WO2014031085A1

WO2014031085A1 - The use of pdgf-bb in a method of enhancing the efficiency of reprogramming of a somatic cell to a pluripotent stem cell

Info

Publication number: WO2014031085A1
Application number: PCT/SG2013/000367
Authority: WO
Inventors: Young-Tae Chang; Seong-Wook Yun; Sung-Jin Park; Nam-Young Kang
Original assignee: Agency for Science Technology and Research Singapore
Current assignee: Agency for Science Technology and Research Singapore
Priority date: 2012-08-24
Filing date: 2013-08-23
Publication date: 2014-02-27
Anticipated expiration: 2015-02-24

Description

The use of PDGF-BB in a method of enhancing the efficiency of reprogramming of a somatic cell to a pluripotent stem cell

Technical field

The present invention generally relates to a method of cell reprogramming. More specifically, the present invention relates to a method for enhancing the efficiency of reprogramming of a somatic cell to a pluripotent stem cell.

Background

Cell reprogramming refers to the process of converting and returning a somatic cell into a pluripotent state. Cell reprogramming is generally achieved by resetting the pattern of gene expression through the ectopic expression of specific reprogramming factors. This cell reprogramming technology was first introduced in 2006 by Yamanaka and colleagues, where they discovered that in-vitro expression of four reprogramming factors, Oct3/4, Sox2 , Klf4, and c-Myc (OSKM) could convert the isolated, . fully differentiated adult mouse and human skin fibroblasts into induced pluripotent stem (iPS) cells. These iPS cells are believed to be morphologically similar to embryonic stem (ES) cells and possess the ability to differentiate into a variety of different somatic cell types. The development of these iPS cells marks an important advancement in stem cell research and holds great promise in its application to regenerative medicine and therapies. In this regard, cell reprogramming technology may allow researchers to obtain pluripotent stem cells without having the associated ethical issues such as the controversial use of embryos. However, the mechanisms of cell reprogramming and natural ligands involved in the processes have not been well understood. Further, current cell reprogramming processes still face issues like low levels of reprogramming efficiency. Generally, less than 1% of transduced cells are reprogrammed to form iPS cells, and the process of establishing iPS cell clones may stretch over a long period of time.

Therefore, there is a need to provide a method of cell reprogramming that overcomes, or at least ameliorates, one or more of the disadvantages described above.

There is also need to provide a method of cell reprogramming, which effectively enhances the reprogramming efficiency of a somatic cell to a pluripotent cell.

Summary

Provided in this disclosure is a method of enhancing reprogramming efficiency of induced pluripotent stem cell from a somatic cell, the method comprises the transfection with reprogramming factors and an administration of a growth factor as disclosed herein.

According to a first aspect, there is provided an in-vitro method of producing an induced pluripotent stem cell from a somatic cell, comprising: a) transfecting said somatic cell with one or more nuclear reprogramming factors selected from the group consisting of an Oct family gene or a gene product, a Sox family gene or a gene product, a Klf family gene or a gene product and a Myc family gene or a gene product; b) contacting said transfected cell with platelet derived growth factor-BB (PDGF-BB) .

According to a second aspect, there is provided a cell culture medium to reprogram a somatic cell into a pluripotent cell; comprising PDGF-BB.

According to a third aspect, there is provided a kit comprising (i) a PDGF-BB and (ii) instructions for use.

According to a fourth aspect, there is provided a nuclear reprogramming factor for a somatic cell, which comprises one or more of

(i) an Oct family gene or a gene product,

(ii) a Sox family gene or a gene product,

(iii) a Klf family gene or a gene product and .

(iv) a Myc family gene or a gene product; and PDGF-BB.

According to a fifth aspect, there is provided a use of a nuclear reprogramming factor as defined above for reprogramming a somatic cell- Definitions

Unless otherwise defined, the technical, scientific and medical terminology used herein has the same meaning as understood by those skilled in the art to which this invention belongs. However, for the purposes of establishing support for various terms that are used in the present application, the following technical comments, definitions and review are provided for reference.

The following words and terms used herein shall have the meaning indicated:

The term ^Preprogramming factor" as used herein refers to any of factors that have the capacity to induce a somatic cell to revert to a pluripotent state. Exemplary reprogramming factors as disclosed herein, include transcription factors such as such as Oct3/4, Sox2 , Klf4 and c- yc, Nanog, Lin28, Glis 1, ESRRB and NR5a2. The reprogramming factors may be utilized singularly or in combination.

The term "gene product" as used herein refers to biochemical material, for example cDNA, RNA or polypeptide or protein that results from the expression of a gene.

The term "growth factor" as used herein refers to a naturally occurring substance capable of stimulating cellular growth, proliferation and cellular differentiation. The growth factor may be a protein or a steroid hormone. Exemplary growth factor, as disclosed herein, include but are not limited to platelet- derived growth factor (PDGF) such as PDGF-AA and PDGF-BB.

PDGF is a type of growth factor which plays a significant role in blood vessel formation (angiogenesis) and the growth of blood vessels from already-existing blood vessel tissue. In chemical terms, platelet-derived growth factor is a dimeric glycoprotein composed of two A (-AA) or two B (-BB) chains or a combination of the two (-AB) .

The term "stem cell" as used herein refers to an undifferentiated cell which has the ability to continuously divide and differentiate into various other kinds of mature functional cells/tissues. For example, a hematopoietic stem cell may give rise to any of the different types of terminally differentiated blood cells. Embryonic stem cells are derived from the embryo and are pluripotent, thus are capable of developing into any organ or tissue type or, at least potentially, into a complete embryo.

The term "induced pluripotent stem cell" as used herein refers to a type of pluripotent stem cell artificially derived from a non-pluripotent cell. An induced pluripotent stem cell is typically an adult somatic cell which has been genetically reprogrammed into a pluripotent stem cell^like state by being forced to express genes and factors important for maintaining the defining properties of natural pluripotent stem cells. Induced pluripotent stem cells are commonly abbreviated as iPS cells or iPSCs.

Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements..

As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the^" stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have^ specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3 , from 1 to , from 1 to 5 , from 2 to 4 , from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Disclosure of Optional Embodiments

Exemplary, non-limiting embodiments of methods and cell culture mediums for enhancing the efficiency of reprogramming of a somatic cell to a pluripotent cell will now be disclosed.

There is provided an in-vitro method of producing an induced pluripotent stem cell from a somatic cell comprising: a) transfecting said somatic cell with one or more nuclear reprogramming factors selected from the group consisting of an Oct family gene or a gene product, a Sox family gene or a gene product, a Klf family gene or a gene product and a Myc family gene or a gene product; b) contacting said transfected cell with PDGF-BB.

Advantageously, the method of the present invention enhances the efficiency of reprogramming of a somatic cell to a pluripotent cell.,

More advantageously, the method of the present invention increases- the induced pluripotent cell production significantly relative to a method of induction that does not comprise contacting the transfected cell with PDGF-BB. The combination of transfecting with the nuclear reprogramming factors as disclosed herein and contacting the somatic cell with the exogenous growth factor PDGF-BB in the disclosed method provides a surprising effect of enhancing the .efficiency of reprogramming and also improving the overall percentage of - iPS cells production, as compared to the method of transfecting with the reprogramming factors disclosed herein alone.

The somatic cell may be contacted by PDGF-BB after transfection by the method of the invention. The presence of exogenous PDGF- BB surprisingly improves, cell reprogramming. The presence of exogenous PDGF-BB after transfection advantageously increases iPS cell generation efficiency. In one embodiment, the induced pluripotent cell generation efficiency may be increased by at least about 3-fold to 6 fold, about 3-fold to 7-fold, about 3- fold to 8-fold, about 3-fold to 9-fold, or about 3-fold to 10- fold. Preferably, the iPS cell generation efficiency may be increased by at least about 3-fold, 4-fold, 5-fold, about 6- fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold relative to a method of induction that does not . comprise contacting the transfected cell with PDGF-BB.

In one embodiment, the iPS cell generation efficiency is increased by at least 3-fold relative to a method of induction that does not comprise contacting the transfected cell with PDGF-BB.

The concentration of PDGF-BB used in the method disclosed herein may be optimized to maximize reprogramming efficiency. The concentration of PDGF-BB may be optimized to be in the range of about lOng/ml to about 15ng/ml, about 5ng/ml to about lOng/ml, about lng/ml to about 5ng/ml, about 0.5ng/ml to about lng/ml or about 0. lng/ml to about lng/ml. Preferably, the concentration of PDGF-BB is about 0. lng/ml, about 0.5 ng/ml, about lng/ml, about 2ng/ml , about 3ng/ml, about 4ng/ml, about 5ng/ml, about -6ng/ml, about 7ng/ml, about 8ng/ml, about 9ng/ml or about lOng/ml.

In one embodiment, the concentration of PDGF-BB to be administered is lng/ml. In another embodiment, the concentration of PDGF-BB to be administered is 5ng/ml .

Likewise, the time point in which the transfected cell is contacted with PDGF-BB may also be optimized to further enhance reprogramming efficiency. The transfected cell may be contacted with PDGF-BB within 24 days from transfection. the cell is contacted with PDGF-BB within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, within 11 days, within 12 days, within 13 days, within 14 days, within 15 days, within 16 days, within 17 days, within 18 days, within 19 days, within 20 days, within 21 days, within 22 days, within 23 days, or within 24 days after transfection. In one embodiment, the cell is contacted with PDGF-BB within 1-3 weeks from transfection, within 2 weeks from transfection, within 3 days to 2 weeks from transfection, or within 3 days to 24 days from transfection.

The somatic cell may be transfected with one or more of the reprogramming factors as disclosed herein. In particular, the somatic cell may be transfected with one or more reprogramming factors selected from the group consisting: an Oct family gene or a gene product, a Sox family gene or a gene product, a Klf family gene or a gene product and a Myc family gene or a gene product. In one embodiment, the Oct family gene or a gene product may be Oct4 or Oct 3, the Sox family gene or a gene product may be Sox2, the Klf family gene or a gene product may be Klf4 and the Myc family gene or a gene product may be c-Myc. The cell may be transfected with Oct4 only, Oct 3 only, Sox 2 only, Klf4 only, c-Myc only, or any one or more combinations of the reprogramming factors above. In one embodiment, the somatic cell is transfected with a combination of c-Myc, Klf4, Sox2 and Oct4.

Further, some of the reprogramming factor gene or, gene product may be substituted with other suitable reprogramming factor. For example, the Myc family gene or gene product and Klf family gene or gene product may be substituted with Nanog and/or Lin28. The Oct family gene or gene product and Klf family gene or gene product may also be substituted with ESRRB and/or NR5a2.

Also disclosed herein is a cell culture medium to reprogram a somatic cell into a pluripotent cell; comprising PDGF-BB . In one embodiment, the cell culture . medium comprises Dulbecco' s Modified Eagle Medium (DMEM). In one embodiment, the cell culture medium further comprises D-glucose. In another embodiment, the concentration of D-glucose is about 3000 ml/L, about 3500 ml/L, about 4000 ml/L, about 4500 ml/L, or 5000 ml/L. In one embodiment, the concentration of D-glucose is about 4500 ml/L. In one embodiment, the cell culture medium comprises DMEM high-glucose. In one embodiment, the cell culture medium comprises L-glutamine, β-mercaptoethanol , non-essential amino acids, leukemia inhibitory factor and DMEM comprising knock-out serum replacement (KOSR) . In one embodiment, the cell culture medium comprises about ImM L-glutamine, about 0. ImM β- mercaptoethanol , about 1% non-essential amino acids, about 10OU/ml leukemia inhibitory factor and DMEM comprising about 20% knock-out serum replacement (KOSR) . In one embodiment, the cell culture medium further comprises PDGF-BB. Also disclosed is a kit comprising: (i) a PDGF-BB and (ii) instructions for^: use. In one embodiment, the kit disclosed herein comprises one or more, nuclear reprogramming factors selected from the group consisting of Oct family gene or a gene product, a Sox family gene or a gene product, a Klf family gene or a gene product and a Myc family gene or a gene product.

Also disclosed is a nuclear reprogramming factor for a somatic cell, which comprises one or more of

(i) an Oct family gene or a gene product,

(ii) a Sox family gene or a gene product,

(iii) a Klf family gene or a gene product and

(iv) a Myc family gene or a gene product; and PDGF-BB.

In one embodiment, the nuclear reprogramming factor described above comprises an Oct family gene or a gene product, a Sox family gene or a gene product, a Klf family gene or a gene product and a Myc family gene or a gene product. In one embodiment, the Oct family gene or gene product is 0ct4 or Oct3. In one embodiment, the Oct family gene or gene product is Oct 4. In one embodiment, the Sox family gene or gene product is Sox2. In one embodiment, the Klf family gene or gene product is Klf4. In one embodiment, the Myc family gene or gene product is c-Myc. In another embodiment, the nuclear reprogramming factor as described above comprises c-Myc, Oct4 , Sox2 and Klf4.

Further, some of the reprogramming factor gene or gene product may be substituted with other suitable reprogramming ^' factor. For example, the Myc family gene or gene product and Klf family gene or gene product may be substituted with Nanog and/or Lin28. The Oct family gene or gene product and Klf family gene or gene product, may also be substituted with ESRRB and/or NR5a2.

Disclosed also is a use of a nuclear reprogramming factor as described above for reprogramming a somatic cell.

Brief Description of Drawings

The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. However, it is to be understood that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

Fig. 1 shows the CDyl stained distinct cell populations observed on the fluorescence microscopy (dark field and bright field) at 3, 5, 7 and 9 days post infection of reprogramming.

Fig. 2 depicts a heat map of gene expression profiles andhierarchical clustering generated using 5-fold differentially expressed genes (DEG) . EF, mouse embryonic fibroblast; S3-C-2, No PDGF treated iPS cell line; S3-1-11, PDGF-BB treated iPS cell line; and mESC, mouse embryonic stem cell were analyzed.

Fig. 3 depicts a Venn diagram showing exclusively expressed 5- fold DEG for early time points of reprogramming into iPS cells. Solid circle indicate cells undergoing reprogramming; dash circle indicates (S3-C-2) , mature iPS cells; and dot circle indicates mES cells.

Fig. 4 depicts a high ranking networks with constructed using Ingenuity Pathway Analysis tool using whole 5- fold DEG (A) and exclusively expressed 5- fold DEG (B) in 3 , 5, 7 and 9 dpi iPS cells. PDGF-BB marked in red appears as a central player in the extracellular space at all the early time points of reprogramming . ·„■ ^' -

Fig. 5 depicts PDGF-B gene expression levels at early time points of reprogramming measured by real time RT-PCR.

Fig. 6A depicts microscopy images of iPS cell colony generated in the presence of PDGF-BB.

Fig. 6B depicts GFP positive colonies in the wells treated with different concentrations of PDGF-BB counted at 24dpi. Data are presented as mean±SD (n=4) .

Fig. 7 depicts graphs comparing the effects of PDGFR inhibitor and PDGF antibody in iPS cell generation. Cells were treated from 3dpi to 24dpi and "the GFP positive colonies were counted at 24dpi. Data are presented in the graphs as mean+SD (n=4) .

Fig. 8 depicts the confirmation of pluripotent stem cell marker SSEA-1 expression by immunocytochemistry in the PDGF-BB treated iPS cell line .

Fig. 9 depicts three germ layer cell types differentiated from PDGF-BB treated iPS cell line (Hematoxylin and eosin staining) . Arrows indicate neural tube; double arrows indicate keratinocytes ; arrow heads indicate smooth muscle cells and double arrow heads indicate intestinal epithelia.

Fig. 10 depicts three germ layer cell types in^' vitro differentiated from PDGF-BB- treated iPS cells - via embryoid body formation.

Fig. 11 depicts mRNA levels of endogenous and exogenous OSKM factors measured by real time RT-PCR. Relative expression levels of endogenous OSKM in the tested cells were compared to those in MEF. For exogenous genes, OSKM-transfected MEF at 6 dpi was used as control. Cells tested were (MEF) mouse embryonic fibroblast; S3-1-1 and S3-1-11, PDGF-BB 1 ng/ml treated cell lines; S3-5-3 and S3-5-11, PDGF-BB 5 ng/ml treated cell lines; S3-C-2, no PDGF treated iPS cell line; and mESC, mouse embryonic stem cell.

Fig. 12 depicts Left:a heat map of gene expression profiles with 5-fold DEG for PDGF treated iPS cell line (S3-1-11) , no PDGF treated iPS cell line (S3-C-2) and mESC; and Right: Correlations between MEF , no PDGF-treated iPS cell line, PDGF-treated iPS cell line and mESC; where r: correlation probability.

Detailed description

Global gene expression analysis revealed molecules playing critical roles at early stages of reprogramming

By using a pluripotent stem cell selective fluorescent probe CDyl developed by the inventors, the cells undergoing reprogramming into induced pluripotent stem (iPS) cell were enriched at different time points for the analysis of global gene expression profiling.

CDyl stains scattered single cells at 3 and 5 days after infection (dpi) of mouse embryonic fibroblasts (MEF) with OSKM factors-expressing retrovirus and cluster at 7 and 9 dpi were observed (Figure 1) .

Because only a small number of cells is stained by CDyl particularly at 3 and 5 dpi, the cells were collected by repeated FACS at 3 , 5, 7 and 9 dpi to isolate sufficient amount of mRNA for DNA microarray. The microarray data were analyzed to select the differentially expressed genes (DEG) , which show distinct differences in a heat map between the cells at early stages of reprogramming and mature iPS cells or ES cells (Figure 2).

With 5-fold difference criteria, 59 (3 dpi) , 164 (5 dpi) , 185 (7 dpi) and 258 (9 dpi) DEG were identified. Among them 37 (3 dpi) , 111 (5 dpi), 109 (7 dpi) and 142 (9 dpi) genes were' exclusively for early time points which were not identified in mature iPS cells or ES cells (Figure 3) .

The networks of these 8 sets of genes were then analyzed for each time point using Ingenuity Pathway Analysis (IPA) network analysis tool to identify specific factors that play important roles in reprogramming. Of particular interest were the factors working from the extracellular space. Among the several networks constructed by grouping the DEG, it was noticed that PDGF-BB appears commonly as a central player in the networks drawn from the largest number of genes at every time point (Figure 4) .

PDGF-BB enhances reprogramming efficiency

As PDGF-BB appeared as the central molecule connecting the DEG in CDyl stained cells at early time points of reprogramming, the effects of PDGF-BB in iPS cell generation were investigated. The expression levels of PDGF -A, -B and -C in CDylbright cells collected at 3, 5, 7 and 9 dpi by quantitative RT-PCR were first measured, which showed 7, 11, 58' and 67-fold increases of only PDGF-B, respectively (Figure 5) .

To figure out the role of exogenous PDGF-BB in reprogramming, OSK -transfected Oct4-GFP MEF were cultured in medium containing 1 ng/ml and 5 ng/ml of recombinant mouse PDGF-BB from the day after plating (3 dpi) to 24 dpi. For a strict control of the PDGF level in the medium, PDGF- free knock out serum replacement (KOSR, Invitrogen) was substituted for FBS . The number of GFP positive colonies counted at 24 dpi in 1 ng/ml PDGF and 5ng/ml PDGF-BB treated groups were 21+4 and 15+3 per well whereas only 7+2 colonies were observed in non-treated control well. Because the higher PDGF-BB concentration of 5 ng/ml did not further increase the iPS cell colony numbers compared to 1 ng/ml, PDGF- BB was applied at a concentration of 1 ng/ml in the following experiments which resulted in 3 to 6-fold increases of iPS cell generation efficiency (Figure 6A,B) .

The critical role of PDGF-BB in reprogramming was further confirmed by the finding that PDGF receptor tyrosine kinase inhibitors such as AG 17, AG 1295, AG 1296 and Imatinib mesylate (Gleevec) and a neutralizing antibody against PDGF-BB reduce the reprogramming efficiency (Figure 7) . iPS cells reprogrammed with PDGF-BB have normal characteristics

To verify that the OCT4-GFP positive cell colonies grown in the medium, containing PDGF-BB are bona fide iPS cells, 4 colonies were ,^■ randomly isolated and continuously passaged for the assessment of their sternness and pluripotency . The expression of pluripotent stem cell marker SSEA-1 was visualized by immunocytochemistry (Figure 8).

The capability of differentiation into all 3 germ layer cell types was demonstrated by in vivo teratoma assay (Figure 9) as well as in vitro embryoid body assay (Figure 10) .

Further, increased expression of endogenous OS M factors and suppression of the exogenously introduced factors were revealed by real time RT-PCR (Figure 11) .

All the 4 cell lines proved to be pluripotent stem cells. These results demonstrate that the GFP expression used as a surrogate marker for. reprogramming in our study is from bona fide iPS cells and the PDGF-BB treatment does not affect the normal characteristics of iPS cells. The global gene expression profile of a PDGF-BB assisted iPS cell line was also found to be very similar to normal iPS and mES cell lines (Figure 12) .

Conclusion

By using a pluripotent stem cell selective fluorescent probe CDyl developed by the inventors, the cells undergoing reprogramming into induced pluripotent stem (iPS) cell were enriched at different time points for the analysis of global gene expression profiling. Network analysis of the differentially expressed genes indicated that PDGF-BB has a key role in iPS cell generation. Exogenously added PDGF-BB during reprogramming increased the iPS cell generation while PDGF signaling inhibitors and neutralizing antibody decreased it. These results suggest that PDGF-BB mediated signaling is important for reprogramming and addition of PDGF-BB into the culture medium increases the efficiency of cell reprogramming into iPS cells.

Applications

It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.

Claims

1. An in-vitro method of producing an induced pluripotent stem cell from a somatic cell comprising: a) transfecting said somatic cell with one or more nuclear reprogramming factors selected from the group consisting of an Oct family gene or a gene product, a Sox family gene or a gene product, a Klf family gene or a gene product and a Myc family gene or a gene product; b) contacting said transfected cell with PDGF-BB.

2. The method of claim 1, wherein the somatic cell is transfected with an Oct family gene or a gene product, a Sox family gene or a gene product, a Klf family gene or a gene product and a Myc family gene or a gene product.

3. The method of claim 1, wherein the cell is -contacted with PDGF-BB within 1-3 weeks from transfection.

4. The method of claim 1, wherein the cell is contacted with PDGF-BB within 3 days to 2 weeks from transfection.

5. The method of - claim 1, wherein the concentration of the PDGF-BB is selected from the group consisting of from lOng/ml to 15ng/ml, 5ng/ml to lOng/ml, lng/ml to 5ng/ml, 0.5ng/ml to lng/ml and 0. lng/ml to lng/ml.

6. The method of claim 1, wherein the concentration of the PDGF-BB is lng/ml.

7. The method of claim 1, wherein the Myc family gene or gene product and Klf family gene or gene product is substituted with Nanog and Lin28.

8 . The method of claim 1, wherein the Oct family gene or gene product and Klf family gene or gene product is substituted with ESRRB and NR5a2.

9. The method of claim 1, wherein the somatic- cell is transfected with c-Myc, Klf , Sox2 and/or Oct4.

-10. The method of claim 1, wherein the induced pluripotent cell production is increased by at least 3-fold in comparison to a method of induction that does not comprise contacting the transfected cell with PDGF-BB.

11. A cell culture medium to reprogram a somatic cell into a pluripotent cell; comprising PDGF-BB.

12. A cell culture medium of claim 11, comprising ImM L- glutamine, 0. ImM β -mercaptoethanol , 1% non-essential amino acids, lOOU/ml leukemia inhibitory factor and DMEM comprising 20% knock-out serum replacement (KOSR)

13. A kit comprising: (i) a PDGF-BB, and .(ii) instructions for use.

14. The kit of claim 13, wherein the kit further comprises one or more nuclear reprogramming factors selected from the group consisting of Oct family gene or a gene product, a Sox family gene or a gene product, a Klf family gene or a gene product and a yc family gene or a gene product.

15. A nuclear reprogramming factor for a somatic cell, which comprises one or more of

(i) an Oct family gene or a gene product,

(ii) a Sox family gene or a gene product,

(iii) a Klf family gene or a gene product and (iv) a Myc family gene or a gene product; and PDGF-BB .

16. The nuclear reprogramming factor of claim 15, wherein the factor comprises an Oct family gene or a gene product, a So family gene or a gene product, a Klf family gene or a gene product and a Myc family gene or a gene product.

17. The nuclear reprogramming factor of claim 15, wherein the factor comprises c-Myc, Oct4, Sox2 and Klf4.

18. The nuclear reprogramming factor of claim 15, wherein the Myc family gene or gene product and Klf family gene or gene product is substituted with Nanog and Lin28.

19. The nuclear reprogramming factor of claim 15, wherein the Oct family gene or gene product and Klf family gene or gene product is substituted with ESRRB and NR5a2.

20. Use of a nuclear reprogramming factor as claimed in claim 15 for reprogramming a somatic cell.