Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors PDF

Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors Kazutoshi Takahashi1 and Shinya Yamanaka1,2,* 1 Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan 2 CREST, Japan Science and Tec...

Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors Kazutoshi Takahashi1 and Shinya Yamanaka1,2,* 1 Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan 2 CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan *Contact: [email protected] DOI 10.1016/j.cell.2006.07.024 SUMMARY or by fusion with ES cells (Cowan et al., 2005; Tada et al., 2001), indicating that unfertilized eggs and ES cells Differentiated cells can be reprogrammed to an contain factors that can confer totipotency or pluripotency embryonic-like state by transfer of nuclear con- to somatic cells. We hypothesized that the factors that tents into oocytes or by fusion with embryonic play important roles in the maintenance of ES cell identity stem (ES) cells. Little is known about factors also play pivotal roles in the induction of pluripotency in that induce this reprogramming. Here, we dem- somatic cells. Several transcription factors, including Oct3/4 (Nichols onstrate induction of pluripotent stem cells et al., 1998; Niwa et al., 2000), Sox2 (Avilion et al., 2003), from mouse embryonic or adult fibroblasts by and Nanog (Chambers et al., 2003; Mitsui et al., 2003), introducing four factors, Oct3/4, Sox2, c-Myc, function in the maintenance of pluripotency in both early and Klf4, under ES cell culture conditions. embryos and ES cells. Several genes that are frequently Unexpectedly, Nanog was dispensable. These upregulated in tumors, such as Stat3 (Matsuda et al., cells, which we designated iPS (induced plurip- 1999; Niwa et al., 1998), E-Ras (Takahashi et al., 2003), otent stem) cells, exhibit the morphology and c-myc (Cartwright et al., 2005), Klf4 (Li et al., 2005), and growth properties of ES cells and express ES b-catenin (Kielman et al., 2002; Sato et al., 2004), have cell marker genes. Subcutaneous transplanta- been shown to contribute to the long-term maintenance tion of iPS cells into nude mice resulted in of the ES cell phenotype and the rapid proliferation of tumors containing a variety of tissues from all ES cells in culture. In addition, we have identified several other genes that are specifically expressed in ES cells three germ layers. Following injection into blas- (Maruyama et al., 2005; Mitsui et al., 2003). tocysts, iPS cells contributed to mouse embry- In this study, we examined whether these factors could onic development. These data demonstrate induce pluripotency in somatic cells. By combining four that pluripotent stem cells can be directly gen- selected factors, we were able to generate pluripotent erated from fibroblast cultures by the addition cells, which we call induced pluripotent stem (iPS) cells, of only a few defined factors. directly from mouse embryonic or adult fibroblast cul- tures. INTRODUCTION Embryonic stem (ES) cells, which are derived from the in- RESULTS ner cell mass of mammalian blastocysts, have the ability to grow indefinitely while maintaining pluripotency and We selected 24 genes as candidates for factors that the ability to differentiate into cells of all three germ layers induce pluripotency in somatic cells, based on our (Evans and Kaufman, 1981; Martin, 1981). Human ES cells hypothesis that such factors also play pivotal roles in the might be used to treat a host of diseases, such as Parkin- maintenance of ES cell identity (see Table S1 in the son’s disease, spinal cord injury, and diabetes (Thomson Supplemental Data available with this article online). For et al., 1998). However, there are ethical difficulties regard- b-catenin, c-Myc, and Stat3, we used active forms, ing the use of human embryos, as well as the problem of S33Y-b-catenin (Sadot et al., 2002), T58A-c-Myc (Chang tissue rejection following transplantation in patients. One et al., 2000), and Stat3-C (Bromberg et al., 1999), respec- way to circumvent these issues is the generation of plu- tively. Because of the reported negative effect of Grb2 ripotent cells directly from the patients’ own cells. on pluripotency (Burdon et al., 1999; Cheng et al., 1998), Somatic cells can be reprogrammed by transferring we included its dominant-negative mutant Grb2DSH2 their nuclear contents into oocytes (Wilmut et al., 1997) (Miyamoto et al., 2004) as 1 of the 24 candidates. Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. 663 Figure 1. Generation of iPS Cells from MEF Cultures via 24 Factors (A) Strategy to test candidate factors. (B) G418-resistant colonies were observed 16 days after transduction with a combination of 24 factors. Cells were stained with crystal violet. (C) Morphology of ES cells, iPS cells (iPS-MEF24, clone 1-9), and MEFs. Scale bars = 200 mm. (D) Growth curves of ES cells, iPS cells (iPS-MEF24, clones 2-1–4), and MEFs. 3 3 105 cells were passaged every 3 days into each well of six-well plates. (E) RT-PCR analysis of ES cell marker genes in iPS cells (iPS-MEF24, clones 1-5, 1-9, and 1-18), ES cells, and MEFs. Nat1 was used as a loading control. (F) Bisulfite genomic sequencing of the promoter regions of Oct3/4, Nanog, and Fbx15 in iPS cells (iPS-MEF24, clones 1-5, 1-9, and 1-18), ES cells, and MEFs. Open circles indicate unmethylated CpG dinucleotides, while closed circles indicate methylated CpGs. To evaluate these 24 candidate genes, we developed ES cells homozygous for the bgeo knockin construct an assay system in which the induction of the pluripotent (Fbx15bgeo/bgeo) were resistant to extremely high concen- state could be detected as resistance to G418 (Figure 1A). trations of G418 (up to 12 mg/ml), whereas somatic cells We inserted a bgeo cassette (a fusion of the b-galactosi- derived from Fbx15bgeo/bgeo mice were sensitive to a nor- dase and neomycin resistance genes) into the mouse mal concentration of G418 (0.3 mg/ml). We expected that Fbx15 gene by homologous recombination (Tokuzawa even partial activation of the Fbx15 locus would result in et al., 2003). Although specifically expressed in mouse resistance to normal concentrations of G418. ES cells and early embryos, Fbx15 is dispensable for the We introduced each of the 24 candidate genes into maintenance of pluripotency and mouse development. mouse embryonic fibroblasts (MEFs) from Fbx15bgeo/bgeo 664 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. Figure 2. Narrowing down the Candidate Factors (A) Effect of the removal of individual factors from the pool of 24 transduced factors on the formation of G418-resistant colonies. Fbx15bgeo/bgeo MEFs were transduced with the indicated factors and selected with G418 for 10 days (white columns) or 16 days (black columns). (B) Effect of the removal of individual factors from the selected 10 factors on the formation of G418-resistant colonies 16 days after transduction. (C) Effect of the transduction of pools of four, three, and two factors on the formation of G418-resistant colonies 16 days after transduction. (D) Morphologies of iPS-MEF4 (clone 7), iPS-MEF10 (clone 6), and iPS-MEF3 (clone 3). Scale bars = 200 mm. embryos by retroviral transduction (Morita et al., 2000). nated these cells iPS-MEF24 for ‘‘pluripotent stem cells in- Transduced cells were then cultured on STO feeder cells duced from MEFs by 24 factors.’’ Reverse transcription in ES cell medium containing G418 (0.3 mg/ml). We did PCR (RT-PCR) analysis revealed that the iPS-MEF24 not, however, obtain drug-resistant colonies with any sin- clones expressed ES cell markers, including Oct3/4, gle factor, indicating that no single candidate gene was Nanog, E-Ras, Cripto, Dax1, and Zfp296 (Mitsui et al., sufficient to activate the Fbx15 locus (Figure 1B; see 2003) and Fgf4 (Yuan et al., 1995) (Figure 1E). Bisulfite also Table S2, which summarizes all of the transduction genomic sequencing demonstrated that the promoters experiments in this study). of Fbx15 and Nanog were demethylated in iPS cells In contrast, transduction of all 24 candidates together (Figure 1F). By contrast, the Oct3/4 promoter remained generated 22 G418-resistant colonies (Figure 1B). Of the methylated in these cells. These data indicate that some 12 clones for which we continued cultivating under selec- combination of these 24 candidate factors induced the tion, 5 clones exhibited morphology similar to ES cells, expression of ES cell marker genes in MEF culture. including a round shape, large nucleoli, and scant cyto- Next, to determine which of the 24 candidates were crit- plasm (Figure 1C). We repeated the experiments and ob- ical, we examined the effect of withdrawal of individual served 29 G418-resistant colonies, from which we picked factors from the pool of transduced candidate genes on 6 colonies. Four of these clones possessed ES cell-like the formation of G418-resistant colonies (Figure 2A). We morphology and proliferation properties (Figure 1D). The identified 10 factors (3, 4, 5, 11, 14, 15, 18, 20, 21, and doubling time of these cells (19.4, 17.5, 18.7, and 18.6 22) whose individual withdrawal from the bulk transduc- hr) was equivalent to that of ES cells (17.0 hr). We desig- tion pool resulted in no colony formation 10 days after Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. 665 transduction and fewer colonies 16 days after transduc- increased acetylation of histone H3 and decreased tion. Combination of these 10 genes alone produced dimethylation of lysine 9 of histone H3 (Figure 3B). CpG more ES cell-like colonies than transduction of all 24 dinucleotides in these promoters remained partially genes did (Figure 2B). methylated in iPS cells (Figure 3C). iPS-MEF4 and iPS- We next examined the formation of colonies after with- MEF10 cells were positive for alkaline phosphatase and drawal of individual factors from the 10-factor pool trans- SSEA-1 (Figure 3D) and showed high telomerase activity duced into MEFs (Figure 2B). G418-resistant colonies did (Figure S2). These results demonstrate that iPS-MEF4 not form when either Oct3/4 (factor 14) or Klf4 (factor 20) and iPS-MEF10 cells are similar, but not identical, to ES was removed. Removal of Sox2 (factor 15) resulted in cells. only a few G418-resistant colonies. When we removed In iPS-MEF3 clones, Ecat1, Esg1, and Sox2 were not c-Myc (factor 22), G418-resistant colonies did emerge, activated (Figure 3A). Nanog was induced, but to a lesser but these had a flatter, non-ES-cell-like morphology. Re- extent than in iPS-MEF4 and iPS-MEF10 clones. Oct3/4 moval of the remaining factors did not significantly affect was weakly activated in iPS-MEF3-3, -5, and -6 but was colony numbers. These results indicate that Oct3/4, Klf4, not activated in the remaining clones. By contrast, E-Ras Sox2, and c-Myc play important roles in the generation and Fgf4 were activated more efficiently in iPS-MEF3 of iPS cells from MEFs. than in iPS-MEF10 or iPS-MEF4. These data confirm Combination of the four genes produced a number of that iPS-MEF3 cells are substantially different from iPS- G418-resistant colonies similar to that observed with the MEF10 and iPS-MEF4 cells. pool of 10 genes (Figure 2C). We continued cultivation of We compared the global gene-expression profiles of 12 clones for each transduction and were able to establish ES cells, iPS cells, and Fbx15bgeo/bgeo MEFs using DNA 4 iPS-MEF4 and 5 iPS-MEF10 clones. In addition, we microarrays (Figure 4A). In addition, we examined could generate iPS cells (iPS-MEF4wt) with wild-type Fbx15bgeo/bgeo MEFs in which the four factors had been c-Myc instead of the T58A mutant (Table S2). These data introduced without G418 selection, immortalized MEFs demonstrate that iPS cells can be induced from MEF expressing K-RasV12, and NIH 3T3 cells transformed culture by the introduction of four transcription factors, with H-RasV12. Pearson correlation analysis revealed Oct3/4, Sox2, c-Myc, and Klf4. that iPS cells are clustered closely with ES cells but sepa- No combination of two factors could induce the forma- rately from fibroblasts and their derivatives (Figure 4A). tion of G418-resistant colonies (Figure 2C). Two combina- The microarray analyses identified genes that were com- tions of three factors—Oct3/4, Sox2, and c-Myc (minus monly upregulated in ES cells and iPS cells, including Klf4) or Klf4, Sox2, and c-Myc (minus Oct3/4)—generated Myb, Kit, Gdf3, and Zic3 (group I, Figure 4B and Table a single, small colony in each case, but these could not be S3). Other genes were upregulated more efficiently in ES maintained in culture. With the combination of Oct3/4, cells, iPS-MEF4, and iPS-MEF10 than in iPS-MEF3 Klf4, and Sox2 (minus c-Myc), we observed the formation clones, including Dppa3, Dppa4, Dppa5, Nanog, Sox2, of 36 G418-resistant colonies, which, however, exhibited Esrrb, and Rex1 (group II). Lower expression of these a flat, non-ES-cell-like morphology. With the combination genes may account for the lack of pluripotency in iPS- of Oct3/4, Klf4, and c-Myc (minus Sox2), we observed MEF3 cells. In addition, we found genes that were upregu- the formation of 54 G418-resistant colonies, of which we lated more prominently in ES cells than in iPS cells, includ- picked 6. Although all 6 clones could be maintained over ing Dnmt3a, Dnmt3b, Dnmt3l, Utf1, Tcl1, and the LIF several passages, the morphology of these cells (iPS- receptor gene (group III). These data confirm that iPS cells MEF3) differed from that of iPS-MEF4 and iPS-MEF10 are similar, but not identical, to ES cells. cells, with iPS-MEF3 colonies exhibiting rough surfaces We examined the pluripotency of iPS cells by teratoma (Figure 2D). These data indicate that the combination of formation (Figure 5A; Table S6 and Figure S3). We ob- Oct3/4, c-Myc, and Klf4 can activate the Fbx15 locus, tained tumors with 5 iPS-MEF10 clones, 3 iPS-MEF4 but the change induced by these three factors alone is dif- clones, 1 iPS-MEF4wt clone, and 6 iPS-MEF3 clones ferent from that seen in iPS-MEF4 or iPS-MEF10 cells. after subcutaneous injection into nude mice. Histological We performed RT-PCR to examine whether ES cell examination revealed that 2 iPS-MEF10 clones (3 and 6), marker genes were expressed in iPS cells (Figure 3A). 2 iPS-MEF4 clones (2 and 7), and the iPS-MEF4wt-4 clone We used primers that would amplify transcripts of the en- differentiated into all three germ layers, including neural dogenous gene but not transcripts of the transgene. iPS- tissues, cartilage, and columnar epithelium. iPS-MEF10- MEF10 and iPS-MEF4 clones expressed the majority of 6 could give rise to all three germ layers even after 30 pas- marker genes, with the exception of Ecat1 (Mitsui et al., sages (Table S6 and Figure S3). We confirmed differentia- 2003). The expression of several marker genes, including tion into neural and muscle tissues by immunostaining Oct3/4, was higher in iPS-MEF4-7, iPS-MEF10-6, and (Figure 5B) and RT-PCR (Figure S4). By contrast, these iPS-MEF10-7 clones than in the remaining clones. Sox2 teratomas did not express the trophoblast marker Cdx2 was only expressed in iPS-MEF10-6. The iPS-MEF4wt (Figure S4). iPS-MEF10-1 tumors differentiated into ecto- clone also expressed many of the ES cell marker genes derm and endoderm, but not mesoderm, and no signs of (Figure S1). Chromatin immunoprecipitation analyses differentiation were observed in tumors derived from the showed that the promoters of Oct3/4 and Nanog had remaining iPS-MEF10 (7 and 10) or from iPS-MEF4-10. 666 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. Figure 3. Gene-Expression Profiles of iPS Cells (A) RT-PCR analysis of ES marker genes in iPS cells, ES cells, and MEFs. We used primer sets that amplified endogenous but not transgenic transcripts. (B) The promoters of Oct3/4 and Nanog were analyzed by ChIP for dimethylation status of lysine 9 of histone H3 and acetylation status of histone H3 in ES cells, MEFs, and iPS cells (MEF4-7 and MEF10-6). Data were quantified by real-time PCR. Shown are the averages and standard deviations of relative values compared to ES cells (n = 3). *p < 0.05; **p < 0.01 compared to MEFs. (C) The promoters of Oct3/4, Nanog, and Fbx15 were analyzed with bisulfite genomic sequencing for DNA methylation status in iPS-MEF4-7 and iPS-MEF10-6. The DNA methylation status of these promoters in ES cells and MEFs is shown in Figure 1F. (D) iPS-MEF4-7 and iPS-MEF10-6 clones were stained with a mouse monoclonal antibody against SSEA-1 (480, Santa Cruz) or with an alkaline phosphatase kit (Sigma). Scale bars = 500 mm (SSEA1) and 1 mm (AP). These data demonstrate that the majority of, but not all, When grown in tissue culture dishes, the embryoid bodies iPS-MEF10 and iPS-MEF4 clones exhibit pluripotency. from iPS-MEF10 and iPS-MEF4 cells attached to the dish In contrast, all tumors derived from iPS-MEF3 clones bottom and initiated differentiation. After 3 days, immu- were composed entirely of undifferentiated cells (Table nostaining detected cells positive for a-smooth muscle ac- S6 and Figure S3). Thus, although the three factors tin (mesoderm marker), a-fetoprotein (endoderm marker), (Oct3/4, c-Myc, and Klf4) could induce the expression of and bIII tubulin (ectoderm marker) (Figure 5D). By contrast, some ES cell marker genes, they were not able to induce embryoid bodies from iPS-MEF3 cells remained undiffer- pluripotency. entiated even when cultured in gelatin-coated dishes (Fig- iPS-MEF10, iPS-MEF4, and iPS-MEF3 cells formed em- ure 5C). These data confirmed pluripotency of iPS-MEF10 bryoid bodies in noncoated plastic dishes (Figure 5C). and iPS-MEF4 and nullipotency of iPS-MEF3 in vitro. Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. 667  Figure 4. Global Gene-Expression Analyses by DNA Microarrays (A) Pearson correlation analysis of 10,517 probes was performed to cluster ES cells, iPS cells (MEF4- 7, MEF10-6, MEF3-2, and MEF3-3), MEFs, MEFs expressing the four factors, immortalized MEFs expressing K-RasV12, and NIH 3T3 cells transformed by H-RasV12. Red indicates increased expression compared to median levels of the eight samples, whereas green means decreased expression. (B) Genes upregulated in ES and/or iPS cells. Genes in group I are genes upregulated in ES cells and iPS cells. Genes in group II are upregulated more in ES cells, iPS-MEF4-7, and iPS-MEF10-6 than in iPS-MEF3 cells. Genes in group III are upregulated more in ES cells than in iPS cells. Lists of genes are shown in Tables S3–S5. 668 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc.  Figure 5. Pluripotency of iPS Cells Derived from MEFs (A) Various tissues present in teratomas de- rived from iPS-MEF4-7 cells. Histology of other teratomas is shown in Figure S3 and Table S6. (B) Immunostaining confirming differentiation into neural tissues and muscles in teratomas derived from iPS-MEF4-7. (C) In vitro embryoid body formation (upper row) and differentiation (lower row). Scale bars = 200 mm. (D) Immunostaining confirming in vitro differen- tiation into all three germ layers. Scale bars = 100 mm. Secondary antibodies were labeled with Cy3 (red), except for a-fetoprotein in iPS- MEF10-6, with which Alexa 488 (green) was used. We next introduced the four selected factors into tail-tip (iPS-TTFgfp4, clones 1–6). In addition, we established fibroblasts (TTFs) of four 7-week-old male Fbx15bgeo/bgeo another iPS-TTFgfp4 (clone 7), in which the cDNA for mice on a C57/BL6-129 hybrid background. We obtained each of the four factors was flanked with two loxP sites 3 G418-resistant colonies, from each of which we could in the transgene. These cells were morphologically indis- establish iPS cells (iPS-TTF4). We also introduced the tinguishable from ES cells (Figure 6A). RT-PCR showed four factors into TTFs from a 12-week-old female that clones 3 and 7 of iPS-TTFgfp4 expressed the majority Fbx15bgeo/bgeo mouse, which also constitutively ex- of ES cell marker genes at high levels and the others at pressed green fluorescent protein (GFP) from the CAG lower levels (Figure 6B). In another attempt, we used either promoter and had a C57/BL6-129-ICR hybrid back- the T58A mutant or the wild-type c-Myc for transduction ground. Of the 13 G418-resistant colonies obtained, we and established 5 iPS-TTFgfp4 clones (clones 8–12) and isolated 6 clones from which we could establish iPS cells 3 iPS-TTFgfp4wt clones (clones 1–3) (Figure S5). RT-PCR Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. 669 Figure 6. Characterization of iPS Cells Derived from Adult Mouse Tail-Tip Fibroblasts (A) Morphology of iPS-TTFgfp4-3 on STO feeder cells. (B) RT-PCR analysis of ES marker gene expression in iPS-TTFgfp4 cells (clones 1–5 and 7). We used primer sets that amplified endogenous but not transgenic transcripts. (C) Contribution of iPS-TTFgfp4-7 and iPS-TTFgfp4-3 cells to mouse embryonic development. iPS cells were microinjected into C57/BL6 blastocysts. Embryos were analyzed with a fluorescence microscope at E7.5 (upper panels, iPS-TTFgfp4-7) or E13.5 (lower panels, iPS-TTFgfp4-3). Scale bars = 200 mm (upper panels) and 2 mm (lower panels). (D) The E13.5 chimeric embryo was sectioned and stained with anti-GFP antibody (brown). Cells were counterstained with eosin (blue). showed that iPS-TTFgfp4wt cells also expressed most contributed to all three germ layers (Figure 6D). We ob- of the ES cell marker genes (Figure S6). served GFP-positive cells in the gonad but could not de- We transplanted 2 iPS-TTF4 and 6 iPS-TTFgfp4 clones termine whether they were germ cells or somatic cells. into nude mice, all of which produced tumors containing With iPS-TTFgfp4-7, we obtained 22 embryos at E7.5, 3 tissues of all three germ layers (Table S6 and Figure S3). of which were positive for GFP. With the 2 clones, we We then introduced 2 clones of iPS-TTFgfp4 cells (clones had 27 pups born, but none of them were chimeric mice. 3 and 7) into C57/BL6 blastocysts by microinjection. With In addition, iPS-TTFgfp4 cells could differentiate into all iPS-TTFgfp4-3, we obtained 18 embryos at E13.5, 2 of three germ layers in vitro (Figure S7). These data demon- which showed contribution of GFP-positive iPS cells strate that the four selected factors could induce pluripo- (Figure 6C). Histological analyses confirmed that iPS cells tent cells from adult mouse fibroblast cultures. 670 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc.  We further characterized the expression of the four fac- acetyltransferase (HAT) complexes, including TRRAP, tors and others in iPS cells. Real-time PCR confirmed that which is a core subunit of the TIP60 and GCN5 HAT com- endogenous expression of Oct3/4 and Sox2 was lower in plexes (McMahon et al., 1998), CREB binding protein iPS cells than in ES cells (Figure S8). However, the total (CBP), and p300 (Vervoorts et al., 2003). Within the mam- amount of the four factors from the endogenous genes malian genome, there may be up to 25,000 c-Myc binding and the transgenes exceeded the normal expression sites (Cawley et al., 2004), many more than the predicted levels in ES cells. In contrast, Western blot analyses number of Oct3/4 and Sox2 binding sites (Boyer et al., showed that the total protein amounts of the four factors 2005; Loh et al., 2006). c-Myc protein may induce global in iPS cells were comparable to those in ES cells (Fig- histone acetylation (Fernandez et al., 2003), thus allowing ure 7A; Figure S8). We could detect Nanog and E-Ras pro- Oct3/4 and Sox2 to bind to their specific target loci. teins in iPS cells, but at lower levels than those in ES cells Klf4 has been shown to repress p53 directly (Rowland (Figures 7A and 7B; Figure S8). The p53 levels in iPS cells et al., 2005), and p53 protein has been shown to suppress were lower than those in MEFs and equivalent to those in Nanog during ES cell differentiation (Lin et al., 2004). We ES cells (Figure 7A; Figure S9). The p21 levels in iPS cells found that iPS cells showed levels of p53 protein lower varied in each clone and were between those in ES cells than those in MEFs (Figure 7A). Thus, Klf4 might contrib- and MEFs (Figure S9). Upon differentiation in vitro, the to- ute to activation of Nanog and other ES cell-specific genes tal mRNA expression levels of Oct3/4 and Sox2 decreased through p53 repression. Alternatively, Klf4 might function but remained much higher than in ES cells. In contrast, as an inhibitor of Myc-induced apoptosis through the re- their protein levels decreased to comparable levels in pression of p53 in our system (Zindy et al., 1998). On the iPS cells and ES cells (Figure 7B). other hand, Klf4 activates p21CIP1, thereby suppressing Southern blot analyses showed that each iPS clone has cell proliferation (Zhang et al., 2000). This antiproliferation a unique transgene integration pattern (Figure 7C). Karyo- function of Klf4 might be inhibited by c-Myc, which sup- typing analyses of the iPS-TTFgfp4 (clones 1, 2, 3, 7, and presses the expression of p21CIP1 (Seoane et al., 2002). 11) and iPS-TTFgfp4wt (clones 1–3) demonstrated that 2 The balance between c-Myc and Klf4 may be important iPS-TTFgfp4 clones and all of the iPS-TTFgfp4wt clones for the generation of iPS cells. showed a normal karyotype of 40XX (Figure 7D), while One question that remains concerns the origin of our the other 3 iPS-TTFgfp4 clones were 39XO, 40XO +10, iPS cells. With our retroviral expression system, we esti- and 40Xi(X). Analyses of PCR-based simple sequence mated that only a small portion of cells expressing the length polymorphisms (SSLPs) demonstrated that iPS- four factors became iPS cells (Figure S11). The low fre- MEF clones have a mixed background of C57/BL6 and quency suggests that rare tissue stem/progenitor cells 129 (Table S7), whereas iPS-TTFgfp clones have a mixed that coexisted in the fibroblast cultures might have given background of ICR, C57/BL6, and 129 (Table S8). Finally, rise to the iPS cells. Indeed, multipotent stem cells have we found that iPS cells could not remain undifferentiated been isolated from skin (Dyce et al., 2004; Toma et al., when cultured in the absence of feeder cells, even with 2001, 2005). These studies showed that
0.067% of the presence of LIF (Figure 7E). These results, together mouse skin cells are stem cells. One explanation for the with the different gene-expression patterns, exclude the low frequency of iPS cell derivation is that the four factors possibility that iPS cells are merely contamination of pre- transform tissue stem cells. However, we found that the existing ES cells. Finally, subclones of iPS cells were pos- four factors induced iPS cells with comparably low effi- itive for alkaline phosphatase and could differentiate into ciency even from bone marrow stroma, which should be all three germ layers in vitro (Figure S10), confirming their more enriched in mesenchymal stem cells and other multi- clonal nature. potent cells (Tables S2 and S6). Furthermore, cells in- duced by the three factors were nullipotent (Table S6 and Figure S3). DNA microarray analyses suggested that DISCUSSION iPS-MEF4 cells and iPS-MEF3 cells have the same origin (Figure 4). These results do not favor multipotent tissue Oct3/4, Sox2, and Nanog have been shown to function stem cells as the origin of iPS cells. as core transcription factors in maintaining pluripotency There are several other possibilities for the low fre- (Boyer et al., 2005; Loh et al., 2006). Among the three, quency of iPS cell derivation. First, the levels of the four we found that Oct3/4 and Sox2 are essential for the gen- factors required for generation of pluripotent cells may eration of iPS cells. Surprisingly, Nanog is dispensable. In have narrow ranges, and only a small portion of cells ex- addition, we identified c-Myc and Klf4 as essential factors. pressing all four of the factors at the right levels can acquire These two tumor-related factors could not be replaced by ES cell-like properties. Consistent with this idea, a mere other oncogenes including E-Ras, Tcl1, b-catenin, and 50% increase or decrease in Oct3/4 proteins induces Stat3 (Figures 2A and 2B). differentiation of ES cells (Niwa et al., 2000). iPS clones The c-Myc protein has many downstream targets that overexpressed the four factors when RNA levels were an- enhance proliferation and transformation (Adhikary and alyzed, but their protein levels were comparable to those in Eilers, 2005), many of which may have roles in the gener- ES cells (Figures 7A and 7B; Figure S8), suggesting that the ation of iPS cells. Of note, c-Myc associates with histone iPS clones possess a mechanism (or mechanisms) that Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. 671 Figure 7. Biochemical and Genetic Analyses of iPS Cells (A) Western blot analyses of the four factors and other proteins in iPS cells (MEF4-7, MEF10-6, TTFgfp4-3, and TTFgfp4-7), ES cells, and MEFs. (B) Changes in RNA (left) and protein (right) levels of Oct3/4, Sox2, and Nanog in iPS cells and ES cells that were undifferentiated on STO feeder cells (U) or induced to differentiated in vitro through embryoid body formation (D). Shown are relative expression levels compared to undifferentiated ES cells. Data of MEFs and TTFs are also shown. RNA levels were determined with real-time PCR using primers specific for endogenous transcripts 672 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. tightly regulates the protein levels of the four factors. We these cells contain multiple loxP sites on multiple chromo- speculate that high amounts of the four factors are re- somes, and, thus, the Cre-mediated recombination would quired in the initial stage of iPS cell generation, but, once cause not only deletion of the transgenes but also inter- they acquire ES cell-like status, too much of the factors and intrachromosomal rearrangements. Studies with are detrimental for self-renewal. Only a small portion of conditional expression systems, such as the tetracycline- transduced cells show such appropriate transgene ex- mediated system, are required to answer this question. pression. Second, generation of pluripotent cells may We showed that the iPS cells can differentiate in vitro require additional chromosomal alterations, which take and in vivo even with the presence of the retroviral vectors place spontaneously during culture or are induced by containing the four factors. We found that Oct3/4 and some of the four factors. Although the iPS-TTFgfp4 clones Sox2 proteins decreased significantly during in vitro differ- had largely normal karyotypes (Figure 7D), we cannot rule entiation (Figure 7B). Retroviral expression has been out the existence of minor chromosomal alterations. Site- shown to be suppressed in ES cells and further silenced specific retroviral insertion may also play a role. Southern upon differentiation by epigenetic modifications, such as blot analyses showed that each iPS clone has
20 retroviral DNA methylation (Yao et al., 2004). The same mechanisms integrations (Figure 7C). Some of these may have caused are likely to play roles in transgene repression in iPS cells silencing or fusion with endogenous genes. Further studies since they express Dnmt3a, 3b, and 3l, albeit at lower will be required to determine the origin of iPS cells. levels than ES cells do (Table S5). In addition, we found Another unsolved question is whether the four factors that iPS cells possess a mechanism (or mechanisms) we identified play roles in reprogramming induced by fu- that lowers protein levels of the transgenes and Nanog sion with ES cells or nuclear transfer into oocytes. Since (Figure 7B; Figure S8). The same mechanism may be the four factors are expressed in ES cells at high levels, enhanced during differentiation. However, silencing of it is reasonable to speculate that they are involved in the Oct3/4 in iPS-TTFgfp4-3 cells was not complete, which reprogramming machinery that exists in ES cells. Our re- may explain our inability to obtain live chimeric mice after sult is also consistent with the finding that the reprogram- blastocyst microinjection of iPS cells. ming activity resides in the nucleus, but not in the cyto- An unexpected finding in this study was the efficient ac- plasm, of ES cells (Do and Scholer, 2004). However, iPS tivation of Fgf4 and Fbx15 by the combination of the three cells were not identical to ES cells, as shown by the global factors devoid of Sox2 since these two genes have been gene-expression patterns and DNA methylation status. It shown to be regulated synergistically by Oct3/4 and is possible that we have missed additional important fac- Sox2 (Tokuzawa et al., 2003; Yuan et al., 1995). It is also tors. One such candidate is ECAT1, although its forced surprising that Nanog is dispensable for induction and expression in iPS cells did not consistently upregulate maintenance of iPS cells. More detailed analyses of iPS ES cell marker genes (Figure S12). cells will enhance our understanding of transcriptional More obscure are the roles of the four factors, especially regulation in pluripotent stem cells. Klf4 and c-Myc, in the reprogramming observed in oo- Our findings may have wider applications, as we have cytes. Both Klf4 and c-Myc are dispensable for preimplan- found that transgene reporters with other ES cell marker tation mouse development (Baudino et al., 2002; Katz genes, such as Nanog, can replace the Fbx15 knockin dur- et al., 2002). Furthermore, c-myc is not detected in oo- ing selection (K. Okita and S.Y., unpublished data). How- cytes (Domashenko et al., 1997). In contrast, L-myc is ex- ever, we still do not know whether the four factors can gen- pressed maternally in oocytes. Klf17 and Klf7, but not Klf4, erate pluripotent cells from human somatic cells. Use of are found in expressed sequence-tag libraries derived c-Myc may not be suitable for clinical applications, and from unfertilized mouse eggs. Klf4 and c-Myc might be the process may require specific culture environments. compensated by these related proteins. It is highly likely Nevertheless, the finding is an important step in controlling that other factors are also required to induce complete pluripotency, which may eventually allow the creation of reprogramming and totipotency in oocytes. pluripotent cells directly from somatic cells of patients. It is likely that the four factors from the transgenes are required for maintaining the iPS cells since the expression EXPERIMENTAL PROCEDURES of Oct3/4 and Sox2 from the endogenous genes remained low (Figure 7B; Figure S8). We intended to prove this by Mice using transgenes flanked by two loxP sites and obtained Fbx15bgeo/bgeo mice were generated with 129SvJae-derived RF8 ES an iPS clone (TTF4gfp4-7). However, we noticed that cells as described previously (Tokuzawa et al., 2003) and were (white columns) or those common for both endogenous and transgenic transcripts (white and black columns). RNA expression levels are shown on logarithmic axes. Protein levels were determined by Western blot normalized with b-actin. Protein levels are shown as the averages and standard deviations on linear axes (n = 4). *p < 0.05 compared to undifferentiated cells. (C) Southern blot analyses showing the integration of transgenes. Genomic DNA isolated from iPS cells and ES cells was digested with EcoRI and BamHI, separated on agarose gel, transferred to a nylon membrane, and hybridized with a Klf4 cDNA probe. (D) Normal karyotype of iPS-TTFgfp4-2 clone. (E) Morphology of ES cells and iPS cells cultured without feeder cells. One thousand cells were cultured on gelatin-coated six-well plates for 5 days, with or without LIF. Scale bars = 200 mm. Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. 673 backcrossed to the C57/BL6 strain for at least five generations. These listed in Table S9. Mutations in b-catenin, c-myc, and Stat3 were intro- mice were used for primary mouse embryonic fibroblast (MEF) and tail- duced by PCR-based site-directed mutagenesis. For forced expres- tip fibroblast (TTF) preparations. To generate Fbx15bgeo/bgeo mice with sion, we amplified the coding regions of candidate genes by RT-PCR, constitutive expression of GFP, an Fbx15bgeo/bgeo mouse (C57/BL6- cloned these sequences into pDONR201 or pENTR-D-TOPO (Invitro- 129 background) was mated with an ICR mouse with the GFP trans- gen), and recombined the resulting plasmids with pMXs-gw by LR gene driven by the constitutive CAG promoter (Niwa et al., 1991). reaction (Invitrogen). The resulting Fbx15bgeo/+,GFP/+ mice were intercrossed to generate Fbx15bgeo/bgeo,GFP/GFP mice. Nude mice (BALB/Jcl-nu) were pur- Teratoma Formation and Histological Analysis chased from CLEA. ES cells or iPS cells were suspended at 1 3 107 cells/ml in DMEM con- taining 10% FBS. Nude mice were anesthetized with diethyl ether. We Cell Culture injected 100 ml of the cell suspension (1 3 106 cells) subcutaneously RF8 ES cells and iPS cells were maintained on feeder layers of mitomy- into the dorsal flank. Four weeks after the injection, tumors were surgi- cin C-treated STO cells as previously described (Meiner et al., 1996). cally dissected from the mice. Samples were weighed, fixed in PBS As a source of leukemia inhibitory factor (LIF), we used conditioned containing 4% formaldehyde, and embedded in paraffin. Sections medium (1:10,000 dilution) from Plat-E cell cultures that had been were stained with hematoxylin and eosin. transduced with a LIF-encoding vector. ES and iPS cells were pas- saged every 3 days. Plat-E packaging cells (Morita et al., 2000), which Bisulfite Genomic Sequencing were also used to produce retroviruses, were maintained in DMEM Bisulfite treatment was performed using the CpGenome modification containing 10% FBS, 50 units/50 mg/ml penicillin/streptomycin, kit (Chemicon) according to the manufacturer’s recommendations. 1 mg/ml puromycin (Sigma), and 100 mg/ml of blasticidin S (Funakoshi). PCR primers are listed in Table S9. Amplified products were cloned For MEF isolation, uteri isolated from 13.5-day-pregnant mice were into pCR2.1-TOPO (Invitrogen). Ten randomly selected clones were se- washed with phosphate-buffered saline (PBS). The head and visceral quenced with the M13 forward and M13 reverse primers for each gene. tissues were removed from isolated embryos. The remaining bodies were washed in fresh PBS, minced using a pair of scissors, transferred Determination of Karyotypes and SSLP by PCR into a 0.1 mM trypsin/1 mM EDTA solution (3 ml per embryo), and Karyotypes were determined with quinacrine-Hoechst staining at the incubated at 37 C for 20 min. After incubation, an additional 3 ml per International Council for Laboratory Animal Science (ICLAS) Monitor- embryo of 0.1 mM trypsin/1 mM EDTA solution was added, and the ing Center (Japan). We obtained PCR primer sequences for SSLP mixture was incubated at 37 C for 20 min. After trypsinization, an equal from the Mouse Genome Informatics website (The Jackson Labora- amount of medium (6 ml per embryo DMEM containing 10% FBS) was tory, http://www.informatics.jax.org). Allele sizes were approximated added and pipetted up and down a few times to help with tissue dis- on the basis of the known allele sizes in various inbred strains. sociation. After incubation of the tissue/medium mixture for 5 min at room temperature, the supernatant was transferred into a new tube. Western Blot Analyses Cells were collected by centrifugation (200 3 g for 5 min at 4 C) and Western blot was performed as previously described (Takahashi et al., resuspended in fresh medium. 1 3 106 cells (passage 1) were cultured 2003). The primary antibodies used were anti-Oct3/4 monoclonal on 100 mm dishes at 37 C with 5% CO2. In this study, we used MEFs antibody (C-10, Santa Cruz), anti-Sox2 antiserum (Maruyama et al., within three passages to avoid replicative senescence. 2005), anti-Klf4 polyclonal antibody (H-180, Santa Cruz), anti-c-Myc To establish TTFs, the tails from adult mice were peeled, minced into polyclonal antibody (A-14, Santa Cruz), anti-Nanog antiserum (Mitsui 1 cm pieces, placed on culture dishes, and incubated in MF-start me- et al., 2003), anti-E-Ras antiserum (Takahashi et al., 2003), anti-p53 dium (Toyobo) for 5 days. Cells that migrated out of the graft pieces polyclonal antibody (FL-393, Santa Cruz), and anti-b-actin monoclonal were transferred to new plates (passage 2) and maintained in DMEM antibody (A5441, Sigma). containing 10% FBS. We used TTFs at passage 3 for iPS cell induction. RT-PCR for Marker Genes Retroviral Infection We performed reverse transcription reactions using ReverTra Ace -a- The day before transduction, Plat-E cells (Morita et al., 2000) were (Toyobo) and the oligo dT20 primer. PCR was done with ExTaq seeded at 8 3 106 cells per 100 mm dish. On the next day, pMXs-based (Takara). Real-time PCR was performed with Platinum SYBR Green retroviral vectors were introduced into Plat-E cells using Fugene 6 qPCR SuperMix-UDG with ROX (Invitrogen) according to manufac- transfection reagent (Roche) according to the manufacturer’s recom- turer’s instructions. Signals were detected with an ABI7300 Real- mendations. Twenty-seven microliters of Fugene 6 transfection re- Time PCR System (Applied Biosystems). Primer sequences are listed agent was diluted in 300 ml DMEM and incubated for 5 min at room tem- in Table S9. perature. Nine micrograms of plasmid DNA was added to the mixture, which was incubated for another 15 min at room temperature. After in- DNA Microarray cubation, the DNA/Fugene 6 mixture was added drop by drop onto Total RNA from ES cells, iPS cells, or MEFs were labeled with Cy3. Plat-E cells. Cells were then incubated overnight at 37 C with 5% CO2. Samples were hybridized to a Mouse Oligo Microarray (G4121B, Agi- Twenty-four hours after transduction, the medium was replaced. lent) according to the manufacturer’s protocol. Arrays were scanned MEFs or TTFs were seeded at 8 3 105 cells per 100 mm dish on mito- with a G2565BA Microarray Scanner System (Agilent). Data were ana- mycin C-treated STO feeders. After 24 hr, virus-containing superna- lyzed using GeneSpring GX software (Agilent). tants derived from these Plat-E cultures were filtered through a 0.45 mm cellulose acetate filter (Schleicher & Schuell) and supple- In Vitro Differentiation of iPS Cells mented with 4 mg/ml polybrene (Nacalai Tesque). Target cells were in- Cells were harvested by trypsinization and transferred to bacterial cul- cubated in the virus/polybrene-containing supernatants for 4 hr to ture dishes in the ES medium without G418 or LIF. After 3 days, aggre- overnight. After infection, the cells were replated in 10 ml fresh gated cells were plated onto gelatin-coated tissue culture dishes and medium. Three days after infection, we added G418 at a final concen- incubated for another 3 days. The cells were stained with anti- tration of 0.3 mg/ml. Clones were selected for 2 to 3 weeks. a-smooth muscle actin monoclonal antibody (N1584, Dako), anti-a- fetoprotein polyclonal antibody (N1501, Dako) or anti-bIII tubulin Plasmid Construction monoclonal antibody (CBL412, Abcam) along with 40 -6-diamidino- To generate pMXs-gw, we introduced a Gateway cassette rfA (Invitro- 2-phenylindole (Sigma). Total RNA derived from plated embryoid gen) into the EcoRI/XhoI site of the pMXs plasmid. Primers used are bodies on day 6 was used for RT-PCR analysis. 674 Cell 126, 663–676, August 25, 2006 ª2006 Elsevier Inc. Chromatin Immunoprecipitation Assay Cartwright, P., McLean, C., Sheppard, A., Rivett, D., Jones, K., and We performed chromatin immunoprecipitation (ChIP) as previously Dalton, S. (2005). LIF/STAT3 controls ES cell self-renewal and pluripo- described (Maruyama et al., 2005). Antibodies used in this experiment tency by a Myc-dependent mechanism. Development 132, 885–896. were anti-dimethyl K9 H3 rabbit polyclonal antibody (ab7312-100, Cawley, S., Bekiranov, S., Ng, H.H., Kapranov, P., Sekinger, E.A., Abcam) and anti-acetyl H3 rabbit polyclonal antibody (06-599, Kampa, D., Piccolboni, A., Sementchenko, V., Cheng, J., Williams, Upstate). PCR primers are listed in Table S9. A.J., et al. (2004). Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread reg- Statistical Analyses ulation of noncoding RNAs. Cell 116, 499–509. Data are shown as averages and standard deviations. We used Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, Student’s t test for protein-level analyses and one-factor ANOVA S., and Smith, A. (2003). Functional expression cloning of nanog, a plu- with Scheffe’s post hoc test for ChIP analyses. All statistical analyses ripotency sustaining factor in embryonic stem cells. 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We are grateful to Tomoko Ichisaka for preparation of mice and Mit- suyo Maeda and Yoshinobu Toda for histological analyses. We thank Do, J.T., and Scholer, H.R. (2004). Nuclei of embryonic stem cells Megumi Kumazaki, Mirei Murakami, Masayoshi Maruyama, and Nor- reprogram somatic cells. Stem Cells 22, 941–949. iko Tsubooka for technical assistance; Masato Nakagawa, Keisuke Domashenko, A.D., Latham, K.E., and Hatton, K.S. (1997). Expression Okita, and Koji Shimozaki for scientific comments; and Yumi Ohuchi of myc-family, myc-interacting, and myc-target genes during preim- for administrative assistance. We also thank Dr. Robert Farese, Jr. plantation mouse development. Mol. Reprod. Dev. 47, 57–65. for RF8 ES cells and Dr. Toshio Kitamura for the Plat-E cells and Dyce, P.W., Zhu, H., Craig, J., and Li, J. (2004). Stem cells with multi- pMX retroviral vectors. This work was supported in part by research lineage potential derived from porcine skin. Biochem. Biophys. Res. grants from the Ministry of Education, Culture, Sports, Science and Commun. 316, 651–658. Technology of Japan to S.Y. This work is also supported in part by Evans, M.J., and Kaufman, M.H. (1981). Establishment in culture of the Takeda Science Foundation, the Osaka Cancer Research Founda- pluripotential cells from mouse embryos. Nature 292, 154–156. tion, the Inamori Foundation, the Mitsubishi Pharma Research Foun- dation, and the Sankyo Foundation of Life Science and by a Grant- Fernandez, P.C., Frank, S.R., Wang, L., Schroeder, M., Liu, S., Greene, in-Aid from the Japan Medical Association to S.Y. K.T. was supported J., Cocito, A., and Amati, B. (2003). Genomic targets of the human by a fellowship from the Japan Society for the Promotion of Science. c-Myc protein. Genes Dev. 17, 1115–1129. Katz, J.P., Perreault, N., Goldstein, B.G., Lee, C.S., Labosky, P.A., Received: April 24, 2006 Yang, V.W., and Kaestner, K.H. (2002). 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