the non-integrating vectors as used herein are rendered replication deficient. Thus, non- integrating vectors have several advantages over retroviral vectors including but not limited to: (1) no disruption of the host genome, and (2) transient expression, and (3) no remaining viral integration products.

[0035] Some non-limiting examples of non-integrating vectors include adenovirus, baculo virus, alphavirus, picornavirus, and vaccinia virus. In one embodiment, the non- integrating viral vector is an adenovirus. The advantages of non-integrating viral vectors further include the ability to produce them in high titers, their stability in vivo, and their efficient infection of host cells.

[0036] While it is known that some non-integrating vectors integrate into the host genome at extremely low frequencies (i.e., 10^~4 to 10^~5), a non-integrating vector, as the term is used herein, refers to vectors having a frequency of integration of less than 0.1% of the total number of infected cells; preferably the frequency of integration is less than 0.01%, less than 0.001%, less than 0.0001%, or less than 0.000001% (or lower) of the total number of infected cells. In one embodiment, the vector does not integrate at all. In another embodiment, the viral integration remnants of the virus are below the detection threshold as assayed by PCR (for nucleic acid detection) or immunoassay (for protein detection). In general, iPS cells produced by the methods described herein should be assayed for an integration event by the viral vector using, for example, PCR-mediated detection of the viral genome prior to administering the iPS cells to a subject. Any iPS cells with detectable integration products should not be administered to a subject.

[0037] The viral titer necessary to achieve a desired (i.e., effective) level of gene expression in a host cell is dependent on many factors, including, for example, the cell type, gene product, culture conditions, co-infection with other viral vectors, and co-treatment with other agents, among others. It is well within the abilities of one skilled in the art to test a range of titers for each virus or combination of viruses by detecting the expression levels of either (a) a marker expression product, or (b) a test gene product. Detection of protein expression in cells can be achieved by several techniques including Western blot analysis, immuno- cytochemistry, and fluorescence-mediated detection, among others. It is contemplated that experiments are first optimized by testing a variety of titer ranges for each cell type under the desired culture conditions. Once an optimal titer of a virus or a cocktail of viruses is determined, then that protocol will be used to induce the reprogramming of somatic cells. [0038] In addition to viral titers, it is also important that the infection and induction times are appropriate with respect to different cells. For example, as discussed in the Examples section herein, initial attempts with an adenoviral vector were deemed unsuccessful due to an inadequate induction time. Upon recognition of this important consideration and considerable lengthening of induction time, induced pluripotent stem cells were produced using an adenoviral vector. With the knowledge provided herein that length of time is an important variable in induced pluripotent stem cell induction, one of skill in the art can test a variety of time points for infection or induction using a non-integrating vector and recover induced pluripotent stem cells from a given somatic cell type.

Reprogramming

[0039] The production of iPS cells is generally achieved by the introduction of nucleic acid sequences encoding stem cell-associated genes into an adult, somatic cell. In general, these nucleic acids have been introduced using retroviral vectors, and expression of the gene products results in cells that are morphologically and biochemically similar to pluripotent stem cells (e.g., embryonic stem cells). For the purposes of this application, the nucleic acid sequences are delivered using a non-integrating viral vector, such as an adenoviral vector. This process of altering a cell phenotype from a somatic cell phenotype to a stem cell-like phenotype is termed "reprogramming".

[0040] Reprogramming can be achieved by introducing a combination of stem cell-associated genes including, for example Oct3/4 (Pouf51), Soxl, Sox2, Sox3, Sox 15, Sox 18, NANOG, KIf 1, Klf2, Klf4, Klf5, c-Myc, 1-Myc, n-Myc and LIN28. In general, successful reprogramming is accomplished by introducing a vector encoding Oct-3/4, a member of the Sox family, a member of the KIf family, and a member of the Myc family to a somatic cell. [0041] In one embodiment of the methods described herein, reprogramming is achieved by delivery of Oct-4, Sox2, c-Myc, and Klf4 constructs to a somatic cell (e.g., fibroblast). In one embodiment, the nucleic acid sequences of Oct-4, Sox2, c-MYC, and Klf4 are delivered using a non-integrating viral vector, such as an adenoviral vector.

[0042] In one embodiment, reprogramming is achieved by introducing more than one non- integrating vector (e.g., 2, 3, 4, or more vectors) to a cell, wherein each vector comprises a nucleic sequence for a different reprogramming factor (e.g., Oct2, Sox2, c-Myc, Klf4, etc). In an alternate embodiment, more than one reprogramming factor is encoded on a non- integrating vector and expression of the reprogramming factors can be controlled using a single promoter, polycistronic promoters, or multiple promoters.

[0043] The use of small molecules to enhance reprogramming is also contemplated for use with the methods described herein. For example, Wnt pathway inhibitors have been shown to enhance cellular reprogramming.

Confirming pluripotency and cell reprogramming

[0044] To confirm the induction of pluripotent stem cells, isolated clones can be tested for the expression of a stem cell marker. Such expression identifies the cells as induced pluripotent stem cells. Stem cell markers can be selected from the non-limiting group including SSEAl, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl. Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides.

[0045] The pluripotent stem cell character of the isolated cells can be confirmed by any of a number of tests evaluating the expression of ES markers and the ability to differentiate to cells of each of the three germ layers. As one example, teratoma formation in nude mice can be used to evaluate the pluripotent character of the isolated clones. The cells are introduced to nude mice and histology is performed on a tumor arising from the cells. The growth of a tumor comprising cells from all three germ layers further indicates that the cells are pluripotent stem cells.

[0046] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus for example, references to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0047] It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

The present invention may be as defined in any one of the following numbered paragraphs.

1. A method for producing an induced pluripotent stem cell from a somatic cell, the method comprising:

(a) contacting a somatic cell with a non-integrating viral vector comprising a nucleic acid sequence encoding at least one reprogramming factor; and

(b) isolating a reprogrammed cell of step (a).

2. The method of paragraph 1, wherein the somatic cell is a human cell.

3. The method of paragraph 1 or 2, wherein the somatic cell is a fibroblast.

4. The method of paragraph 1 or 2, wherein the somatic cell is a hepatocyte.

5. The method of paragraph 1, wherein the reprogramming factor is selected from the group consisting of Oct4, Sox2, c-Myc and Klf4. 6. The method of paragraph 1, wherein the non-integrating viral vector comprises an adenoviral vector.

7. The method of paragraph 1, further comprising the step of passaging the cells.

8. The method of paragraph 1, wherein the induced pluripotent stem cell is free from viral integration remnants.

9. The method of paragraph 1, wherein production of the induced pluripotent stem cell is evidenced by detection of a stem cell marker.

10. The method of paragraph 9, wherein the stem cell marker is selected from the group consisting of SSEAl, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, Oct4, SOX2, and Natl.

11. An induced pluripotent stem cell produced by the method of paragraph 1.

12. Use of a non-integrating vector comprising a nucleic acid sequence encoding at least one reprogramming factor for producing an induced pluripotent stem cell from a somatic cell.

13. The use of paragraph 12, wherein the non-integrating vector is an adenoviral vector.

14. The use of paragraph 12, wherein the somatic cell is a human cell.

15. The use of paragraph 12, wherein the somatic cell is a fibroblast or a hepatocyte.

16. The use of paragraph 12, wherein the reprogramming factor is selected from the group consisting of Oct4, Sox2, c-Myc, and Klf4.

17. Use of a cell produced by the method of paragraph 1 for the treatment of a disease or disorder.

EXAMPLES Example 1: Results

[0048] The production of iPS cells from mouse somatic cells using an adenoviral vector, which permits transient, high-level expression of exogenous genes without integrating into the host genome, was attempted. Specifically, cDNAs for Oct4, Sox2, c-Myc and Klf4 were cloned into replication-incompetent adenoviral vectors deleted for El and E3 under the control of the hCMV IE promoter (Figure 5). Initial attempts to reprogram tail-tip fibroblasts (TTFs) into iPS cells with adenoviruses expressing the four reprogramming factors were unsuccessful, possibly owing to the rapid dilution of the virally encoded proteins from the cells. Alternatively, it is noted that the time of induction of iPS cells using an adenoviral vector is considerably longer than the induction time when using a retroviral vector. This was not recognized in early experiments, thus it was originally thought that adenoviral vectors could not be used for the induction of iPS cells. For example, iPS cells begin to be visible at approximately 2 weeks using a retroviral vector, however at 2 weeks of induction there are no visible iPS cells when using an adenoviral vector. The appearance of iPS cells did not occur until 4-6 weeks after initial infection with an adenoviral vector. Primary experiments with the adenoviral vector were therefore determined to be unsuccessful when observed at the 2 week time point.

[0049] It has been previously shown that viral encoded Oct4 expression can be substituted by a doxycycline inducible Oct4 allele (Oct4IND) (Maherali, N., et al (2007), supra) and it has been reported that endodermal derivatives such as liver cells require lower numbers of viral integrations than fibroblasts to be reprogrammed (Aoi, T., et al (2008), supra). It was thus reasoned that fetal liver cells carrying the inducible Oct4 transgene might be more amenable to adenoviral reprogramming. Therefore approximately 500,000 adherent Oct4IND fetal liver cells were infected with adenoviruses expressing Sox2, Klf4 and c-Myc at multiplicities of infection (MOI, = number of virus per cell) of 20-50. This led to an infection efficiency of approximately 40-50% of cells with each factor and an estimated infection efficiency of 20- 30% of cells expressing all three adenoviral transgenes (Figure 6A,6E). After culture of infected fetal liver cells in the presence of doxycycline, a total of nine iPS-like colonies emerged after 24-30 days, which expressed the pluripotency markers Sox2 and SSEA-I and could be expanded into stable ES cell-like lines, similar to iPS cells produced with retro- or lentiviral vectors (Figure 2A and data not shown). These adenoviral iPS (Adeno-iPS) cells continued to grow in the absence of doxycycline, indicating that transgenic Oct4 expression is no longer required.

[0050] Further experiments were designed to test if postnatal tail fibroblasts carrying the Oct4-inducible allele were equally amenable to reprogramming into Adeno-iPS cells. Fibroblasts required MOIs of 50-250 to achieve an infection efficiency of -30% for each vector, resulting in an estimated 10-20% infection efficiency for triple-infected cells (Figure 6B, 6E). Infection of more than 1,000,000 neonatal Oct4IND TTFs harboring an Oct4-GFP reporter with adenoviral myc, Klf4 and Sox2 vectors in the presence of doxycycline gave rise to a single GFP+ colony, that grew into a stable, doxycycline independent line. [0051] To explore adult cell types that do not require transgenic Oct4 expression, hepatocytes were chosen. Hepatocytes are highly permissive for adenoviral infection (Li, Q., et al., (1993) Hum Gene Ther 4:403-9; Yamada, S., et al., (2006) Endocr J 53:789-95). Indeed, MOIs of 1- 4 were sufficient to infect 70-80% of these cells with individual vectors, with an estimated 50-60% of cells expressing all four viral reprogramming factors (Figure 6C, 6D, 6E). After incubation of approximately 500,000 adult hepatocytes isolated from mice lacking the Oct4- inducible allele with adenoviruses expressing c-Myc, Klf4, Oct4 and Sox2, three colonies were obtained, all of which could be expanded into stable ES-like cell lines expressing the pluripotency markers Oct-4 and SSEAl. This demonstrates that iPS-like cells can be produced from adult cells by adenoviral vectors in the absence of an endogenously integrated transgene.

[0052] Next, it was tested whether Adeno-iPS cells had re-established pluripotency at the molecular level by examining the activity of ES cell-specific markers. Expression analysis for the endogenous Oct4, Klf4, Sox2, c-Myc and Nanog genes by Q-PCR gave signals that were indistinguishable from those of ES cells, consistent with faithful molecular reprogramming (Figure 2B). In agreement with this, the Oct4 and Nanog promoters became demethylated in Adeno-iPS cells to a similar extent as seen in ES cells, while they remained hypermethylated in cultured fibroblasts and hepatocytes, indicating that Adeno-iPS cells had undergone successful epigenetic reprogramming (Figure 2C and Figure 7). In contrast to freshly infected fibroblasts, viral transcripts could no longer be detected in any of the Adeno-iPS lines tested, suggesting that the viral vectors had been diluted from the cells over time (Figure 2D). Adenoviral vectors can integrate into the genome of host cells at extremely low frequencies (Harui, A., et al., (1999) / Virol 73:6141-6). To exclude the possibility of permanent viral integration, PCR analysis of genomic DNA isolated from Adeno-iPS clones was performed with primers recognizing the different cDNA expression cassettes. [0053] While adenoviral vector DNA, used as a positive control, readily produced PCR signals, PCR products from genomic DNA were not amplified from any of the Adeno-iPS cells (Figure 3B). Southern blot analysis using the cDNAs of the four viral vectors as probes confirmed the PCR results and yielded no evidence for the continuous presence of the adenoviral sequences in the Adeno-iPS cells while the single-copy Oct4 transgenic allele integration into the CoIlA locus could be readily detected in iPS clones generated from Oct4IND cells (Figure 8).

[0054] To rule out genomic integration of adenoviral sequences other than the cDNAs, we performed Southern blot analysis using the BamHI-digested full-length vector as a probe. Again, there was not detectable signal for exogenous viral sequences in the genomes of Adeno-iPS cells, whereas adenoviral sequences were detectable in HEK cells, consistent with a previous report (Louis, N., et al (1997) Virology 233:423-9) (Figure 3C). Moreover, the pBluescript (pBS)-derived portion of the adenoviral vector probe cross -hybridized with the Oct4 transgene, which also carries the pBluescript backbone, giving rise to a specific ~3kb signal in the iPS lines derived from fetal liver and tail-tip fibroblasts, thus serving as an internal positive control (Figure 3C).

[0055] In order to ascertain the developmental potential of Adeno-iPS cells, cells were injected into the flanks of SCID mice. All cell lines tested produced teratomas after 3-4 weeks, which upon histological examination showed differentiation into representative cell types of the three germ layers including muscle, cartilage, and epithelial cells, thus demonstrating the pluripotency of Adeno-iPS cells (Figure 4A-C). In addition, Adeno-iPS cells generated apparently normal postnatal chimeras indicating that the iPS cells were likely truly pluripotent- i.e., restrictions of developmental potential were not observed in these experiments. One chimera, obtained after blastocyst injection of Adeno-iPS cells labeled with a lentivirus expressing the red fluorescent protein tdTomato, was sacrificed at birth to examine the contribution of Adeno-iPS cells to different tissues. As shown in Figure 4D-I, a high degree of chimerism was seen in multiple tissues including the lungs, brain and heart. Adeno-iPScells gave rise to high-degree coat color chimeras (Figure 4J,K) and differentiated into functional germ cells as evidenced by the derivation of GFP+ blastocysts after breeding of a male chimera generated with TTF-derived Adeno-iPS cells with a wildtype female (Figure 4L-O). Together, these results indicate that Adeno-iPS cells share the same developmental potential as iPS cell obtained with integrating viruses or ES cells. [0056] The efficiency of deriving iPS-like cells from fetal liver cells, TTFs and hepatocytes (see Figure 9 for an illustration of the derivation process from the different cell types) was extremely low, ranging from 0.0001% to 0.001% (Table 1). This frequency is lower than that obtained with integrating viruses and without wishing to be bound by theory is probably due to the fact that many cells do not maintain viral expression for long enough to trigger entry into a state sustained by endogenous pluripotency factors (Brambrink, T., et al., (2008) Cell Stem Cell 2:151-159; Stadtfeld, M., et al., (2008) Cell Stem Cell doi:10.1016/jstem.2008.02.001). This conclusion is supported by qPCR analysis for adenoviral gene expression, which is gradually lost in dividing fibroblasts (Figure 10). It is contemplated that the low efficiency of adenoviral reprogramming can be increased by the use of chemical compounds as has been reported for retroviral reprogramming (Huangfu, D., et al., (2008) Nat Biotechnol; Mikkelsen, T.S., et al (2008) Nature; Shi, Y., et al (2008) Cell Stem Cell 2:525-8).

[0057] Interestingly, DNA content analysis showed that about 23% (3 out of 13) of the 13 Adeno-iPS lines were tetraploid, which is not seen with retro- or lentiviral vectors (Figure 11 and Table 2). It was speculated that adenoviral reprogramming either induces cell fusion or, alternatively, selects for rare tetraploid cells pre-existing in the starting cell populations. Indeed, it has been shown that the frequency of polyploid hepatocytes increases with age (Gupta, S., et al (2000) Semin Cancer Biol 10:161-171).

[0058] These results are the first demonstration for the generation of iPS cells without the use of integrating viruses by employing either a combination of adenoviruses and an inducible transgene or adenoviruses alone. This work shows that insertional mutagenesis is not required for in vitro reprogramming, and it provides a platform for studying the biology of iPS cells lacking any viral integrations. For example, this model can be used to assess if iPS cells and ES cells are indeed equivalent at the molecular and functional levels. This comparison has not been possible so far because viral transgenes are expressed at low levels in iPS cells and their progeny, which may affect their molecular signatures as well as their differentiation behavior and developmental potential. Transient reprogramming approaches, like this one, will likely have important implications in cell therapy, as it can allow for the generation of patient-specific cells lacking potentially harmful genetic elements.

Example 2: Materials and Methods Adenoviral vectors and infection

[0059] For the cloning of the adenoviral vectors, EcoRI fragments containing the cDNAs for c-Myc (constitutively active human T58A variant), Klf4, Oct4 or Sox2 were subcloned into the EcoRI site of pH1HG-Ad2 (obtained from the Harvard Gene Therapy Initiative). A fragment containing the cDNA sequence was isolated from the resulting plasmids by Mfel/Pacl digestion and used for electroporation of BJ5183 cells together with linearized vector pAd-Clal (http://hgti.med.harvard.edu/InfoRepository.php3). Correct clones were identified by HindIII digestion. Viral constructs were transfected into 293A cells, and viral particles purified by two cycles of CsCl gradient and titered by optical absorbance (Takahashi, K., et al (2007), supra). Cells were infected with the indicated MOIs for 1 hour at 37°C, 5% CO2, followed by two washes with PBS and continued culture in the respective culture medium.

Isolation of primary cells

[0060] Adherent fetal liver cultures were established as previously described (Takahashi, K., et al (2003), supra). Briefly, embryos were harvested at embryonic day E13.5 and livers dissected, washed with HBSS and incubated for 10-15 minutes at 37°C in 0.2% collagenase IV (Invitrogen), ImM EDTA and ImM MgC12. Single-cell suspensions were prepared by repetitive pipetting and filtering through a 40 μm cell strainer. Cells were resuspended in DMEM containing 10% FBS, IOng/ml EGF, 10 ng/ml bFGF, 20 ng/ml TGFα, 0.5 μg/ml insulin and 40 ng/ml HGF and plated onto collagen I-treated culture plates (Becton Dickinson). Adult hepatocytes were isolated from 2-4 month-old mice by two-step collagenase perfusion protocol using Blendzyme 3 (Roche) (Wernig, M., et al (2007), supra). The perfused and isolated liver was freed of gall bladder and connective tissue and digested for 5-7 minutes. Digestion was stopped by addition of ice-cold DMEM containing FBS, followed by mechanically shearing of the liver capsule to release the dissociated cells. The resulting cell suspension was poured through a 100 μm cell strainer and centrifuged at 600 rpm for 1 minute. The pellet was considered as the hepatocytes preparation. Hepatocytes were cultured in DMEM containing 10% FBS, 10-7 M dexamethasone, 10 ng/ml EGF, 0.5 μg/ml insulin on collagen I-treated plates. Fibroblast cultures from tail-tip biopsies of neonatal mice were established as previously described (Okita, K., (2007), supra).

Southern blot analysis

[0061] 10-12 micrograms of genomic DNA was digested with BamHI, separated on a 0.85% agarose gel and blotted onto HybondXL membrane (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com). For cDNA blots, probes were prepared by digesting retroviral pMX vectors containing the four reprogramming cDNAs (Maherali 2007) with NcoIVBamHI (for c-myc) or EcoRI (for KIf 4, Oct4 and Sox2). For the whole vector blot, pAD-Clal was digested with BamHI. Labeling with 32P-α-dCTP was done using the Prime-It® II Random Labeling Kit from Stratagene following the manufacturers' instructions.

PCR analyses for adenoviral integration

[0062] PCR reactions were set up using 100 ng of genomic DNA isolated from the adeno iPS clones or 1 pg of the four different adenoviral vector plasmid DNAs (corresponding to the equivalent of 1 integration per genome sequence) using the primers described in

Table 3. The PCR reaction consisted of 30 cycles of 95°C for 30 seconds,

62°C for 45 seconds and 72°C for 30 seconds. The products were resolved on a 2% agarose gel. Teratoma formation

[0063] iPS cells were harvested by trypsinization and injected into the flanks of NOD/SCID mice, using ~5 million cells per injection. Mice were sacrificed 3 weeks later and teratomas isolated and processed for histological analysis.

Production of chimeric mice

[0064] Female BDFl mice were superovulated with PMS and hCG and mated to BDFl stud males. Zygotes were isolated from females with a vaginal plug 24 hour after hCG injection. After 3 days of in vitro culture in KSOM media, blastocysts were identified, injected with iPS cells and transferred into pseudopregnant recipient females. Pups were delivered by Cesarean section at day 19.5 and nurtured by foster mothers.

Immunofluorescence analysis

[0065] iPS cells were cultured on pretreated cover slips, fixed with 4% PFA and permeabilized with 0.5% Triton X-100. The cells were then stained with primary antibodies against mθct4

(Santa Cruz, sc-8628), mSox2 (Chemicon, AB5603), HA (Abeam, abl8181, for c-myc detection) and FLAG (Sigma, Fl 804, for Klf4 detection) followed by staining with the respective secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 546

(Invitrogen). Nuclei were counterstained with DAPI (Invitrogen). Cells were imaged using a

Leica DMI4000B inverted fluorescence microscope equipped with a Leica DFC350FX camera. Images were processed and analyzed using Adobe Photoshop software.

RNA isolation and real-time quantitative PCR analysis

[0066] RNA was isolated from cells using the TriPure reagent (Roche) followed by RNA clean up with the RNeasy Minikit (Qiagen). cDNA was produced with the First Strand cDNA

Synthesis Kit (Roche). Real-time quantitative PCR reactions were set up in triplicates with the Brilliant II SYBR Green QPCR Master Mix (Stratagene) and run on a Mx3000P

QPCR System (Stratagene). Primer sequences are listed in Table 4.

Bisulfite sequencing

[0067] Bisulfite treatment of DNA was performed with the EpiTect Bisulfite Kit (Qiagen) according to manufacturer's instructions. Oct4 and Nanog promotor regions were amplified as previously described (Takahashi 2006). Amplified PCR products were purified by using gel filtration columns, cloned into the pCR4-TOPO vector (Invitrogen), and sequenced with M 13 forward and reverse primers.

Table 2: Characteristics of Adeno-iPS clones

Claims

(b) isolating a reprogrammed cell of step (a).

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

3. The method of claim 1 or 2, wherein the somatic cell is a fibroblast.

4. The method of claim 1 or 2, wherein the somatic cell is a hepatocyte.

5. The method of claim 1, wherein the reprogramming factor is selected from the group consisting of Oct4, Sox2, c-Myc and Klf4.

6. The method of claim 1, wherein the non-integrating viral vector comprises an adenoviral vector.

7. The method of claim 1, further comprising the step of passaging the cells.

8. The method of claim 1, wherein the induced pluripotent stem cell is free from viral integration remnants.

9. The method of claim 1, wherein production of the induced pluripotent stem cell is evidenced by detection of a stem cell marker.

10. The method of claim 9, wherein the stem cell marker is selected from the group consisting of SSEAl, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, Oct4, SOX2, and Natl.

11. An induced pluripotent stem cell produced by the method of claim 1.

13. The use of claim 12, wherein the non-integrating vector is an adenoviral vector.

14. The use of claim 12, wherein the somatic cell is a human cell.

15. The use of claim 12, wherein the somatic cell is a fibroblast or a hepatocyte.

16. The use of claim 12, wherein the reprogramming factor is selected from the group consisting of Oct4, Sox2, c-Myc, and Klf4.

17. Use of a cell produced by the method of claim 1 for the treatment of a disease or disorder.