WO2013062140A1 - Method for efficiently inducing differentiation of pluripotent stem cells into hepatic lineage cells - Google Patents

Abstract

The present invention provides a method for producing a cell belonging to an endodermal lineage, including inducing differentiation of a blood cell-derived iPS cell, specifically, a method for producing a cell belonging to an endodermal lineage, inducing the following steps (1) and (2): (1) a step of producing an iPS cell by contacting a nuclear reprogramming substance with a blood cell (2) a step of inducing differentiation of the iPS cell obtained in step (1) into a cell belonging to an endodermal lineage. In addition, the present invention provides a method for inducing differentiation of a human pluripotent stem cell into a CXCR4 positive endoderm cell, including (1) cultivating a human pluripotent stem cell in a single-cell state in the presence of activin A and Wnt3a for 4-10 days and (2) cultivating in the co-presence of 0.1-0.8 mM sodium butyrate for 0-10 days during the culture period.

Description

DESCRIPTION

METHOD FOR EFFICIENTLY INDUCING DIFFERENTIATION OF PLURIPOTENT STEM CELLS INTO HEPATIC LINEAGE CELLS Technical Field of the Invention

The present invention relates to a method for efficiently inducing differentiation of a pluripotent stem cell into a hepatocyte. More particularly, the present invention relates to a method for efficiently inducing differentiation of an induced pluripotent stem cell (hereinafter to be referred to as iPS cell) into a hepatocyte by using an iPS cell derived from a blood cell, and a method for efficiently inducing differentiation of a pluripotent stem cell into a hepatocyte. Background of the Invention

In recent years, mouse and human iPS cells have been established one after another. Yamanaka et al . induced iPS cells by introducing the Oct3/4, Sox2, Klf4 and c-Myc genes into fibroblasts derived from a mouse and human, and forcing the cells to express the genes [WO 2007/069666 Al; Takahashi, K. and Yamanaka, S., Cell, 126: 663-676 (2006)]. On the other hand, a group of Thomson et al. produced human iPS cells using Nanog and Lin28 in place of Klf4 and c-Myc (4, 5) .

The iPS cells obtained as mentioned above can be

differentiated into the cells of respective tissues, and the obtained differentiated cells can be a tool for in vitro drug discovery screening and a cell to be transplanted for

regenerative medicine. Therefore, many attempts have been actively made at present in the world to establish a method for inducing differentiation of an iPS cell into cells of various lineages.

Hepatocyte produced from a human iPS cell also becomes a useful resource for the development of pharmaceutical products and regenerative medicine. However, a method for inducing differentiation of an iPS cell into a mature hepatocyte in vitro has not been established yet. While various attempts have been made to improve the differentiation method (6-16), whether or not there exists a human iPS cell appropriate for hepatocyte differentiation, namely, the relationship between hepatocyte differentiation and derived cell, has not been studied yet. Moreover, there is no report on the use of a blood cell-derived human iPS cell for the differentiation of human iPS cell into hepatocyte (6-16).

Cited references:

1. WO 2007/069666 Al

2. Takahashi, K. and Yamanaka, S., Cell, 126: 663-676 (2006)

3. Takahashi, K. et al., Cell, 131: 861-872 (2007)

4. WO 2008/118820 A2

5. Yu, J. et al., Science, 318: 1917-1920 (2007)

6. Song, Z. et al., Cell Res., 19(11): 1233-1242 (2009) ^" 7. Sullivan, G.J. et al . , Hepatology, 51(1): 329-335 (2010)

8. Touboul, T. et al., Hepatology, 52(5): 1754-1765 (2010)

9. Si-Tayeb, K. et al., Hepatology, 51(1): 297-305 (2010)

10. Rashid, S.T. et al., J. Clin. Invest., 120(9): 3127-3136 (2010)

11. Ghodsizadeh, A. et al., Stem Cell Rev., 6(A): 622-632 (2010)

12. Inamura, M. et al., Mol. Ther. , 19(2): 400-407 (2011)

13. Jozefczuk, J. et al . , Stem Cells Dev., 20(1): 1259-1275 (2011) .

14. Ohi, Y. et al., Nat. Cell Biol., 13(5): 541-549 (2011)

15. Nagae, G. et al., Hum. Mol. Genet., 20(14): 2710-2721 (2011)

16. Takata, A. et al., Hepatol. Int., Feb 6. (2011) [Epub ahead of print]

Summary of the Invention

An object of the present invention is to_. search for an iPS cell suitable for hepatocyte differentiation, with which to provide a method for inducing differentiation of a human iPS cell into a hepatocyte efficiently and with good reproducibility.

To achieve the above-mentioned object, the present inventors first somewhat modified the existing methods of ES cell (Hay, D.C. et al., Stem Cells, 26(4): 894-902 (2008); Hay, D.C. et al., Proc. Natl. Acad. Sci . USA, 105(34): 12301-12306 (2008)) to induce differentiation of various human iPS cell lines into hepatocytes. As a result, it has been clarified that the differentiation ability into hepatocyte and the level of differentiation vary depending on the iPS cell lines.

Then, the present inventors developed a new method by improving an existing method (Hay, D.C. et al., Stem Cells (2008), supra; Hay, D.C. et al., Proc. Natl. Acad. Sci. USA (2008), supra) to perform induction of differentiation of human iPS cell into hepatocyte more efficiently and with good reproducibility. To be specific, since an undifferentiated human iPS cell colony is directly used to induce

differentiation in the existing method, nonuniformity of the colony size influences the differentiation induction

efficiency and reproducibility thereafter. Therefore, the present inventors developed a new method capable of inducing superior differentiation of a human iPS cell in a single-cell state into a hepatocyte by adjusting the number of days of the first step (Step 1) of differentiation induction and

concentration of sodium butyrate to be used in Step 1.

Then, the present inventors studied the differentiation tendency (differentiation propensity) toward hepatocyte by using this new differentiation induction method and 22 kinds of human iPS cell lines established from various somatic cell origins by the retrovirus method or episomal method. As a result, it has unexpectedly been found that a blood cell- derived iPS cell shows a significantly high differentiation ability (albumin secretion potential) toward hepatocyte as compared to iPS cells derived from fibroblast and dental pulp stem cell, and human ES cells. Particularly, such tendency was high in an iPS cell derived from peripheral blood mononuclear cell (PBMC) . iPS cell maintains the memory (epigenetic memory) of the somatic cell origin from which it was derived and, in fact, a report has documented that a blood cell-derived iPS cell is easily differentiated into a blood cell {Nature, 467, 285-290 (2010) ) . Also from such aspect, the present finding is epoch-making and overturns conventional theories.

The present inventors have conducted further studies based on these findings, which resulted in the completion of the present invention.

That is, the present invention provides the following.

[1] A method for producing a cell belonging to an endodermal lineage, comprising inducing differentiation of a blood cell- derived induced pluripotent stem (iPS) cell.

[2] A method for producing a cell belonging to an endodermal lineage, comprising the following steps (1) and (2):

(1) a step of producing an iPS cell by contacting a nuclear reprogramming substance with a blood cell

(2) a step of inducing differentiation of the iPS cell

obtained in step (1) into a cell belonging to an endodermal lineage.

[3] The method according to the above-mentioned [1] or [2], wherein the blood cell is a peripheral blood mononuclear cell or a cord blood cell.

[4] The method according to the above-mentioned [1] or [2], wherein the cell belonging to the endodermal lineage is a hepatic lineage cell.

[5] The method according to the above-mentioned [4], wherein the hepatic lineage cell is a hepatocyte.

[6] The method according to the above-mentioned [2], wherein the nuclear reprogramming substance comprises Oct3/4, Klf4 and Sox2, or a nucleic acid encoding the same.

[7] The method according to any of the above-mentioned [l]-[6], wherein the blood cell is derived from human.

[8] Use of a blood cell as a somatic cell source of an iPS cell for production of a cell belonging to an endodermal lineage .

[9] The use according to the above-mentioned [8], wherein the blood cell is a peripheral blood mononuclear cell or a cord blood cell.

[10] The use according to the above-mentioned [8], wherein the cell belonging to the endodermal lineage is a hepatic lineage cell .

[11] The use according to the above-mentioned [10] , wherein the hepatic lineage cell is a hepatocyte.

[12] The use according to any of the above-mentioned [8] -[11], wherein the blood cell is derived from human.

[13] A method for inducing differentiation of a human

pluripotent stem cell into a CXCR4 positive endoderm cell, characterized by the following (1) and (2) :

(1) cultivating a human pluripotent stem cell in a single-cell state in the presence of activin A and Wnt3a for 4-10 days

(2) cultivating in the co-presence of 0.1-0.8 mM sodium butyrate for 0-10 days during the culture period in the aforementioned (1).

[14] The method according to the above-mentioned [13], wherein the culture period in the aforementioned (1) is 7 days, and the cell is cultivated in the presence of 0.5 mM sodium butyrate for 0-6 days in the aforementioned (2) .

[15] The method according to the above-mentioned [13] or [14], wherein the human pluripotent stem cell is a human iPS cell or a human ES cell.

[16], The method according to the above-mentioned [13] or [14] , wherein the human pluripotent stem cell is a blood cell- derived human iPS cell, and sodium butyrate is not co-present during the culture period in the aforementioned (1) .

According to the present invention, differentiation of iPS cell into cells belonging to an endodermal lineage such as hepatocyte can be induced efficiently by using a blood cell- derived iPS cell as a cell source. Particularly, since an iPS cell derived from a peripheral blood mononuclear cell shows high differentiation propensity toward hepatocyte, an iPS cell having hepatic differentiation propensity equal to or. more than that of a liver-derived iPS cell can be obtained by ordinary blood sampling, without using a method including inducing an iPS cell from a hepatocyte collected from a hepatic tissue, which is highly burdensome for the donor.

Moreover, since a hepatocyte having mature hepatocyte-like function can be induced, a cell better reflecting the

physiological functions of hepatocyte in a living organism than established hepatocyte can be prepared without a

quantitative limitation unlike primary tissue culture, and the cell can be utilized as a high-throughput in vitro screening system for the evaluation of efficacy and toxicity in drug discovery.

Brief Description of the Drawings

Fig. 1 shows directed hepatic differentiation of hiPSCs and hESCs. (A) Schematic presentation of directed hepatic differentiation protocol in this study. (B) Time course expression of undifferentiated and hepatocyte differentiation marker genes in the hepatic differentiated hiPSCs (201B6) . Embryoid body (EB) was -obtained with a floating culture for 8 days followed by a monolayer culture on gelatin-coated plate for another 8 days, and used as positive control of early lineage differentiated markers. HepG2 and adult liver were used as positive control for hepatic markers.

Fig. 2 shows marked hepatic differentiation diversity among hESC and sibling hiPSC lines. (A) Percentage of albumin positive cells by flow cytometry analysis after 17 days of hepatic differentiation. (B) Real time-PCR analysis of liver- related and undifferentiated gene expression of hepatic differentiated hiPSCs and hESCs. The graph represent the fold expression of genes relative to KhESl-dayl7. ND = not determined. (C) Albumin secretion potential of hepatic differentiated hiPSCs and hESCs at day 17 and other control cells analyzed by ELISA. (D) Comparison of the ammonia clearance activity after 17 days of hepatic differentiation. Error bars indicate standard deviation (n=3) .

Fig. 3 shows characterization of hepatic differentiated hiPSCs (201B6) and hESCs (KhES3) . (A) RT-PCR analysis of various CYP450 expression, ABC transporters and enzyme of gluclonidation in hepatic differentiated hiPSC and hESCs at day 17. (B) Periodic acid-Schiff (PAS) staining was performed to detect intracellular glycogen, (bar 100 μπι) (C) Assessment of liver-specific cytochrome P450 (CYP3A4) metabolic activity of hepatic differentiated hiPSCs and hESCs at day 17. Error bars indicate standard deviation (n=3) .

Fig. 4 shows close comparison of hepatic differentiation propensity between sibling hiPSC lines, 201B6 and 201B7. (A) Shematic presentation of modified protocol for hepatic differentiation. In this protocol, hiPSCs/hESCs were

enzymatically digested into single cells and plated on the Matrigel-coated dish. For endoderm cell induction, cells were cultivated with activin A, nt3a for 7 days. 0.5 mM sodium butyrate (NaB) was supplemented from day 1 for various durations (0 day - 6 days) . (B) Albumin secretion level after 21 days of hepatic differentiation. Error bars indicate standard deviation (n=3) . (C) Percentage of CXCR4 positive endoderm cells after 7 days of hepatic differentiation analyzed by flow cytometry. Error bars indicate standard deviation (n=3) . (D) Immunostaining of SOX17 and OCT3/4 after 7 days of hepatic differentiation. NaB was added for 3 days. (bar 100 urn)

Fig. 5 shows time course of endodermal and hepatic differentiation from single hiPSCs. Phase contrast images of endodermal differentiation from single hiPSCs (201B6, 201B7) and hepatic differentiation with further cultivation. 0.5 mM NaB was added for 3 days (day 1 - day 3) . (bar 100 μπι) At day 21 of hepatic differentiation, albumin staining was performed.

Fig. 6 shows characterization of undifferentiated cells, CXCR4 positive cells at day 7 and hepatic differentiated cells at day 21 derived from 201B6 and 201B7 hiPS clones by

microarray analysis. Microarray-based gene expression analysis showed that the global gene expression pattern including 10 liver-related transcription factors (orange dots and letters) is similar between 201B6 and 201B7 hiPSC lines at both

undifferentiated state and CXCR4 positive cells at day 7.

(A) (B) The green lines indicate the diagonal and 2-fold

changes between the two samples.

Fig. 7 shows comparison of albumin secretion potential of hepatic differentiated cells at day 21 among various

hiPSC/hESC lines. Various hiPSC lines were divided into 4 groups according to the original donor cell types. (A)

Duration of NaB administration (Od, 3d or 6d) , in which

highest albumin secretion was observed. (B) Albumin secretion potential of hepatic differentiated hiPSCs/hESCs at day 21 was analyzed by ELISA. In each clone, highest data among different NaB administration period (Od, 3d or 6d) was used for analysis.

Fig. 8 shows comparison of endoderm and hepatic

differentiation propensity among various hiPSC/hESC lines. (A) Percentage of CXCR4 positive endoderm cells at day 7 by flow cytometry analysis. (B) Albumin secretion potential of hepatic differentiated hiPSCs and hESCs at day 21 was analyzed by

ELISA. In each clone, data was obtained at different NaB administration period (Od, 3d and 6d during first 7 days of endodermal differentiation) . Error bars indicate standard deviation (n=3) . retro: retrovirus vector, epi : episomal vector *: Data was not obtained due to significant cell death or poor cell growth.

Detailed Description of the Invention

The present invention provides a method for producing a cell belonging to an endodermal lineage comprising inducing differentiation of a blood cell-derived iPS cell.

(I) Preparation of iPS cells

An iPS cell can be prepared by transferring a nuclear reprogramming substance to a somatic cell.

(a) Sources of somatic cells

The somatic cell to be used as a starting material for the production of an iPS cell in the present invention may be derived from any mammal such as human, mouse, monkey, bovine, swine, rat, dog and the like, as long as it is derived from blood. Specific examples of the blood cell include peripheral blood mononuclear cell (PBMC, also referred to as peripheral mononuclear cell (PM C) ) and cord blood cell . (CB cell). In addition, T cells, B cells, NK cells, NKT cells and the like obtained by separation from peripheral blood mononuclear cells can also be used as a somatic cell source in the present invention. PBMC . can be prepared by diluting the peripheral blood collected from a mammal with PBS etc. as necessary, and separating a mononuclear cell layer by Ficoll density gradient centrifugation . In addition, T cells, B cells, NK cells, NKT cells and the like can be isolated by flow cytometry and the like using an antibody against a cell surface marker specific to each of them.

The choice of mammal individual as a source of somatic cells is not particularly limited; however, when the endoderm cells as a product are to be used for the treatment of diseases such as liver disfunction in humans, it is preferabl from the viewpoint of prevention of graft rejection and/or GvHD, that blood cells are patient's own cells or collected from another person having the same or substantially the same HLA type as that of the patient. "Substantially the same HLA type" as used herein means that the HLA type of donor matches with that of patient to the extent that the transplanted cell which have been obtained by inducing differentiation of iPS cells derived from the donor's blood cells, can be engrafted when they are transplanted to the patient with use of

immunosuppressor and the like. For example, it includes an HLA type wherein major HLAs (the three major loci of HLA-A, HLA-B and HLA-DR or four loci further including HLA-Cw) are

identical (hereinafter the same meaning shall apply) and the like. When the endoderm cells are not to be administered (transplanted) to a human, but used as, for example, a source of cells for screening for evaluating a patient's drug

susceptibility or adverse reactions, it is likewise necessary to collect the blood cells from the patient or another person with the same genetic polymorphism correlating with the drug susceptibility or adverse reactions.

Blood cells separated from a mammal can be pre-cultured using a medium known per se suitable for the cultivation thereof, depending on the kind of the cells. Examples of such media include, but are not limited to, a minimal essential medium (MEM) containing about 5 to 20% fetal calf serum,

Dulbecco's modified Eagle medium (DMEM) , RPMI1640 medium, 199 medium, F12 medium, and the like. When using, for example, a transfection reagent such as a cationic liposome in contacting the cell with nuclear reprogramming substance (s) and iPS cell establishment efficiency improver (s), it is sometimes

preferable that the medium be previously replaced with a serum-free medium to prevent a reduction in the transfer efficiency.

(b) Nuclear reprogramming substances

In the present invention, "a nuclear reprogramming substance" refers to any substance (s) capable of inducing an iPS cell from a somatic cell, which may be composed of any substance such as a proteinous factor or a nucleic acid that encodes the same (including forms incorporated in a vector) , or a low-molecular compound. When the nuclear reprogramming substance is a proteinous factor or a nucleic acid that encodes the same, the following combinations, for example, are preferable (hereinafter, only the names for proteinous factors are shown) .

(1) Oct3/4, Klf4, c-Myc

(2) Oct3/4, Klf4, c-Myc, Sox2 (Sox2 is replaceable with Soxl, Sox3, Soxl5, Soxl7 or Soxl8; Klf4 is replaceable with Klfl, Klf2 or Klf5; c-Myc^' is replaceable with T58A (active mutant) , N-Myc, or L-Myc)

(3) Oct3/4, Klf4, c-Myc, Sox2, Fbxl5, Nanog, Eras, ECAT15-2, Tell, β-catenin (active mutant S33Y)

(4) Oct3/4, Klf4, c-Myc, Sox2, TERT, SV40 Large T antigen

(hereinafter SV40LT)

(5) Oct3/4, Klf4, c-Myc, Sox2, TERT, HPV16 E6

(6) Oct3/4, Klf4, c-Myc, Sox2, TERT, HPV16 E7

(7) Oct3/4, Klf4, c-Myc, Sox2, TERT, HPV6 E6, HPV16 E7

(8) Oct3/4, Klf4, c-Myc, Sox2, TERT, Bmil

[For more information on the factors shown above, see WO

2007/069666 (for information on replacement of Sox2 with Soxl8 and replacement of Klf4 with Klfl or Klf5 in the combination (2) above, see Nature Biotechnology, 26, 101-106 (2008)); for the combination "Oct3/4, Klf4, c-Myc, Sox2", see also Cell, 126, 663-676 (2006), Cell, 131, 861-872 (2007) and the like; for the combination "Oct3/4, Klf2 (or Klf5) , c-Myc, Sox2", see also Nat. Cell Biol., 11, 197-203 (2009); for the combination "Oct3/4, Klf4, c-Myc, Sox2, hTERT, SV40 LT", see also Nature, 451, 141-146 (2008).]

(9) Oct3/4, Klf4, Sox2 (see Nature Biotechnology, 26, 101-106 (2008) )

(10) Oct3/4, Sox2, Nanog, Lin28 (see Science, 318, 1917-1920 (2007) )

(11) Oct3/4, Sox2, Nanog, Lin28, hTERT, SV40LT (see Stem Cells, 26, 1998-2005 (2008))

(12) Oct3/4, Klf4, c-Myc, Sox2, Nanog, Lin28 (see Cell

Research (2008) 600-603)

(13) Oct3/4, Klf4, c-Myc, Sox2, SV40LT (see also Stem Cells, 26, 1998-2005 (2008))

(14) Oct3/4, Klf4 isee Nature 454:646-650 (2008), Cell Stem Cell, 2:525-528 (2.008))

(15) Oct3/4, c-Myc (see Nature 454:646-650 (2008))

(16) Oct3/4, Sox2 (see Nature, 451, 141-146 (2008),

WO2008/118820)

(17) Oct3/4, Sox2, Nanog (see WO2008/118820)

(18) Oct3/4, Sox2, Lin28 (see WO2008/118820)

(19) Oct3/4, Sox2, c-Myc, Esrrb (Here, Essrrb can be

substituted by Esrrg, see Nat. Cell Biol., 11, 197-203 (2009))

(20) Oct3/4, Sox2, Esrrb (see Nat. Cell Biol., 11, 197-203 (2009))

(21) Oct3/4, Klf4, L-Myc

(22) Oct3/4, Nanog

(23) Oct3/4

(24) Oct3/4, Klf4, c-Myc, Sox2, Nanog, Lin28, SV40LT (see

Science, 324: 797-801 (2009))

In (l)-(24) above, Oct3/4 may be replaced with another member of the Oct family, for example, OctlA, Oct6 or the like. Sox2 (or Soxl, Sox3, Soxl5, Soxl7, Soxl8) may be replaced with another member of the Sox family, for example, Sox7 or the like. In place of Klf4, other members of the Klf family, for example, Klfl, Klf2 and Klf5, or known Klf4 substitutes, for example, members of the Esrr family such as Esrrb, Esrrg and the like, members of the IRX family such as IRX1, IRX2, IRX3, IRX4, IRX5, IRX6 and the like, members of the GLIS family such as GLISl, GLIS2, GLIS3 and the like, members of the PTX family such as PITX1, PITX2, PITX3 and the like, and DMRTB1 can also be used. Furthermore, in (1) to (24) above, when c-Myc or

Lin28 is included as a nuclear reprogramming factor, L-Myc or Lin28B can be used in place of c-Myc or Lin28, respectively. Members of the GLIS family such as GLISl and GLIS3 may also be used in place of c-Myc.

Any combination that does not fall in (1) to (24) above but comprises all the constituents of any one of (1) to (24) above and further comprises an optionally chosen other

substance can also be included in the scope of "nuclear reprogramming substances" in the present invention. Provided that the somatic cell to undergo nuclear reprogramming is endogenously expressing one or more of the constituents of any one of (1) to (24) above at a level sufficient to cause

nuclear reprogramming, a combination of only the remaining constituents excluding the one or more constituents can also be included in the scope of "nuclear reprogramming substances" in the present invention.

Among these combinations, at least one, preferably two or more, more preferably three or more selected from Oct3/4, Sox2, Klf4, c-Myc or L-Myc, Nanog, Lin28 or Lin28B and GLIS1 or

GLIS3 can be mentioned as examples of preferable nuclear reprogramming substance.

Particularly, when the iPS cells obtained are to be used for therapeutic purposes, a combination of the three factors Oct3/4, Sox2 and Klf [combination (9) above] are preferably used. When the iPS cells obtained are not to be used for therapeutic purposes (e.g., used as an investigational tool for drug discovery screening and the like) , three factors of Oct3/4, Sox2 and Klf4, as well as four factors additionally further containing c-Myc/L-Myc, five factors additionally further containing Lin28/Lin28B, six factors additionally further containing GLIS1/GLIS3, seven factors additionally further containing Nanog, and the like can be recited as

examples .

Mouse and human cDNA sequence information on the

aforementioned proteinous factors can be acquired by referring to the NCBI accession numbers mentioned in WO 2007/069666 (in the publication, Nanog is mentioned with the designation

"ECAT4"; mouse and human cDNA sequence information on L-Myc, Lin28, Lin28B, GLIS1, GLIS3, Esrrb and Esrrg can be acquired by referring to the following NCBI accession numbers,

respectively) ; those skilled in the art are easily able to isolate these cDNAs. Name of gene Mouse Human

L-Myc . NM_008506 NM_001033081

Lin28 NM_145833 NM_024674

Lin28b NM_001031772 NM_001004317

GLIS1 NM_147221 NM_147193

GLIS3 NM_175459 NM_001042413

Esrrb NM_011934 NM_004452

Esrrg NM_011935 NM_001438

A proteinous factor for use as a nuclear reprogramming substance can be prepared by inserting the cDNA obtained into an appropriate expression vector, introducing the vector into a host cell, and recovering the recombinant proteinous factor from the cultured cell or its conditioned medium. Meanwhile, when the nuclear reprogramming substance used is a nucleic acid that encodes a proteinous factor, the cDNA obtained is inserted into a viral vector, plasmid vector, episomal vector etc. to construct an expression vector, and the vector is subjected to the step of nuclear reprogramming.

(c) Method of transferring a nuclear reprogramming substance to a somatic cell

Transfer of a nuclear reprogramming substance to a somatic cell (i.e. blood cell) can be achieved using a method known per se for protein transfer into a cell, provided that the substance is a proteinous factor. In view of human clinical applications, it is preferable that the starting material iPS cell be also prepared without gene manipulation.

Such methods include, for example, the method using a protein transfer reagent, the method using a protein transfer domain (PTD) - or cell penetrating peptide (CPP)- fusion protein, the microinjection method and the like. Protein transfer reagents are commercially available, including those based on a cationic lipid, such as BioPOTER Protein Delivery Reagent (Gene Therapy Systems), Pro-Ject™ Protein Transfection Reagent (PIERCE) and ProVectin (IMGENEX) ; those based on a lipid, such as Profect-1 (Targeting Systems) ; those based on a membrane-permeable peptide, such as Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif), GenomONE (ISHIHARA SANGYO KAISHA, LTD.) utilizing HVJ envelope (inactivated hemagglutinating virus of Japan) and the like. The transfer 5 can be achieved per the protocols attached to these reagents, a common procedure being as described below. Nuclear

reprogramming substance (s) is (are) diluted in an appropriate solvent (e.g., a buffer solution such as PBS or HEPES) , a transfer reagent is added, the mixture is incubated at room 10 temperature for about 5 to 15 minutes to form a complex, this complex is added to cells after exchanging the medium with a serum-free medium, and the cells are incubated at 37°C for one to several hours. Thereafter, the medium is removed and replaced with a serum-containing medium.

15 Developed PTDs include those using transcellular domains of proteins such as drosophila-derived AntP, HIV-derived TAT (Frankel, A. et al, Cell 55, 1189-93 (1988) or Green, M. & Loewenstein, P. M. Cell 55, 1179-88 (1988)), Penetratin

(Derossi, D. et al, J. Biol. Chem. 269, 10444-50 (1994)),

20 Buforin II (Park, C. B. et al. Proc. Natl Acad. Sci. USA 97, 8245-50 (2000)), Transportan (Pooga, M. et al. FASEB J. 12, 67-77 (1998)), MAP (model amphipathic peptide) (Oehlke, J. et al. Biochim. Biophys. Acta. 1414, 127-39 (1998)), K-FGF (Lin, Y. Z. et al. J. Biol. Chem. 270, 14255-14258 (1995) ) , Ku70

25 (Sawada, M. et al. Nature Cell Biol. 5, 352-7 (2003)), Prion (Lundberg, P. et al. Biochem. Biophys. Res. Commun. 299, 85-90 (2002)), pVEC (Elmquist, A. et al . Exp. Cell Res. 269, 237-44 (2001)), Pep-1 (Morris, M. C. et al. Nature Biotechnol. 19, 1173-6 (2001)), Pep-7 (Gao, C. et al. Bioorg. Med. Chem. 10,

30 4057-65 (2002)), SynBl (Rousselle, C. et al. Mol. Pharmacol.

57, 679-86 (2000)), HN-I (Hong, F. D. & dayman, G L. Cancer Res. 60, 6551-6 (2000)), and HSV-derived VP22. CPPs derived from the PTDs include polyarginines such as 11R {Cell Stem Cell, 4,381-384 (2009)) and 9R (Cell Stem Cell, 4, 472-476

35 (2009) ) . A fused protein expression vector incorporating cDNA of a nuclear reprogramming substance and PTD or CPP sequence is prepared, and recombination expression is performed using the vector. The fused protein is recovered and used for transfer. Transfer can be performed in the same manner as above except that a protein transfer reagent is not added.

Microinjection, a method of placing a protein solution in a glass needle having a tip diameter of about 1 um, and injecting the solution into a cell, ensures the transfer of the protein into the cell.

Other useful methods of protein transfer include

electroporation, the semi-intact cell method [ ano, F. et al. Methods in Molecular Biology, Vol. 322, 357-365(2006)], transfer using the r-t peptide [Kondo, E. et al., Mol . Cancer Ther. 3(12), 1623-1630(2004)] and the like.

The protein transferring operation can be performed one or more optionally chosen times (e.g., once or more to 10 times or less, or once or more to 5 times or less and the like) . Preferably, the transferring operation can be performed twice or more (e.g., 3 times or 4 times) repeatedly. The time interval for repeated transferring operation is, for example, 6 hours to 7 days, preferably 12 to 48 hours or 7 days.

However, taking into account the efficiency of

establishment of iPS cells, nuclear reprogramming substance may also be used preferably in the form of a nucleic acid that encodes a proteinous factor, rather than the factor as it is. The nucleic acid may be a DNA or an RNA, or a DNA/RNA chimera, and may be double-stranded or single-stranded. Preferably, the nucleic acid is a double-stranded DNA, particularly a cDNA.

A cDNA of a nuclear reprogramming substance is inserted into an appropriate expression vector comprising a promoter capable of functioning in a host somatic cell. Useful

expression vectors include, for example, viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus and Sendai virus, plasmids for the expression in animal cells (e.g., pAl-11, pXTl, pRc/CMV, pRc/RSV, pcDNAI/Neo) and the like.

A vector for this purpose can be chosen as appropriate according to the intended use of the iPS cell to be obtained. Useful vectors include adenovirus vector, plasmid vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, Sendai virus vector, episomal vector and the like/^"-'

Examples of promoters used in expression vectors include the EFla promoter, the CAG promoter, the SRa promoter, the SV40 promoter, the LTR promoter, the C V (cytomegalovirus) promoter, the RSV (Rous sarcoma virus) promoter, the MoMuLV (Moloney mouse leukemia virus) LTR, the HSV-TK (herpes simplex virus thymidine kinase) promoter and the like, with preference given to the EFla promoter, the CAG promoter, the MoMuLV LTR, the CMV promoter, the SRa promoter and the like.

The expression vector may contain as desired, in addition to a promoter, an enhancer, a polyadenylation signal, a

selectable marker gene, a SV40 replication origin and the like. Examples of selectable marker genes include the dihydrofolate reductase gene, the neomycin resistant gene, the puromycin resistant gene and the like.

The nucleic acids as nuclear reprogramming substances (reprogramming genes) may be separately integrated into

different expression vectors, or 2 kinds or more, preferably 2 to 3 kinds, of genes may be incorporated into a single

expression vector. Preference is given to the former case with the use of a retrovirus or lentivirus vector, which offer high gene transfer efficiency, and to the latter case with the use of a plasmid, adenovirus, or episomal vector and the like.

Furthermore, an expression vector incorporating two kinds or more of genes and another expression vector incorporating one gene alone can be used in combination.

In the context above, when a plurality of genes are incorporated in one expression vector, these genes can

preferably be inserted into the expression vector via an intervening sequence enabling polycistronic expression. By using an intervening sequence enabling polycistronic

expression, it is possible to more efficiently express a plurality of genes incorporated in one kind of expression vector. Useful sequences enabling polycistronic expression include, for example, the 2A sequence of foot-and-mouth disease virus {PLoS ONE 3, e2532, 2008, Stem Cells 25, 1707, 2007), IRES sequence (U.S. Patent No. 4,937,190) and the like, with preference given to the 2A sequence.

An expression vector harboring a nucleic acid as a nuclear reprogramming substance can be introduced into a cell by a technique known per se according to the choice of the vector. In the case of a viral vector, for example, a plasmid containing the nucleic acid is introduced into an appropriate packaging cell (e.g., Plat-E cells) or a complementary cell line (e.g., 293-cells) , the viral vector produced in the culture supernatant is recovered, and the vector is infected to the cell by a method suitable for the viral vector. For example, specific means using a retroviral vector are

disclosed in WO2007/69666, Cell, 126, 663-676 (2006) and Cell _r 131, 861-872 (2007). Specific means using a lentivirus vector is disclosed in Science, 318, 1917-1920 (2007). When PGC-like cells induced from iPS cells are utilized for regenerative medicine such as treatment of infertility and gene therapy of germ cells, an expression (reactivation) of a reprogramming gene potentially increases the risk of carcinogenesis in germ cells or reproductive tissues regenerated from PGC-like cells derived from iPS cells; therefore, a nucleic acid encoding a nuclear reprogramming substance is preferably expressed transiently, without being integrated into the chromosome of the cells. From this viewpoint, use of an adenoviral vector, whose integration into chromosome is rare, is preferred.

Specific means using an adenoviral vector is disclosed in Science, 322, 945-949 (2008) . Because an adeno-associated viral vector is also low in the frequency of integration into chromosome, and is lower than adenoviral vectors in terms of cytotoxicity and inflammation-inducibility, it can be

mentioned as another preferred vector. Because Sendai viral vector is capable of being stably present outside the

5 chromosome, and can be degraded and removed using an siRNA as required, it is preferably utilized as well. Regarding a Sendai viral vector, one described in J. Biol. Chem. , 282, 27383-27391 (2007) and JP-3602058 B can be used.

When a retroviral vector or a lentiviral vector is used,

10 even if silencing of the transgene has occurred, it possibly becomes reactivated; therefore, for example, a method can be used preferably wherein a nucleic acid encoding a nuclear reprogramming substance is cut out using the Cre-loxP system, when becoming unnecessary. That is, with loxP sequences

15 arranged on both ends of the nucleic acid in advance, iPS

cells are induced, thereafter the Cre recombinase is allowed to act on the cells using a plasmid vector or adenoviral vector, and the region sandwiched by the loxP sequences can be cut out. Because the enhancer-promoter sequence of the LTR U3

20 region possibly upregulates a^' host gene in the vicinity

thereof by insertion mutation, it is more preferable to avoid the expression regulation of the endogenous gene by the LTR outside of the loxP sequence remaining in the genome without being cut out, using a 3' -self-inactivating (SIN) LTR prepared

25 by deleting the sequence, or substituting the sequence with a polyadenylation sequence such as of SV40. Specific means using the Cre-loxP system and SIN LTR is disclosed in Chang et al., Stem Cells, 27: 1042-1049 (2009) .

Meanwhile, being a non-viral vector, a plasmid vector can

30 be transferred into a cell using the lipofection method,

liposome method, electroporation method, calcium phosphate co- precipitation method, DEAE dextran method, microinjection method, gene gun method and the like. Specific means using a plasmid as a vector are described in, for example, Science,

35 322, 949-953 (2008) and the like. When a plasmid vector, an adenovirus vector and the like are used, the transfection can be performed once or more optionally chosen times (e.g., once to 10 times, once to 5 times or the like) . When two or more kinds of expression vectors are introduced into a somatic cell, it is preferable that these all kinds of expression vectors be concurrently introduced into a somatic cell; however, even in this case, the transfection can be performed once or more optionally chosen times (e.g., once to 10 times, once to 5 times or the like) , preferably the transfection can be repeatedly performed twice or more (e.g., 3 times or 4 times).

Also when an adenovirus or a plasmid is used, the

transgene can get integrated into chromosome; therefore, it is eventually necessary to confirm the absence of insertion of the gene into chromosome by Southern blotting or PCR. For this reason, like the aforementioned Cre-loxP system, it can be advantageous to use a means wherein the transgene is

integrated into chromosome, thereafter the gene is removed. In another preferred mode of embodiment, a method can be used wherein the transgene is integrated into chromosome using a transposon, thereafter a transposase is allowed to act on the cell using a plasmid vector or adenoviral vector so as to completely eliminate the transgene from the chromosome. As examples of preferable transposons, piggyBac, a transposon derived from a lepidopterous insect, and the like can be mentioned. Specific means using the piggyBac transposon is disclosed in Kaji, K. et al . , Nature, 458: 771-775 (2009), Woltjen et al., Nature, 458: 766-770 (2009).

Another preferred non-recombination type vector is an episomal vector autonomously replicable outside the chromosome. A specific procedure for using an episomal vector is disclosed by Yu et al. in Science, 324, 797-801 (2009). As required, an expression vector may be constructed by inserting a

reprogramming gene into an episomal vector having loxP

sequences placed in the same orientation at both the 5' and 3' sides of the vector element essential for the replication of the episomal vector, and this may be transferred into a somatic cell.

Examples of the episomal vector include vectors

comprising a sequence required for its autonomous replication, derived from EBV, SV40 and the like, as a vector element.

Specifically, the vector element required for its autonomous replication is a replication origin or a gene that encodes a protein that binds to the replication origin to regulate its replication; examples include the replication origin oriP and EBNA-1 gene for EBV, and the replication origin ori and SV40 large T antigen gene for SV40.

The episomal expression vector contains a promoter that controls the transcription of the reprogramming gene. The promoter used can be the same promoter as the above. The episomal expression vector may further comprise an enhancer, poly-A addition signal, selection marker gene and the like as desired, as described above. Examples of selection marker gene include the dihydrofolate reductase gene, neomycin resistance gene and the like.

An episomal vector can be introduced into a cell using, for example, lipofection method, liposome method,

electroporation method, calcium phosphate co-precipitation method, DEAE dextran method, microinjection method, gene gun method and the like. Specifically, the method described in Science, 324: 797-801 (2009), for example, can be used.

Whether or not the vector element required for

replication of reprogramming gene has been removed from the iPS cell can be determined by performing Southern blot

analysis or PCR analysis using a part of the vector as a probe or primer, with an episome fraction isolated from the iPS cell as the template, to examine for the presence or absence of a band or the length of the band detected. An episome fraction can be prepared using a method well known in the art, for example, the method described in Science, 324: 797-801 (2009) . When the nuclear reprogramming substance is a low- molecular compound, introduction thereof into a somatic cell can be achieved by dissolving the substance at an appropriate concentration in an aqueous or non-aqueous solvent, adding the solution to a medium suitable for cultivation of somatic cells isolated from human or mouse [e.g., minimal essential medium (MEM) comprising about 5 to 20% fetal bovine serum, Dulbecco' s modified Eagle medium (DMEM) , RPMI1640 medium, 199 medium, F12 medium, and the like] so that the nuclear reprogramming substance concentration will fall in a range that is

sufficient to cause nuclear reprogramming in somatic cells and does not cause cytotoxicity, and culturing the cells for a given period. The nuclear reprogramming substance

concentration varies depending on the kind of nuclear

reprogramming substance used, and is chosen as appropriate over the range of about 0.1 nM to about 100 nM. Duration of contact is not particularly limited, as far as it is

sufficient to cause nuclear reprogramming of the cells;

usually, the nuclear reprogramming substance may be allowed to be co-present in the medium until a positive colony emerges. (d) iPS cell establishment efficiency improvers

In recent years, various substances that improve the efficiency of establishment of iPS cells, which has

traditionally been low, have been proposed one after another. When brought into contact with a somatic cell together with the aforementioned nuclear reprogramming substances, these establishment efficiency improvers are expected to further raise the efficiency of establishment of iPS cells.

Examples of iPS cell establishment efficiency improvers include, but are not limited to, histone deacetylase (HDAC) inhibitors [e.g., valproic acid (VPA) {Nat. Biotechnol. ,

26{1): 795-797 (2008)), low-molecular inhibitors such as trichostatin A, sodium butyrate, MC 1293, and M344, nucleic acid-based expression inhibitors such as siRNAs and shRNAs against HDAC (e.g., HDAC1 siRNA Smartpool^® (Millipore) , HuSH 29mer shRNA Constructs against HDAC1 (OriGene) and the like) , and the like], DNA methyltransferase inhibitors (e.g., 5'- azacytidine) [Nat. Biotechnol., 26{1) 795-797 (2008)], G9a histone methyltransferase inhibitors [e.g., low-molecular inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), nucleic acid-based expression inhibitors such as siRNAs and shRNAs against G9a (e.g., G9a siRNA (human) (Santa Cruz Biotechnology) and the like) and the like] , L-channel calcium agonists (e.g., Bayk8644) [Cell Stem Cell, 3, 568-574 (2008)], p53 inhibitors [e.g., siRNA and shRNA against p53 (Cell Stem Cell, 3, 475-479 (2008)), UTF1 [Cell Stem Cell, 3, 475-479 (2008)], Wnt Signaling activator (e.g., soluble Wnt3a) [Cell Stem Cell, 3, 132-135 (2008)], 2i/LIF [2i is an

inhibitor of mitogen-activated protein kinase signaling and glycogen synthase kinase-3, PloS Biology, 6(10), 2237-2247 (2008)], ES cell-specific miRNA [for example, miR-302-367 cluster (Mol. Cell. Biol, doi : 10.1128/MCB .00398-08 ) , miR-302 (RNA (2008) 14: 1-10), miR-291-3p, miR-294 and miR-295 (for above, Nat. Biotechnol. 27: 459-461 (2009))], 3'- phosphoinositide-dependent kinase-1 (PDK1) activator [for example, PS48 (Cell Stem Cell, 7: 651-655 (2010))] and the like. As mentioned above, the nucleic acid-based expression inhibitors may be in the form of expression vectors harboring a DNA that encodes an siRNA or shRNA.

Among the constituents of the aforementioned nuclear reprogramming substances, SV40 large T and the like, for example, can also be included in the scope of iPS cell

establishment efficiency improvers because they are deemed not essential, but auxiliary, factors for somatic cell nuclear reprogramming. In the situation of the mechanisms for nuclear reprogramming remaining unclear, the auxiliary factors, which are not essential for nuclear reprogramming, may be

conveniently considered as nuclear reprogramming substances or iPS cell establishment efficiency improvers. Hence, because the somatic cell nuclear reprogramming process is understood as an overall event resulting from contact of nuclear

reprogramming substance (s) and iPS cell establishment

efficiency improver (s) with a somatic cell, it seems

unnecessary for those skilled in the art to always distinguish between the nuclear reprogramming substance and the iPS cell establishment efficiency improver.

Contact of an iPS cell establishment efficiency improver with a somatic cell can be achieved as described above for each of three cases: (a) the improver is a proteinous factor, (b) the improver is a nucleic acid that encodes the proteinous factor, and (c) the improver is a low-molecular compound.

An iPS cell establishment efficiency improver may be brought into contact with a somatic cell simultaneously with a nuclear reprogramming substance, or either one may be

contacted in advance, as far as the efficiency of

establishment of iPS cells from the somatic cell is

significantly improved, compared with the absence of the improver. In an embodiment, for example, when the nuclear reprogramming substance is a nucleic acid that encodes a proteinous factor and the iPS cell establishment efficiency improver is a chemical inhibitor, the iPS cell establishment efficiency improver can be added to the medium after the cell is cultured for a given length of time after the gene transfer treatment, because the nuclear reprogramming substance

involves a given length of time lag from the gene transfer treatment to the mass-expression of the proteinous factor, whereas the iPS cell establishment efficiency improver is capable of rapidly acting on the cell. In another embodiment, when a nuclear reprogramming substance and an iPS cell

establishment efficiency improver are both used in the form of a viral, plasmid or episomal vector, for example, both may be simultaneously introduced into the cell.

(e) Improving the establishment efficiency by culture

conditions

The efficiency of establishment of iPS cells can be further improved by culturing the somatic cells therefor under hypoxic conditions in the step of nuclear reprogramming of the cells. The term hypoxic conditions as used herein means that the oxygen concentration in the ambient atmosphere during cell culture is significantly lower than that in the air.

Specifically, such conditions include lower oxygen

concentrations than the oxygen concentrations in the ambient atmosphere of 5-10% CO₂/95-90% air, which is commonly used for ordinary cell culture; for example, oxygen concentrations of 18% or less in the ambient atmosphere are applicable.

Preferably, the oxygen concentration in the ambient atmosphere is 15% or less (e.g., 14% or less, 13% or less, 12% or less, 11% or less and the like), 10% or less (e.g., 9% or less, 8% or less, 7% or less, 6% or less and the like) , or 5% or less (e.g., 4% or less, 3% or less, 2% or less and the like). The oxygen concentration in the ambient atmosphere is preferably 0.1% or more (e.g., 0.2% or more, 0.3% or more, 0.4% or more and the like), 0.5% or more (e.g., 0.6% or more, 0.7% or more, 0.8% or more, 0.9% or more and the like), or 1% or more (e.g., 1.1% or more, 1.2% or more, 1.3% or more, 1.4% or more and the like) .

There is no limitation on how to create hypoxic

conditions in a cellular environment; the easiest of suitable methods is to culture cells in a C0₂ incubator that allows control of oxygen concentrations. Such C0₂ incubators are commercially available from a number of manufacturers of equipment (e.g., C0₂ incubators for hypoxic culture

manufactured by Thermo Scientific, Ikemoto Scientific

Technology, Juji Field Inc., and Wakenyaku Co., Ltd. can be used) .

The timing of beginning cell culture under hypoxic conditions is not particularly limited, as far as it does not interfere with improving the efficiency of establishment of iPS cells compared with that obtained at a normal oxygen concentration (20%) . The starting time may be before or after contact of nuclear reprogramming substances with a somatic cell, and may be at the same time as the contact. For example, it is preferable that cell culture under hypoxic conditions be begun just after contacting a nuclear reprogramming substance with a somatic cell, or after a given time (e.g., 1 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8 or 9) days) following the contact.

The duration of cell culture under hypoxic conditions is not particularly limited, as far as it does not interfere with improving the efficiency of establishment of iPS cells

compared with that obtained at a normal oxygen concentration (20%); examples include, but are not limited to, between 3 days or more, 5 days or more, 7 days or more or 10 days or more, and 50 days or less, 40 days or less, 35 days or less or 30 days or less. The preferred duration of cell culture under hypoxic conditions also varies depending on the oxygen

concentration in the ambient atmosphere; those skilled in the art can adjust as appropriate the duration of cell culture according to the oxygen concentration used. In an embodiment of the present invention, when iPS cell candidate colonies are selected with drug resistance as an indicator, it is

preferable that a normal oxygen concentration be restored from hypoxic conditions by the start of drug selection.

Furthermore, the preferred starting time and duration of cell culture under hypoxic conditions also vary depending on the choice of nuclear reprogramming substances used, the efficiency of establishment of iPS cells under conditions involving a normal oxygen concentration, and other factors.

After the nuclear reprogramming substance (s) (and iPS cell establishment efficiency improver (s) ) is (are) brought into contact with the cell, the cell can be cultured under conditions suitable for the cultivation of, for example, ES cells. In the case of mouse cells, the cultivation is carried out with the addition of Leukemia Inhibitory Factor (LIF) as a differentiation suppressor to an ordinary medium. Meanwhile, in the case of human cells, it is desirable that basic fibroblast growth factor (bFGF) and/or stem cell factor (SCF) be added in place of LIF. Usually, the cells are cultured in the co-presence of mouse embryo-derived fibroblasts (MEFs) treated with radiation or an antibiotic to terminate the cell division thereof, as feeder cells. Usually, STO cells and the like are commonly used as MEFs, but for inducing iPS cells, SNL cells [McMahon, A. P. & Bradley, A. Cell 62, 1073-1085

(1990)] and the like are commonly used. Co-culture with feeder cells may be started before contact of the nuclear

reprogramming substance, at the time of the contact, or after the contact (e.g., 1-10 days later).

A candidate colony of iPS cells can be selected by a method with drug resistance and reporter activity as

indicators, and also by a method based on visual examination of morphology. As an example of the former, a colony positive for drug resistance and/or reporter activity is selected using a recombinant somatic cell wherein a drug resistance gene and/or a reporter gene is targeted to the locus of a gene highly expressed specifically in pluripotent cells (e.g.,

Fbxl5, Nanog, Oct3/4 and the like, preferably Nanog or Oct3/4) . Examples of such recombinant somatic cells include blood cells from a mouse having the Pgeo (which encodes a fusion protein of β-galactosidase and neomycin phosphotransferase) gene knocked- in to the Fbxl5 locus [Takahashi & Yamanaka, Cell, 126, 663- 676 (2006) ] , blood cells from a transgenic mouse having the green fluorescent protein (GFP) gene and the puromycin

resistance gene integrated in the Nanog locus [Okita et al., Nature, 448, 313-317 (2007)] and the like. Meanwhile, examples of the method of selecting candidate colonies based on visual examination of morphology include the method described by

Takahashi et al. in Cell, 131, 861-872 (2007). Although the method using reporter cells is convenient and efficient, it is desirable from the viewpoint of safety that colonies be

selected by visual examination when iPS cells are prepared for the purpose of human treatment. When the three factors Oct3/4, Klf4 and Sox2 are used as nuclear reprogramming substances, the number of clones established decreases but the resulting colonies are mostly of iPS cells of high quality comparable to ES cells, so that iPS cells can efficiently be established even without using reporter cells.

The identity of the cells of a selected colony as iPS cells can be confirmed by positive responses to a Nanog (or Oct3/4) reporter (puromycin resistance, GFP positivity and the like) as well as by the formation of a visible ES cell-like colony, as described above. However, to ensure higher accuracy, it is possible to perform tests such as analyzing the

expression of various ES cell-specific genes and transplanting the cells selected to a mouse and confirming the formation of teratomas .

(2) Induction of differentiation from iPSCs to endoderm cells The blood cell-derived iPS cell produced as mentioned above can be differentiated into a cell belonging to an

endodermal lineage by any existing differentiation induction method. Here, the "cell belonging to the endodermal lineage" is specifically classified into a hepatic lineage cell, a pancreatic lineage cell, and other lineage cell. Specific examples of the hepatic lineage cell include hepatocyte, bile duct epithelial cell and the like. Specific examples of the pancreatic lineage cell include endocrine pancreas cell (β cell etc.), exocrine pancreas cell, pancreatic duct epithelial cell and the like. Specific examples of other lineage cell include gastrointestinal tract epithelial cell, alveolar

epithelial cell, thyroid follicular epithelial cell and the like. Preferably, the cell belonging to the endodermal lineage efficiently induced to differentiate from a blood cell-derived iPS cell is a hepatic lineage cell, more preferably a

hepatocyte .

Examples of the basic medium for differentiation

induction include, but are not limited to, serum-free minimum essential medium (MEM), Dulbecco's modified Eagle medium (DMEM) , RPMI1640 medium, 199 medium, F12 medium and a mixed medium thereof, any of the aforementioned media supplemented with an appropriate concentration of a well-known

conventionally-used medium additive (e.g., serum albumin, 2- mercaptoethanol, insulin, transferrin, sodium selenite,

ethanolamine, antibiotic (e.g., penicillin, streptomycin) etc.) and the like. A serum may or may not be added as

appropriate to a medium according to the differentiation induction method to be used.

Cultivation is performed by seeding iPS cells in a culture container known per se (e.g., 10 cm cell culture dish coated with gelatin, atrigel, collagen etc., or seeded with a suitable feeder cell, and the like) at a cell density of, for example, about 3- about 10 x 10⁴ cells/mL, preferably about 4- about 8 x 10⁴ cells/mL (about 3- about 10 x 10⁵ cells/10 cm dish, preferably about 4- about 8 x 10⁵ cells/10 cm dish, and cultivating in an incubator at 5% C0₂/95% air, about 30- about. 40°C, preferably about 37°C.

Examples of the known differentiation induction method of iPS cell into hepatocyte include the methods described in Song, Z. et al., Cell Res., 15(11): 1233-1242 (2009); Sullivan, G.J. et al., Hepatology, 52(1): 329-335 (2010); Touboul, T. et al., Hepatology, 52(5): 1754-1765 (2010); Si-Tayeb, K. et al.,

Hepatology, 52(1): 297-305 (2010); Rashid, S.T. et al . , J.

Clin. Invest., 120(9): 3127-3136 (2010); Ghodsizadeh, A. et al., Stem Cell Rev., 6(A): 622-632 (2010); Inamura, M. et al., Mol. Ther., 19(2): 400-407 (2011); Jozefczuk, J. et al., Stem Cells Dev., 20(1): 1259-1275 (2011); Ohi, Y. et al . , Nat. Cell Biol., 13(5): 541-549 (2011); Nagae, G. et al., Hum. Mol.

Genet., 20(14): 2710-2721 (2011); Takata, A. et al., Hepatol. Int., Feb 6. (2011) [Epub ahead of print] and the like.

It is also possible to use a differentiation induction method of ES cell into hepatocyte and, for example, the

methods described in Hay, D.C. et al., Stem Cells, 26(A): 894- 902 (2008); Hay, D.C. et al . , Proc. Natl. Acad. Sci. USA, 105(34): 12301-12306 (2008) and the like can be used directly or after partial alteration.

Conventionally, as a method for inducing differentiation of an ES cell into a cell belonging to an endodermal lineage, a method including forming embryoid body (EB) by floating culture is general. For example, using expression of

Brachyury (T) , which is an early mesodermal marker, as an index, early mesoderm is induced by the EB formation method, and concentrated by flow cytometry, activin A is added under serum-free conditions and attachment culture is performed, whereby differentiation induction into endoderm is performed (Development, 131: 1651-1662 (2004)). In addition, using expression of Foxa2 and T, which are mesendoderm/endoderm markers, as an index, differentiation of anterior primitive streak cells from which endoderm is developed in normal

development can be induced (Proc. Natl. Acad. Sci. USA, 103: 16806-16811 (2006) ) . Moreover, endoderm can also be induced without via EB by cultivating ES cell in an activin A- containing serum-free medium (Nat. Biotechnol., 23: 1542-1550 (2005); Nat. Biotechnol., 23: 1534-1541 (2005)).

Since a signal from mesoderm is considered to be

essential in the development process of endoderm, mesendoderm and endoderm can be induced by normal development, without via EB formation, by using a mesoderm-derived cultured cell (e.g., M15 cell which is a mouse fetal kidney-derived cultured cell etc.) as a feeder cell (Stein Cells, 26: 874-885 (2008)). As hormonal factors, activin A and bFGF are added to a medium to efficiently induce differentiation into endoderm, since bFGF transfers ES cell from an undifferentiated state to a

differentiation state and then activin A promotes

differentiation into mesendoderm while inhibiting

differentiation- into ectoderm, and further, activin A and bFGF promote differentiation of mesendoderm into endoderm. On the other hand, BMP can be added to the primary medium since it inhibits, like activin A, differentiation of ES cell into ectoderm, but is desirably removed from the medium after expression of an early mesoderm marker since it promotes differentiation of mesendoderm into mesoderm. After endoderm induction, differentiation into a-fetoprotein (AFP) positive hepatic progenitor cell can be promoted by substituting the serum with Knockout Serum Replacement (KSR) . Hepatic

differentiation can be further promoted by adding

dexamethasone, a hepatocyte growth factor (HGF) and oncostatin M (OS ) . Finally, albumin positive hepatocyte and DBA positive bile duct cell can be induced, and increase of glycogen storage, expression of drug-metabolizing enzyme (cytochrome P450 group) can also be confirmed (Genes Cells, 13: 731-746 (2008) ) .

Other examples of the differentiation induction method of ES cell into hepatic, lineage cell include, but are not limited to, a method including forming EB in a medium containing activin A and BMP4, and performing an attachment culture {Nat. Biotechnol., 24: 1402-1411 (2006)), a method including

differentiating a hepatic progenitor cell, concentrating same and co-culturing liver-derived mesenchymal cell (Exp. Cell Res., 309: 68-77 (2005)) and the like.

On the other hand, examples of known differentiation induction method of ES cell into a pancreatic lineage cell include a method including an attachment culture after

embryoid body formation (Stem Cells, 22: 1205-1217 (2004)), a method including separating T positive cell {Development, 131: 1651-1662 (2004)) or T and Foxa2 positive cell (Proc. Natl. Acad. Sci. USA, 103: 16806-16811 (2006)), followed by an attachment culture, a method including, in co-culture with the above-mentioned M15 cell, continuing culture in the presence of a serum and a hormonal factor (activin A and bFGF) after endoderm induction (Stem Cells, 26: 874-885 (2008) ) and the like.

Moreover, examples of known differentiation induction method of ES cell into other lineage cell include a method including inducing differentiation of thyroid cell by

attachment culture after EB formation {Endocrinology, 147:

3007-3015 (2006) ) , a method including inducing differentiation of pneumocyte by attachment culture in an activin-containing serum-free medium after EB formation (Cloning Stem Cells, 10: 49-64 (2008)), a method including inducing differentiation of pneumocyte by culturing a cell extract of mouse lung

epithelial cell line after EB formation (Stem Cells, 23: 712- 718 (2005)), a method including inducing enterocyte by an EB formation method (Stem Cells, 24: 2618-2626 (2006)) and the like .

All the above-mentioned methods for ES cell can be utilized for differentiation induction of iPS cell into hepatocyte in the present invention.

In one preferable embodiment of the present invention, a method of inducing differentiation of a cell belonging to an endodermal lineage is provided in which differentiation is not induced by using a colony already grown to some level as in the conventional methods but by using an iPS cell in a single- cell state. Use of iPS cells as a colony, namely, in a cell mass state, is defective in that the size and nonuniformity of the colony influence differentiation induction efficiency and reproducibility. In the case of a human iPS cell, however, when it is used in a single-cell state for differentiation induction, problems of easy occurrence of apoptosis and the like are caused. In addition, when the conditions are the same as those for conventional methods of differentiation induction using a colony, new appropriate culture conditions need to be determined since the sensitivity of the hormonal factors to be added to the medium varies markedly.

In the existing methods used for human ES cells (Hay, D.C. et al., Stem Cells, 26(4): 894-902 (2008); Hay, D.C. et al., Proc. Natl. Acad. Sci. USA, 105(34): 12301-12306 (2008)), in step 1, a colony grown to subconfluence is cultured in a serum-free medium containing activin A (100 ng/ml), Wnt3a (50 ng/ml) and sodium butyrate (1 mM) for 1 day, sodium butyrate is removed and the colony is further cultivated for 2 days. This step induces differentiation into an endoderm cell characterized by CXCR4 positive (and E-cadherin positive) . To increase induction efficiency of differentiation into a cell belonging to an endodermal lineage thereafter, it is

considered a necessary condition to increase the proportion of CXCR4 positive cells in this step.

In the present invention, differentiation of single- celled iPS cell into CXCR4 positive endoderm cell can be induced with high efficiency by prolonging the culture period in an activin A and Wnt3a-containing medium and adjusting concentration and addition period of sodium butyrate. The single-celled iPS cell can be prepared by dissociating an iPS cell colony prepared as mentioned above by pipetting and the like in a suitable cell dissociation solution (e.g., 0.1% collagenase, 0.1-0.2% EDTA, 0.05% trypsin/1 mM EDTA, Ca²⁺- treated trypsin/collagenase, DISPASE, Accutase™) .

In step 1 of the differentiation induction protocol of the present invention, single-cell state iPS cell is

cultivated in the presence of activin A and Wnt3a for not less than 4 days, preferably not less than 5 days, more preferably not less than 6 days, and not more than 10 days, preferably not more than 9 days, more preferably not more than 8 days. A particularly preferable culture period is about 7 days. As the medium, any of the above-mentioned basic media containing 0- 20%, preferably 0-5%, of serum can be used. More preferred is serum-free. The concentration of activin A to be added to the medium is, without limitation, for example, not less than 50 ng/ml, preferably not less than 70 ng/ml, more preferably not less than 80 ng/ml, and not more than 200 ng/ml, preferably not more than 150 ng/ml, more preferably not more than 120 ng/ml. Particularly preferably, it is about 100 ng/ml. The concentration of nt3a is, without limitation, for example, not less than 10 ng/ml, preferably not less than 20 ng/ml, more preferably not less than 30 ng/ml, and not more than 100 ng/ml, preferably not more than 80 ng/ml, more preferably not more than 70 ng/ml. Particularly preferably, it is about 50 ng/ml.

Although there is a report stating that sodium butyrate

(hereinafter to be also referred to as NaB) is important for differentiation of human ES cell into endoderm, it has been clarified that it causes cell death when single-celled human iPS cell is used at a concentration of 1 m . In step 1 of the differentiation induction protocol of the present invention, the concentration of NaB to be added to the medium is, without limitation, for example, not less than 0.2 mM, preferably not less than 0.3 mM, more preferably not less than 0.4 mM, and not more than 0.8 mM, preferably not more than 0.7 mM, more preferably not more than 0.6 mM. Particularly preferably, it is about 0.5 mM.

It has also been shown that the sensitivity to NaB varies greatly between iPS cell clones depending on the kind of the somatic cell to be derived from. In the case of blood cell- derived iPS cell showing high differentiation efficiency into a cell belonging to an endodermal lineage, many clones show marked cell death even in the presence of 0.5 mM NaB, and the probability of highly efficient induction into CXCR4 positive endoderm cell becomes the highest when cultured in the absence of NaB through step 1. On the other hand, in the case of cutaneous cell-derived iPS cell and ES cell, the probability of highly efficient induction into CXCR4 positive endoderm cell becomes high when cultured in the presence of NaB during a part or the entirety of the culture period in step 1. While the period of NaB addition varies depending on the culture period of step 1, it can be appropriately determined between one day and the whole culture period (e.g., 10 days) . For example, when an iPS cell for which addition of NaB does not induce cell death of the level that exerts an adverse

influence on the differentiation induction is subjected to the cultivation of step 1 for 7 days, the addition period of NaB is, for example, not less than 1 day, preferably not less than 2 days, and not more than 7 days, preferably not more than 6 days. When a cell is cultivated in the presence of NaB during a part of the culture period in step 1, the timing of addition of NaB to a medium can be optionally selected, and NaB can be preferably added to a medium one day after the start of the culture in step 1. While NaB may be intermittently added to a medium, it can be preferably added continuously to a medium for a given period.

Since single-celled human iPS/ES cell easily induces apoptosis, a ROCK inhibitor (e.g., Y27632 etc.) can be added to the medium as an apoptosis inhibitor. Since a ROCK

inhibitor may exert an unpreferable influence on the cell, it is desirably removed from the medium after the period

requiring suppression of apoptosis. For example, ROCK

inhibitor can be removed one day after the start of the

culture in step 1.

The novel differentiation induction protocol of the present invention can also be used for inducing

differentiation of not only iPS cell but also other

pluripotent stem cells such as ES cell into CXCR4 positive endoderm cell. Examples of other pluripotent stem cells for which the protocol can be used include, but are not limited to, ES cell, embryonic germ (EG) cell derived from primordial germ cell, multipotent germline stem (mGS) cell isolated from

testis tissue during the process of establishment and culture of GS cell, multipotent adult progenitor cell (MAPC) isolated from the bone marrow and the like.

Pluripotent stem cells can be acquired by methods known per se. For example, available methods of preparing ES cells include, but are not limited to, methods in which a mammalian inner cell mass in the blastocyst stage is cultured [see, for example, Manipulating the Mouse Embryo: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)] and methods in which an early embryo prepared by somatic cell nuclear transfer is cultured [Wilmut et al., Nature, 385, 810 (1997); Cibelli et al., Science, 280, 1256 (1998); Iritani et al., Protein, Nucleic Acid and Enzyme, 44, 892 (1999); Baguisi et al., Nature Biotechnology, 17, 456 (1999); Wakayama et al., Nature, 394, 369 (1998); Wakayama et al., Nature Genetics, 22, 127 (1999); Wakayama et al., Proc. Natl. Acad. Sci. USA, 96, 14984 (1999); RideoutHI et al., Nature Genetics, 24, 109

(2000)]. Also, ES cells can be obtained from various public and private depositories and are commercially available. For example, human ES cell lines HI and H9 can be obtained from WiCell Institute of University of Wisconsin and KhES-1, -2 and -3 can be obtained from Institute for Frontier Medical

Sciences, Kyoto University. When ES cells are produced by somatic cell nuclear transfer, the kinds and sources of

somatic cells are the same as those used for producing iPS cells mentioned below.

The CXCR4 positive endoderm cell obtained as mentioned above can be further induced to differentiate into a cell belonging to each endodermal lineage by a method known per se. For example, differentiation induction into hepatocyte can be performed according to the method described in Hay, D.C. et al., Stem Cells, 26(4): 894-902 (2008); Hay, D.C. et al., Proc. Natl. Acad. Sci. USA, 205(34): 12301-12306 (2008). Briefly put, CXCR4 positive cell can be differentiated up to a

differentiation state showing properties characteristic of mature hepatocyte (TAT-, TD02-, ASGRl-positive, highly

expressing cytochrome P450 group, synthesizing and storing glycogen, PAS positive etc.) by cultivating in the presence of 1% DMSO and 20% KSR for 7 days and further, in the presence of 20 ng/ml HGF and 20 ng/ml OSM for 7 days. Induction of

differentiation into cell belonging to endodermal lineage other than hepatocyte can be performed by any of the above- mentioned methods selected as appropriate.

(3) Cell population containing cell belonging to endodermal lineage derived from blood cell-derived iPS cell

The present invention also provides a cell population containing a cell belonging to an endodermal lineage, which is derived from blood cell-derived iPS cell and produced by the aforementioned (2) . The cell population may be a purified cell population (e.g., hepatocyte, endocrine pancreas cell etc.), or one or more kinds of other cell types may be co-present. (4) Use of cell belonging to endodermal lineage derived from blood cell-derived iPS cell

The thus-established cell belonging to endodermal lineage and derived from blood cell-derived iPS cell can be used for various purposes. For example, a stem cell therapy by

autogeneic or allogeneic transplantation, wherein hepatic lineage or pancreatic lineage cell differentiated from an iPS cell induced by using the blood collected from a patient with hepatic disease or pancreatic disease or another person with the same or substantially the same HLA type as that of the patient is transplanted to the patient to regenerate liver or pancreas becomes possible. Furthermore, since a hepatic lineage or pancreatic lineage cell differentiated from an iPS cell derived from a patient's blood cell is considered to better reflect the actual state of the cell of the patient's liver or pancreas than does the corresponding existing cell line of the liver or pancreas, it can also be suitably used for an in vitro evaluation system for the effectiveness and toxicity of a therapeutic drug for a hepatic disease or a pancreatic disease. Moreover, it can be preferably used as a tool for pathological studies of hepatic diseases and

pancreatic diseases with unclarified etiology.

The cell belonging to endodermal lineage of the present invention (including a cell belonging to an endodermal

lineage-containing cell population, hereinafter the same) is produced as a parenteral preparation, which is preferably injection, suspension, drip infusion and the like, by mixing with a pharmaceutically acceptable carrier and the like according to a conventional means. Examples of the pharmaceutically acceptable carrier to be contained in the parenteral preparation include aqueous liquids for injection such as saline, isotonic solution containing glucose and other auxiliary agents (e.g., D-sorbitol, D-mannitol, sodium

chloride and the like) and the like. The preparation of the present invention may also be mixed with, for example,

buffering agents (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride,

procaine hydrochloride and the like), stabilizers (e.g., human serum albumin, polyethylene glycol and the like) ,

preservatives, antioxidants and the like.

When the preparation of the present invention is

formulated as an aqueous suspension, cells belonging to an endodermal lineage only need to be suspended in the above- mentioned aqueous liquid at about 1.0*10⁶- about l.OxlO⁷

cells/ml..

The thus-obtained preparation is stable and of low

toxicity, and therefore, can be safely administered to mammals such as human and the like. While the administration method is not particularly limited, it is preferably injection or drip administration, and intravenous administration, intraarterial administration, intramuscular administration (topical

administration to affected part) and the like can be mentioned. While the dose of the preparation of the present invention varies depending on the subject of administration, treatment target site, symptom, administration method and the like, for example, it is generally preferable to administer about

1.0x10^s- about Ι^χΙΟ⁷ cells in a hepatocyte amount per dose to a hepatitis patient (body weight 60 kg) by intravenous injection about 4- about 8 times at about 1- about 2 week intervals.

The present invention is explained in more detail in the following by referring to Examples, which are not to be

construed as limitative. Examples

[Materials & Methods]

Cell Culture

Human iPS cells (hiPSCs) and human ES Cells (hESCs) were maintained on feeder layers of mitomycin C-treated SNL cells in Primate ES medium (ReproCELL) supplemented with 4 ng/ml recombinant human basic fibroblast growth factor (bFGF, WAKO) as previously described (Takahashi K et al, Cell, 131, 861- 872(2007)). HepG2 and HuH7 (human hepatoma cell lines) were cultured in Dulbecco' s modified Eagle's medium (DMEM, Nacalai Tesque) containing 10% fetal bovine serum (FBS) .

Human IPS and ES Cell lines

201B2, 201B6 and 201B7 lines were generated by

introducing four transcription factors (OCT3/4, SOX2, KLF4 and C- YC) into human dermal fibroblasts (HDFs, Lotl388, 36-year- old female) , whereas the 253G1 and 253G4 lines were

established using three factors devoid of c- YC. Detail information of other human iPS cell lines is described in Table 1.

Human ES cell lines KhESl and KhES3 were obtained from

Institute for Frontier Medical Sciences, Kyoto University, HI and H9 were obtained from WiCell, and ES03, ES04 and ES06 were obtained from Wicell.

Table 1. HiPSC information used in Examples

Oct3/4, S: Sox2, K: Klf4, sh-p53: p53 shRNA Hepatic Differentiation in Vitro

For hepatic differentiation of hiPSCs and hESCs, we applied a previously reported hepatic differentiation protocol for hESCs (Hay, D.C. et al., Stem Cells, 26(4): 894-902

(2008); Hay, D.C. et al . , Proc. Natl. Acad. Sci. USA, 105 (34): 12301-12306 (2008)) with some modifications. Briefly,

undifferentiated hiPSCs and hESCs were seeded on Matrigel (growth-factor reduced, BD Biosciences ) -coated plate and cultured in mouse embryonic fibroblast (MEF) -conditioned

Primate ES cell medium supplemented with 4 ng/ml bFGF. When hiPSCs and hESCs reached nearly 70% confluence, the medium was replaced with RPMI1640 (Nacalai Tesque) containing 1><B27 supplement (Invitrogen) , 100 ng/ml activin A (PeproTech), 50 ng/ml Wnt3a (R&D systems) and 1 mM sodium butyrate (NaB) (Sigma) , and the cells were cultured for 1 day. On the following 2 days, sodium butyrate was omitted from the medium. After 3 days culture in serum-free activin A based medium, the medium was replaced with knockout-Dulbecco' s modified Eagle's medium -(KO-DMEM) containing 20% knockout serum replacement (KSR) , 1 mM L-glutamine, 1% nonessential amino acids, 0.1 mM 2-mercaptoethanol (all from Invitrogen) and 1% DMSO (Sigma) (differentiation medium) , and the cells were cultured for 7 days. Finally, the cells were cultured in Hepatocyte culture medium (Lonza) supplemented with 20 ng/ml hepatocyte growth factor (HGF, PeproTech) and 20 ng/ml oncostatin M (OSM,

PeproTech) (maturation medium) for another 7 days. The medium was changed daily during the differentiation period. The culture protocol is summarized in Fig. 1A.

For endoderm cell induction from single hiPSCs and hESCs, hiPSCs/hESCs were incubated with Accutase (Innovative Cell

Technologies) for 20 minutes and dissociated into single cells by pipetting. The cells were resuspended with RP I1640 medium containing 1*B27 supplement, 100 ng/ml activin A, 50 ng/ml nt3a and ΙΟμΜ Y27632 ( AKO) , and seeded on Matrigel-coated culture dish at 1*10⁵ cells/cm² (day 0) . From next day, Y27632 was omitted from the medium and 0.5 mM NaB was added in the culture medium for 0-6 days. For hepatic differentiation, further cultivation was performed with differentiation medium and maturation medium described above from day 7 to 13, and from day 14 to 20, respectively. The culture protocol is summarized in Fig. 4A.

RNA Isolation and Polymerase Chain Reaction (PCR)

Total RNA was purified with Trizol reagent (Invitrogen). One microgram of total RNA was used for reverse transcription reaction with ReverTraAce-a (Toyobo) and dT20 primer,

according to the manufacturer's instructions. Reverse

Transcription (RT) -PCR was performed with ExTaq (Takara) .

Real-time PCR analysis was carried out with SYBR Premix Ex Taq II (Takara) and run on a StepOne™ Real-Time PCR System (Applied Biosystems) . The mean of duplicate measurements was normalized against that of housekeeping gene (GAPDH) for the same sample.

Primer sequences are shown in Table 2.

Table 2. Primer information for RT-PCR and real-time PCR

Immunohistoc emical S ainings

Cells were fixed with PBS containing 4% paraformaldehyde for 10 minutes at room temperature. After washing with PBS, nonspecific binding was blocked with PBS containing 5% normal goat or donkey serum (Chemicon) , 1% bovine serum albumin (BSA, Nacalai Tesque) , and 0.1% Triton X-100 for 45 minutes at room temperature. Primary antibodies included HNF4A (1:500,

santacruz sc-6556) , AFP (1:200, DAKO 0008), ALBUMIN (1:200, Bethyl . A80-229A) , AlAT (1:50, Invitrogen 180002), SOX17 (1:300, R&D systems AF1924) and OCT3/4 (1:100, santacruz sc-5279) .

Secondary antibodies used were cyanine3 (Cy3 ) -conjugated anti- goat IgG (1:500, Chemicon) for HNF4A, Alexa488-conjugated anti-rabbit IgG (1:500, Invitrogen) for AFP, Alexa488- conjugated anti-goat IgG (1:500, Invitrogen) for ALBUMIN and SOX17, Cy3-conjugated anti-rabbit IgG (1:500, Chemicon) for AlAT and Cy3-con ugated anti-mouse IgG (1:500, Chemicon) for OCT3/4. Nuclei were stained with 1 g/ml Hoechst 33342

( Invitrogen) .

Flow Cytometry

For analyzing albumin-positive cells, hepatic ^'

differentiated hiPSCs/hESCs were dissociated in 0.25% Trypsin- EDTA (Invitrogen) and then resuspended in 2% FBS/PBS.

Collected cell suspensions were fixed with PBS containing 4% paraformaldehyde, permeabilized with ^"0.1% Triton X-10,0, and stained with the anti-albumin antibody described above and the Alexa488-conjugated secondary antibody. For analyzing CXCR4- positive cells, endoderm differentiated hiPSCs/hESCs were dissociated in Accutase and stained with PE-conjugated anti- CXCR4 antibody (R&D systems FAB170P) . Analysis was performed by FACS Aria II flow cytometer (BD Biosciences) .

Functional Analysis of Hepatic differe tiated hiPSCs/hESCs in Vitro

To evaluate glycogen production and storage of hepatic differentiated hiPSCs and hESCs, Periodic acid-Shiff (PAS) staining was performed. The cultured cells were fixed in 3.3% formalin for 10 minutes, and intracellular glycogen was stained using a PAS staining solution (Muto Pure Chemicals), according to the manufacturer's instructions. For albumin secretion assay, the culture media of differentiated cells after 24 h incubation was collected and measured with the Human Albumin ELISA Quantitation Kit (Bethyl Laboratories Inc.) according to the manufacturer's protocol. To examine cellular ability to metabolize ammonia, cells were cultured for 24 h in phenol red-free DMEM (Nacalai Tesque) supplemented with 1.5 mM ammonium chloride (Nacalai Tesque). Ammonia concentrations in the culture media were measured using an Ammonia-Test Wako kit (Wako Pure Chemical) , according to the manufacturer's protocol. To evaluate cytochrome P450 3A4 activity, P450-Glo CYP3A4 Assay Kit (Luciferin-IPA, Promega) was used according to the manufacturer's protocol. Read out was performed with Centro LB 960 detection system (BERTHOLD) . The CYP450 activity was expressed as relative light units per 50 μL of medium.

DNA methylation analysis

Bisulfite treatment of genomic DNA was carried out using

EZ DNA Methylation™ Kit (Zymo) according to the manufacturer's protocol. For pyrosequencing, the bisulfite treated DNA was amplified by PyroMark PCR kit (QIAGEN) . Pyrosequencing analysis was performed by PyroMarkQ96 ID system (QIAGEN) according to standard procedures. Genomic DNAs of human heart, liver and brain were purchased from BioChain.

Primer sequences are shown in Table 3.

Table 3 . Primer information for pyrosequencing

F— orward primer

R— biotinated reverse primer

S— sequence primer

[Results]

In vitro directed differentiation of hiPSCs and hESCs to hepatic lineage cells

To generate hepatic dif ferentiated cells from hiPSCs and hESCs , we initially used a previously reported dif ferentiation protocol that had been designed for hESCs with some modifications, which comprises the following steps:

Step 1: undifferentiated hiPSCs/hESCs grown until reaching nearly 70% confluence were cultivated in serum-free medium supplemented with 100 ng/ml Activin A, 50 ng/ml nt3a and 1 mM sodium butyrate (NaB) toward endoderm lineage for 3 days (NaB was removed from the medium for the last two days) ;

Step 2: the cells were cultured in the differentiation medium containing 1% dimethyl sulfoxide (DMSO) and 20% knockout serum replacement (KSR) for 7 days; and

Step 3: medium was changed to the maturation medium supplemented with 20 ng/ml oncostatin M (OSM) and 20 ng/ml hepatocyte growth factor (HGF) for further maturation for another 7 days.

At first, we investigated five "sibling" hiPS clones which had generated from the same fibroblast culture of a single individual donor and two hES clones. Three hiPS clones (201B2, B6 and B7) were generated with retroviral transduction of four reprogramming factors (OCT3/4, SOX2, KLF4 and C-MYC) to adult human dermal fibroblasts (aHDFs) and two clones (253G1 and G4) with three factors devoid of C-MYC to the same aHDFs. Two hESC lines (KhESl and 3) established in Japan were used as control.

After total 17 days of differentiation period, definite areas of hepatocyte-like cell morphology: large cuboidal cell shape with prominent compact nuclei, were observed in 3 clones (201B2, 201B6 and KhES3) . Some binuclear cells indicating mature hepatic phenotype were also found in those clones.

Whereas, the other clones (201B7, 253G1, 253G4 and KhESl) showed only a few hepatocyte-like cell clusters. Flow

cytometry analysis also revealed that the percentage of albumin-positive cells were higher in morphologically good- hepatic-differentiating clones (201B2, 201B6 and KhES3) than poor-hepatic-differentiating clones (201B7, 253G1, 253G4 and KhESl) (Fig. 2A) .

Liver-related gene expression analysis of hepatic differentiated hiPSC and hESC

Using representative good-hepatic-differentiating hiPS clone 201B6, we examined the time course gene expression during the differentiation period. Reverse transcription PCR (RT-PCR) analysis revealed that ES cell markers, including OCT3/4 and NANOG, were gradually diminished in a time

dependent manner. Treatment with high concentration of Activin A, Wnt3a and NaB led to marked upregulation of the mesendoderm marker, BRACHYURY, which had expressed until day 10. The definitive endoderm markers, such as FOXA2 and SOX17, were detected from day 3, followed by the gradual increase of liver-related gene expressions (HNF4A, A1AT (alpha 1- antitrypsin) , AFP and ALBUMIN) . Finally, mature hepatic

markers including TAT (tyrosine aminotransferase) , TD02

(tryptophan 2, 3-dioxygenase) and ASGR1 (asialoglycoprotein receptor 1) were detected and elevated to a substantial level at day 17 (Fig. IB) . Taken together, investigated gene

expression profiles during the protocol roughly recapitulated developmental process of the liver and were consistent with the previous, report using hESCs .

Real time-PCR analysis revealed that after 17 days of hepatic differentiation, good-differentiating clones (201B2, 201B6 and KhES3) showed higher expression of liver-related genes (HNF4A, A1AT, AFP, ALBUMIN, TD02, ASGR1) than poor- differentiating clones (201B7, 253G1, 253G4 and KhESl) .

Undifferentiated marker OCT3/4 was downregulated in all clones (Fig. 2B) .

We also evaluated the mRNA expression of various

cytochrome P450 (CYP450) enzymes, which play central roles in drug metabolism in human liver, indicating mature phenotype of hepatocytes. Hepatic differentiated cells at day 17 from good- differentiating clones (hiPS(201B6) and hES (KhES3) )

represented various type of CYP450 mRNA expression, such as CYP1A1, CYP2C9, CYP2C19, CYP2D6 CYP3A4 and CYP7A1. Furthermore, some mature hepatocyte markers, ABCC2(MRP2) and ABCB11 (MDR/TAP) ; ATP-binding cassette (ABC) transporter supporting bile acid export in apical surface of the

hepatocyte and UGT1A1 which encodes an enzyme of the

glucuronidation pathway, were also detected (Fig. 3A) .

Immunofluorescent staining also confirmed that

differentiated cells expressed liver-related markers: HNF4A at day 10, and AFP, ALBUMIN and A1AT at day 17.

Functional analyses of hepatic differentiated hiPSC and hESC

Next, we examined whether hepatic differentiated hiPSCs and hESCs in vitro possessed liver-related functional

activities. To assess glycogen synthesis and storage, Periodic acid-Shiff (PAS) staining was performed. At day 17, hepatic differentiated cells from good-differentiating clones

(hiPS(201B6) and hES (KhES3) ) were positive for PAS staining indicating mature hepatic phenotype (Fig. 3B) . Albumin

secretion level in the culture media and ammonia clearance capacity of the cells at day 17 from good-hepatic- differentiating clones (201B2, 201B6 and KhES3) was comparable to those obtained from human hepatoma cell lines (HepG2.and HuH7) . On the other hand, reduced ability of albumin

production and ammonia removal was observed in poor-hepatic- differentiating clones (201B7, 253G1, 253G4 and KhESl) (Fig. 2C, 2D) . ^" We also tested CYP activity of those differentiated cells. Hepatic differentiated cells from good-differentiating clones (hiPS(201B6) and hES (KhES3 ) ) at day 17 showed CYP3A4 activity similar to the level of HepG2 (Fig. 3C) .

Taken together, in vitro differentiated cells from hiPSCs and hESCs showed several functional hepatocyte-like properties in markedly different degrees among the five "sibling" hiPS and the two hES clones.

Novel endoderm cell induction protocol from single hiPS and hES cells

Previously reported protocols for hepatic directed differentiation from hiPSCs or hESCs start with addition of differentiation medium onto undifferentiated hiPSC/hESC colonies already grown to some extent. In such way, size of colonies at the starting point cannot be strictly controlled, resulting that cell-to-cell interaction and cellular responses to growth factors supplemented in differentiation medium may be skewed and impair reproducibility among experiments. To overcome this problem, we designed a novel protocol which starts with single hiPSCs/hESCs and can generate endoderm cells stably and efficiently (Fig. 4A) . Briefly, hiPSC/hESC colonies are dissociated with Accutase into single cells by pipetting and seeded on the Matrigel-coated culture dish with serum-free medium containing 100 ng/ml Activin A and 50 ng/ml Wnt3a. 10 μg/ml ROCK inhibitor (Y27632) was added to the culture medium for the first day. In this protocol, endoderm differentiation period was set for 7 days. Because NaB has been reported to be important for endoderm differentiation of hESCs, we added NaB to the culture medium from day 1. Addition of 1 mM NaB showed significant cell death. Accordingly, we set the concentration of NaB at 0.5 mM and varied the

administration period from 1 day to 6 days. After 7 days of differentiation, we .assessed endoderm differentiation by the percentage of CXCR4-positive cells with flow cytometry. In addition, we measured albumin secretion capacity after

subsequent cultivation: 7 days for differentiation medium followed by another 7 days for maturation medium (Fig. 4A) .

At first, we focused on the "sibling" hiPS clones, 201B6 and 201B7, derived from the same aHDFs culture of a single donor. They showed quite opposite hepatic differentiating behavior in the conventional protocol as described above:

201B6 and 201B7 exhibited representative good- and poor- hepatic differentiation, respectively. With this new protocol, sibling clones 201B6 and 201B7 again showed quite different results. Time course images of endoderm and hepatic

differentiation (0.5 mM NaB was administrated for 3 days.) were shown in Fig. 5, indicating that more albumin-positive hepatocytes were observed in derivatives of 201B6 than those of 201B7 at the end of the differentiation protocol (day 21) . We found that NaB administration affected hepatic

differentiation, and a few days of NaB treatment was necessary even in good-hepatic differentiation clone, 201B6. Albumin secretion level of 201B6 at day 21 was also far higher than that of 201B7 in any period of NaB administration (Fig. 4B) , indicating that 201B6 favors differentiate into hepatic lineage cells whereas 201B7 is refractory to hepatic

differentiation.

However, in contrast to the result of final hepatic differentiation, more CXCR4 positive endoderm cells were obtained at day 7 in 201B7 than 201B6 in any duration of NaB administration, although the administration period of NaB also affected the percentage of CXCR4 positive cells at day 7 (Fig. 4C) . Immunostaining analysis at day 7 also showed that more SOX17, an endoderm marker, -positive cells and less OCT3/4, undifferentiated marker, -positive cells were obtained in 201B7 than 201B6 with 3 days of NaB administration (Fig. 4D) .

Together, a new differentiation protocol starting from single hiPSCs/hESCs demonstrated differences in hepatic differentiation propensity between sibling hiPS clones: low efficient CXCR4-positive cell induction followed by higher efficient hepatic differentiation in 201B6 and vice versa in 201B7.

Methylation analysis of liver-related transcription factors in various clones

Although sibling hiPS clones, 201B6 and 201B7,

theoretically has the identical genetic background, these clones showed significant difference in behaviors in in vitro hepatic differentiation. Then, we presumed that these

differences may be attributed to epigenetic modification. We selected 10 transcription factors known to be closely related to liver development, and investigated DNA methylation status of their promoter regions in their undifferentiated status. We added three more siblings hiPS clones (201B2, 253G1 and 253G4) and four hES clones (KhESl, KhES3, HI and H9) to the analysis. Pyrosequencing analysis of undifferentiated cells showed no significant difference among sibling hiPS- clones with various hepatic differentiation propensity: promoter regions of HNF1A and HNF4A were highly methylated, whereas promoters of HNF1B, FOXA2 (HNF3B), HNF6, SOX17, GATA4 , GATA6, HHEX and CEBPA were unmethylated in all the hiPSCs/hESCs . Next, we sorted out CXCR4-positive cells of 201B6 and 201B7 at day 7 by flow cytometry, and analyzed DNA methylation status of the 10 liver-related transcription factors. As a result, we did not find significant difference between these clones even in the CXCR4-positive population.

Global gene expression analysis between 201B6 and 201B7 sibling hiPS clones

To compare global gene expression profile of sibling hiPS clones, 201B6 and 201B7, we performed microarray analysis.

Similar gene expression pattern was observed between these hiPS clones in CXCR4-positive cell population at day 7 as well as in undifferentiated status (Fig. 6). 10 liver-related transcription factors described above also showed similar expression level between 201B6 and 201B7 both in

undifferentiated and CXCR4 positive cells, compatible to the results of pyrosequencing described above.

Peripheral blood-derived hiPSCs favor hepatic differentiation With this new protocol efficiently generating CXCR4- positive endoderm cells, we compared endoderm and hepatic differentiation propensity among various hiPSC lines with different type of donor cell origin, method of iPS generation or combination of reprogramming factors. We examined hiPSC lines derived from aHDFs (aHDF-iPSC) , dental pulp cells (DP- iPSC) , peripheral blood cells (PB-iPS) and cord blood cells (CB-.iPS) . We also tested 7 hESC lines. Detail information of hiPSC and hESC lines is described in Table 1. Albumin

secretion level into culture media at day 21 was. used as a representative marker of hepatic properties for screening, because .the results of several tests were almost consistent in the analyses using 5 hiPSCs and 2 hESCs (Fig. 2 and Fig. 3), and measurement of albumin concentration in media by ELISA is convenient for analysis of large number of samples. We thought NaB requirement might be different in various clones, hence we applied 0.5 mM NaB for three different time periods during the first 7 days of endoderm differentiation (0 day (no

administration) , 3 days (dayl-3) or 6 days (dayl-6) ) .

NaB requirement for generating CXCR4-positive endoderm cells at day 7 was quite different among the original cell types. In nearly half of PB-iPS and CB-iPS clones, NaB

administration caused significant cell death. Generally, NaB was dispensable for endodermal and hepatic differentiation in PB-iPS and CB-iPS clones, whereas 3 or 6 days of NaB

administration was necessary to yield high percentage of

CXCR4-positive endoderm cells at day 7 and high level of albumin secretion at day 21 in aHDF-hiPS clones. HESC lines showed various NaB requirements (Fig. 7A and Fig. 8) . DP-iPSC lines did not respond to this differentiation protocol due to significant cell death or poor cell growth. PB-iPS clones showed significantly high level of albumin secretion compared to aHDF-iPS, DP-iPS and hES clones (Fig. 7B) , suggesting that peripheral blood can be suitable donor cell candidate for generating iPSCs aimed to hepatocyte differentiation.

While the present invention has been described with emphasis on preferred embodiments, it is obvious to those skilled in the art that the preferred embodiments can be modified. The present invention intends that the present invention can be embodied by methods other than those

described in detail in the present specification. Accordingly, the present invention encompasses all modifications

encompassed in the gist and scope of the appended "CLAIMS."

In addition, the contents disclosed in any publication cited herein, including patents and patent applications, are hereby incorporated in their entireties by reference, to the extent that they have been disclosed herein.

This application is based on US provisional patent application No. 61/552,791, the contents of which are incorporated in full herein.

Claims

1. A method for producing a cell belonging to an endodermal lineage, comprising inducing differentiation of a blood cell- derived induced pluripotent stem (iPS) cell.

2. A method for producing a cell belonging to an endodermal lineage, comprising the following steps (1) and (2):

(1) a step of producing an iPS cell by contacting a nuclear reprogramming substance with a blood cell

(2) a step of inducing differentiation of the iPS cell

obtained in step (1) into a cell belonging to an endodermal lineage .

3. The method according to claim 1 or 2, wherein the blood cell is a peripheral blood mononuclear cell or a cord blood cell .

4. The method according to claim 1 or 2, wherein the cell belonging to the endodermal lineage is a hepatic lineage cell.

5. The method according to claim 4, wherein the hepatic lineage cell is a hepatocyte.

6. The method according to claim 2, wherein the nuclear reprogramming substance comprises Oct3/4, Klf4 and Sox2, or a nucleic acid encoding the same.

7. The method according to any one of claims 1-6, wherein the blood cell is derived from human.

8. Use of a blood cell as a somatic cell source of an iPS cell for production of a cell belonging to an endodermal lineage.

9. The use according to claim 8, wherein the blood cell is a peripheral blood mononuclear cell or a cord blood cell.

10. The use according to claim 8, wherein the cell belonging to the endodermal lineage is a hepatic lineage cell.

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11. The use according to claim 10, wherein the hepatic lineage cell is a hepatocyte.

12. The use according to any one of claims 8-11, wherein the blood cell is derived from human.

13. A method for inducing differentiation of a human

pluripotent stem cell into a CXCR4 positive endoderm cell, characterized by the following (1) and (2) :

(1) cultivating a human pluripotent stem cell in a single-cell state in the presence of activin A and nt3a for 4-10 days (2) cultivating in the co-presence of 0.1-0.8 mM sodium butyrate for 0-10 days during the culture period in (1) above.

14. The method according to claim 13, wherein the culture period in (1) above is 7 days, and the cell is cultivated in the presence of 0.5 mM sodium butyrate for 0-6 days in (2) above .

15. The method according to claim 13 or 14, wherein the human pluripotent stem cell is a human iPS cell or a human ES cell.

16. The method according to claim 13 or 14, wherein the human pluripotent stem cell is a blood cell-derived human iPS cell, and sodium butyrate is not co-present during the culture period in (1) above.