@article{okita_generation_2008,
	title = {Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors},
	volume = {322},
	url = {http://www.sciencemag.org/cgi/content/abstract/322/5903/949},
	doi = {10.1126/science.1164270},
	abstract = {Induced pluripotent stem {(iPS)} cells have been generated from mouse and human somatic cells by introducing Oct3/4 and Sox2 with either Klf4 and {c-Myc} or Nanog and Lin28 using retroviruses or lentiviruses. Patient-specific {iPS} cells could be useful in drug discovery and regenerative medicine. However, viral integration into the host genome increases the risk of tumorigenicity. Here, we report the generation of mouse {iPS} cells without viral vectors. Repeated transfection of two expression plasmids, one containing the complementary {DNAs} {(cDNAs)} of Oct3/4, Sox2, and Klf4 and the other containing the {c-Myc} {cDNA,} into mouse embryonic fibroblasts resulted in {iPS} cells without evidence of plasmid integration, which produced teratomas when transplanted into mice and contributed to adult chimeras. The production of virus-free {iPS} cells, albeit from embryonic fibroblasts, addresses a critical safety concern for potential use of {iPS} cells in regenerative medicine.
},
	number = {5903},
	journal = {Science},
	author = {Keisuke Okita and Masato Nakagawa and Hong Hyenjong and Tomoko Ichisaka and Shinya Yamanaka},
	month = nov,
	year = {2008},
	pages = {949--953}
},

@article{lovell-badge_many_2007,
	title = {Many ways to pluripotency},
	volume = {25},
	issn = {1087-0156},
	url = {http://dx.doi.org/10.1038/nbt1007-1114},
	doi = {10.1038/nbt1007-1114},
	number = {10},
	journal = {Nat Biotech},
	author = {Robin {Lovell-Badge}},
	month = oct,
	year = {2007},
	pages = {1114--1116}
},

@article{huangfu_induction_2008,
	title = {Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds},
	volume = {26},
	issn = {1087-0156},
	url = {http://dx.doi.org/10.1038/nbt1418},
	doi = {10.1038/nbt1418},
	number = {7},
	journal = {Nat Biotech},
	author = {Danwei Huangfu and Rene Maehr and Wenjun Guo and Astrid Eijkelenboom and Melinda Snitow and Alice E Chen and Douglas A Melton},
	month = jul,
	year = {2008},
	pages = {795--797}
},

@article{takahashi_induction_2007,
	title = {Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors},
	volume = {131},
	url = {http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4R70J0T-1&_user=108429&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059713&_version=1&_urlVersion=0&_userid=108429&md5=b7b1084d20b42dc0b8551c4c499942cc},
	doi = {10.1016/j.cell.2007.11.019},
	abstract = {{1Department} of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan {2CREST,} Japan Science and Technology Agency, Kawaguchi 332-0012, Japan {3Gladstone} Institute of Cardiovascular Disease, San Francisco, {CA} 94158, {USA} {4Institute} for Integrated {Cell-Material} Sciences, Kyoto University, Kyoto 606-8507, Japan Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem {(iPS)} cells, capable of germline transmission, from mouse somatic cells by transduction of four defined transcription factors. Here, we demonstrate the generation of {iPS} cells from adult human dermal fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and {c-Myc.} Human {iPS} cells were similar to human embryonic stem {(ES)} cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that {iPS} cells can be generated from adult human fibroblasts.   We showed that induced pluripotent stem {(iPS)} cells can be generated from mouse embryonic fibroblasts {(MEF)} and adult mouse tail-tip fibroblasts by the retrovirus-mediated transfection of four transcription factors, namely Oct3/4, Sox2, {c-Myc,} and Klf4 {(Takahashi} and Yamanaka, 2006). Mouse {iPS} cells are indistinguishable from {ES} cells in morphology, proliferation, gene expression, and teratoma formation. Furthermore, when transplanted into blastocysts, mouse {iPS} cells can give rise to adult chimeras, which are competent for germline transmission {([Maherali} et al., 2007], {[Okita} et al., 2007] and {[Wernig} et al., 2007]). These results are proof of principle that pluripotent stem cells can be generated from somatic cells by the combination of a small number of factors. In the current study, we sought to generate {iPS} cells from adult human somatic cells by optimizing retroviral transduction in human fibroblasts and subsequent culture conditions. These efforts have enabled us to generate {iPS} cells from adult human dermal fibroblasts and other human somatic cells, which are comparable to human {ES} cells in their differentiation potential in vitro and in teratomas. Induction of {iPS} cells from mouse fibroblasts requires retroviruses with high transduction efficiencies {(Takahashi} and Yamanaka, 2006). We, therefore, optimized transduction methods in adult human dermal fibroblasts {(HDF).} We first introduced green fluorescent protein {(GFP)} into adult {HDF} with amphotropic retrovirus produced in {PLAT-A} packaging cells. As a control, we introduced {GFP} to mouse embryonic fibroblasts {(MEF)} with ecotropic retrovirus produced in {PLAT-E} packaging {cells(Morita} et al., 2000). In {MEF,} more than 80\% of cells expressed {GFP} {(Figure} S1). In contrast, less than 20\% of {HDF} expressed {GFP} with significantly lower intensity than in {MEF.} To improve the transduction efficiency, we introduced the mouse receptor for retroviruses, Slc7a1 {(Verrey} et al., 2004) (also known as {mCAT1),} into {HDF} with lentivirus. We then introduced {GFP} to {HDF-Slc7a1} with ecotropic retrovirus. This strategy yielded a transduction efficiency of 60\%, with a similar intensity to that in {MEF.} The protocol for human {iPS} cell induction is summarized in Figure {1A.} We introduced the retroviruses containing human Oct3/4, Sox2, Klf4, and {c-Myc} into {HDF-Slc7a1} {(Figure} {1B;} 8 × 105 cells per 100 mm dish). The {HDF} derived from facial dermis of 36-year-old Caucasian female. Six days after transduction, the cells were harvested by trypsinization and plated onto mitomycin C-treated {SNL} feeder cells {(McMahon} and Bradley, 1990) at 5 × 104 or 5 × 105 cells per 100 mm dish. The next day, the medium {(DMEM} containing 10\% {FBS)} was replaced with a medium for primate {ES} cell culture supplemented with 4 ng/ml basic fibroblast growth factor {(bFGF).} Approximately two weeks later, some granulated colonies appeared that were not similar to {hES} cells in morphology {(Figure} {1C).} Around day 25, we observed distinct types of colonies that were flat and resembled {hES} cell colonies {(Figure} {1D).} From 5 × 104 fibroblasts, we observed 10 {hES} cell-like colonies and 100 granulated colonies (7/122, 8/84, 8/171, 5/73, 6/122, and 11/213 in six independent experiments, summarized in Table S1). At day 30, we picked {hES} cell-like colonies and mechanically disaggregated them into small clumps without enzymatic digestion. When starting with 5 × 105 fibroblasts, the dish was nearly covered with more than 300 granulated colonies. We occasionally observed some {hES} cell-like colonies in between the granulated cells, but it was difficult to isolate {hES} cell-like colonies because of the high density of granulated cells. The nature of the {non-hES-like} cells remains to be determined. The {hES-like} cells expanded on {SNL} feeder cells with the primate {ES} cell medium containing {bFGF.} They formed tightly packed and flat colonies {(Figure} {1E).} Each cell exhibited morphology similar to that of human {ES} cells, characterized by large nuclei and scant cytoplasm {(Figure} {1F).} As is the case with {hES} cells, we occasionally observed spontaneous differentiation in the center of the colony {(Figure} {1G).} These cells also showed similarity to {hES} cells in feeder dependency {(Figure} S2). They did not attach to gelatin-coated tissue-culture plates. By contrast, they maintained an undifferentiated state on Matrigel-coated plates in {MEF-conditioned} primate {ES} cell medium, but not in nonconditioned medium. Since these cells were similar to {hES} cells in morphology and other aspects noted above, we will refer to the selected cells after transduction of {HDF} as human {iPS} cells, as we describe the molecular and functional evidence for this claim. Human {iPS} cells clones established in this study are summarized in Table S2. In general, except for a few cells at the edge of the colonies, human {iPS} cells did not express stage-specific embryonic antigen {(SSEA)-1} {(Figure} {1H).} In contrast, they expressed {hES} cell-specific surface {antigens(Adewumi} et al., 2007), including {SSEA-3,} {SSEA-4,} tumor-related antigen {(TRA)-1-60,} {TRA-1-81} and {TRA-2-49/6E} (alkaline phosphatase), and {NANOG} protein {(Figures} {1I–1N).} {RT-PCR} showed human {iPS} cells expressed many undifferentiated {ES} cell-marker genes {(Adewumi} et al., 2007), such as {OCT3/4,} {SOX2,} {NANOG,} growth and differentiation factor 3 {(GDF3),} reduced expression 1 {(REX1),} fibroblast growth factor 4 {(FGF4),} embryonic cell-specific gene 1 {(ESG1),} developmental pluripotency-associated 2 {(DPPA2),} {DPPA4,} and telomerase reverse transcriptase {(hTERT)} at levels equivalent to or higher than those in the {hES} cell line H9 and the human embryonic carcinoma cell line, {NTERA-2} {(Figure} {2A).} By western blotting, proteins levels of {OCT3/4,} {SOX2,} {NANOG,} {SALL4,} {E-CADHERIN,} and {hTERT} were similar in human {iPS} cells and {hES} cells {(Figure} {2B).} Although the expression levels of Klf4 and {c-Myc} increased more than 5-fold in {HDF} after the retroviral transduction (not shown), their expression levels in human {iPS} cells were comparable to those in {HDF} {(Figures} {2A} and {2B),} indicating retroviral silencing. {RT-PCR} using primers specific for retroviral transcripts confirmed efficient silencing of all the four retroviruses {(Figure} {2C).} {DNA} microarray analyses showed that the global gene-expression patterns are similar, but not identical, between human {iPS} cells and {hES} cells {(Figure} {2D).} Among 32,266 genes analyzed, 5,107 genes showed more than 5-fold difference in expression between {HDF} and human {iPS} cells {(Tables} S3 and S4), whereas 6083 genes between {HDF} and {hES} cells showed {\textgreater}5-fold difference in expression {(Tables} S5 and S6). In contrast, a smaller number of genes (1,267 genes) showed {\textgreater}5-fold difference between human {iPS} cells and {hES} cells {(Tables} S7 and S8). Bisulfite genomic sequencing analyses evaluating the methylation statuses of cytosine guanine dinucleotides {(CpG)} in the promoter regions of pluripotent-associated genes, such as {OCT3/4,} {REX1,} and {NANOG,} revealed that they were highly unmethylated, whereas the {CpG} dinucleotides of the regions were highly methylated in parental {HDFs} {(Figure} {3A).} These findings indicate that these promoters are active in human {iPS} cells. Luciferase reporter assays also showed that human {OCT3/4} and {REX1} promoters had high levels of transcriptional activity in human {iPS} cells and {EC} cells {(NTERA-2)} but not in {HDF.} The promoter activities of ubiquitously expressed genes, such as human {RNA} polymerase {II} {(PolII),} showed similar activities in both human {iPS} cells and {HDF} {(Figure} {3B).} We also performed chromatin immunoprecipitation to analyze the histone modification status in human {iPS} cells {(Figure} {3C).} We found that histone H3 lysine 4 was methylated whereas H3 lysine 27 was demethylated in the promoter regions of Oct3/4, Sox2, and Nanog in human {iPS} cells. We also found that human {iPS} cells showed the bivalent patterns of development-associated genes, such as Gata6, Msx2, Pax6, and Hand1. These histone modification statuses are characteristic of {hES} cells {(Pan} et al., 2007). As predicted from the high expression levels of {hTERT,} human {iPS} cells showed high telomerase activity {(Figure} {4A).} They proliferated exponentially for as least 4 months {(Figure} {4B).} The calculated population doubling time of human {iPS} cells were 46.9 ± 12.4 (clone {201B2),} 47.8 ± 6.6 {(201B6)} and 43.2 ± 11.5 {(201B7)} hours. These times are equivalent to the reported doubling time of {hES} cells {(Cowan} et al., 2004). To determine the differentiation ability of human {iPS} cells in vitro, we used floating cultivation to form embryoid bodies {(EBs)} {(Itskovitz-Eldor} et al., 2000). After 8 days in suspension culture, {iPS} cells formed ball-shaped structures {(Figure} {5A).} We transferred these embryoid body-like structures to gelatin-coated plates and continued cultivation for another 8 days. Attached cells showed various types of morphologies, such as those resembling neuronal cells, cobblestone-like cells, and epithelial cells {(Figures} {5B–5E).} Immunocytochemistry detected cells positive for {βIII-tubulin} (a marker of ectoderm), glial fibrillary acidic protein {(GFAP,} ectoderm), α-smooth muscle actin {(α-SMA,} mesoderm), desmin (mesoderm), α-fetoprotein {(AFP,} endoderm), and vimentin (mesoderm and parietal endoderm) {(Figures} {5F–5K).} {RT-PCR} confirmed that these differentiated cells expressed forkhead box A2 {(FOXA2,} a marker of endoderm), {AFP} (endoderm), cytokeratin 8 and 18 (endoderm), {SRY-box} containing gene 17 {(SOX17,} endoderm), {BRACHYURY} (mesoderm), Msh homeobox 1 {(MSX1,} mesoderm), microtubule-associated protein 2 {(MAP2,} ectoderm), and paired box 6 {(PAX6,} ectoderm) {(Figure} {5L).} In contrast, expression of {OCT3/4,} {SOX2,} and {NANOG} was markedly decreased. These data demonstrated that {iPS} cells could differentiate into three germ layers in vitro. We next examined whether lineage-directed differentiation of human {iPS} cells could be induced by reported methods for {hES} cells. We seeded human {iPS} cells on {PA6} feeder layer and maintained them under differentiation conditions for 2 weeks {(Kawasaki} et al., 2000). Cells spread drastically, and some neuronal structures were observed {(Figure} {6A).} Immunocytochemistry detected cells positive for tyrosine hydroxylase and {βIII} tubulin in the culture {(Figure} {6B).} {PCR} analysis revealed expression of dopaminergic neuron markers, such as {aromatic-L-amino} acid decarboxylase {(AADC),} member 3 {(DAT),} choline acetyltransferase {(ChAT),} and {LIM} homeobox transcription factor 1 beta {(LMX1B),} as well as another neuron marker, {MAP2} {(Figure} {6C).} In contrast, {GFAP} expression was not induced with this system. On the other hand, the expression of {OCT3/4} and {NANOG} decreased markedly, whereas Sox2 decreased only slightly {(Figure} {6C).} These data demonstrated that {iPS} cells could differentiate into neuronal cells, including dopaminergic neurons, by coculture with {PA6} cells. We next examined directed cardiac differentiation of human {iPS} cells with the recently reported protocol, which utilizes activin A and bone morphogenetic protein {(BMP)} 4 {(Laflamme} et al., 2007). Twelve days after the induction of differentiation, clumps of cells started beating {(Figure} {6D} and Movie S1). {RT-PCR} showed that these cells expressed cardiomyocyte markers, such as troponin T type 2 cardiac {(TnTc);} myocyte enhancer factor {2C} {(MEF2C);} myosin, light polypeptide 7, regulatory {(MYL2A);} myosin, heavy polypeptide 7, cardiac muscle, beta {(MYHCB);} and {NK2} transcription factor-related, locus 5 {(NKX2.5)} {(Figure} {6E).} In contrast, the expression of Oct3/4, Sox2, and Nanog markedly decreased. Thus, human {iPS} cells can differentiate into cardiac myocytes in vitro. To test pluripotency in vivo, we transplanted human {iPS} cells (clone {201B7)} subcutaneously into dorsal flanks of immunodeficient {(SCID)} mice. Nine weeks after injection, we observed tumor formation. Histological examination showed that the tumor contained various tissues {(Figure} 7), including gut-like epithelial tissues (endoderm), striated muscle (mesoderm), cartilage (mesoderm), neural tissues (ectoderm), and keratin-containing epidermal tissues (ectoderm). {PCR} of genomic {DNA} of human {iPS} cells showed that all clones have integration of all the four retroviruses {(Figure} {S3A).} Southern blot analysis with a {c-Myc} {cDNA} probe revealed that each clone had a unique pattern of retroviral integration sites {(Figure} {S3B).} In addition, the patterns of 16 short tandem repeats were completely matched between human {iPS} clones and parental {HDF} {(Table} S9). These patterns differed from any established {hES} cell lines reported on National Institutes of Health website (http://stemcells.nih.gov/research/nihresearch/scunit/genotyping.htm). In addition, chromosomal G-band analyses showed that human {iPS} cells had a normal karyotype of {46XX} (not shown). Thus, human {iPS} clones were derived from {HDF} and were not a result of cross-contamination. Whether generation of human {iPS} cells depends on minor genetic or epigenetic modification awaits further investigation. In addition to {HDF,} we used primary human fibroblast-like synoviocytes {(HFLS)} from synovial tissue of 69-year-old Caucasian male and {BJ} cells, a cell line established from neonate fibroblasts {(Table} S1 and S2). From {HFLS} (5 × 104 cells per 100 mm dish), we obtained more than 600 hundred granulated colonies and 17 {hES} cell-like colonies {(Table} S1). We picked six colonies, of which only two were expandable as {iPS} cells {(Figure} S4). Dishes seeded with 5 × 105 {HFLS} were covered with granulated cells, and no {hES} cell-like colonies were distinguishable. In contrast, we obtained 7 to 8 and 100 {hES} cell-like colonies from 5 × 104 and 5 × 105 {BJ} cells, respectively, with only a few granulated colonies {(Table} S1). We picked six {hES} cell-like colonies and generated {iPS} cells from five colonies {(Figure} S4). Human {iPS} cells derived from {HFLS} and {BJ} expressed {hES} cell-marker genes at levels similar to or higher than those in {hES} cells {(Figure} S5). They differentiated into all three germ layers through {EBs} {(Figure} S6). {STR} analyses confirmed that {iPS-HFLS} cells and {iPS-BJ} cells were derived from {HFLS} and {BJ} fibroblasts, respectively {(Tables} S10 and S11). In this study, we showed that {iPS} cells can be generated from adult {HDF} and other somatic cells by retroviral transduction of the same four transcription factors with mouse {iPS} cells, namely Oct3/4, Sox2, Klf4, and {c-Myc.} The established human {iPS} cells are similar to {hES} cells in many aspects, including morphology, proliferation, feeder dependence, surface markers, gene expression, promoter activities, telomerase activities, in vitro differentiation, and teratoma formation. The four retroviruses are strongly silenced in human {iPS} cells, indicating that these cells are efficiently reprogrammed and do not depend on continuous expression of the transgenes for self renewal. {hES} cells are different from mouse counterparts in many respects {(Rao,} 2004). {hES} cell colonies are flatter and do not override each other. {hES} cells depend on {bFGF} for self renewal {(Amit} et al., 2000), whereas mouse {ES} cells depend on the {LIF/Stat3} pathway {([Matsuda} et al., 1999] and {[Niwa} et al., 1998]). {BMP} induces differentiation in {hES} cells {(Xu} et al., 2005) but is involved in self renewal of mouse {ES} cells {(Ying} et al., 2003). Despite these differences, our data show that the same four transcription factors induce {iPS} cells in both human and mouse. The four factors, however, could not induce human {iPS} cells when fibroblasts were kept under the culture condition for mouse {ES} cells after retroviral transduction (data not shown). These data suggest that the fundamental transcriptional network governing pluripotency is common in human and mice, but extrinsic factors and signals maintaining pluripotency are unique for each species. Deciphering of the mechanism by which the four factors induce pluripotency in somatic cells remains elusive. The function of Oct3/4 and Sox2 as core transcription factors to determine pluripotency is well documented {([Boyer} et al., 2005], {[Loh} et al., 2006] and {[Wang} et al., 2006]). They synergistically upregulate “stemness” genes, while suppressing differentiation-associated genes in both mouse and human {ES} cells. However, they cannot bind their targets genes in differentiated cells because of other inhibitory mechanisms, including {DNA} methylation and histone modifications. We speculate that {c-Myc} and Klf4 modifies chromatin structure so that Oct3/4 and Sox2 can bind to their targets {(Yamanaka,} 2007). Notably, Klf4 interacts with p300 histone acetyltransferase and regulates gene transcription by modulating histone acetylation {(Evans} et al., 2007). The negative role of {c-Myc} in the self renewal of {hES} cells was recently reported {(Sumi} et al., 2007). They showed that forced expression of {c-Myc} induced differentiation and apoptosis of human {ES} cells. This is great contrast to the positive role of {c-Myc} in mouse {ES} cells {(Cartwright} et al., 2005). During {iPS} cell generation, transgenes derived from retroviruses are silenced when the transduced fibroblasts acquire {ES-like} state. The role of {c-Myc} in establishing {iPS} cells may be as a booster of reprogramming rather than a controller of maintenance of pluripotency. We found that each {iPS} clone contained three to six retroviral integrations for each factor. Thus, each clone had more than 20 retroviral integration sites in total, which may increase the risk of tumorigenesis. In the case of mouse {iPS} cells, 20\% of mice derived from {iPS} cells developed tumors, which were attributable, at least in part, to reactivation of the {c-Myc} retrovirus {(Okita} et al., 2007). This issue must be overcome to use {iPS} cells in human therapies. We have recently found that {iPS} cells can be generated without Myc retroviruses, albeit with lower efficiency {(M.} Nakagawa, M. Koyanagi, and {S.Y.,} unpublished data). Nonretroviral methods to introduce the remaining three factors, such as adenoviruses or cell-permeable recombinant proteins, should be examined in future studies. Alternatively, one might be able to identify small molecules that can induce {iPS} cells, without gene transfer. As is the case with mouse {iPS} cells, only a small portion of human fibroblasts that had been transduced with the four retroviruses acquired {iPS} cell identity. We obtained 10 {iPS} cells colonies from 5 × 104 transduced {HDF.} From a practical point of view, this efficiency is sufficiently high, since multiple {iPS} cell clones can be obtained from a single experiment. From a scientific point of view, however, the low efficiency raises several possibilities. First, the origin of {iPS} cells may be undifferentiated stem or progenitor cells coexisting in fibroblast culture. Another possibility is that retroviral integration into some specific loci may be required for {iPS} cell induction. Finally, minor genetic alterations, which could not be detected by karyotype analyses, or epigenetic alterations are required for {iPS} cell induction. These issues need to be elucidated in future studies. Our study has opened an avenue to generate patient- and disease-specific pluripotent stem cells. Even with the presence of retroviral integration, human {iPS} cells are useful for understanding disease mechanisms, drug screening, and toxicology. For example, hepatocytes derived from {iPS} cells with various genetic and disease backgrounds can be utilized in predicting liver toxicity of drug candidates. Once the safety issue is overcome, human {iPS} cells should also be applicable in regenerative medicine. Human {iPS} cells, however, are not identical to {hES} cells: {DNA} microarray analyses detected differences between the two pluripotent stem cell lines. Further studies are essential to determine whether human {iPS} cells can replace {hES} in medical applications. {HDF} from facial dermis of 36-year-old Caucasian female and {HFLS} from synovial tissue of 69-year-old Caucasian male were purchased from Cell Applications, Inc. When received, the population doubling was less than 16 in {HDF} and 5 in {HFLS.} We used these cells for the induction of {iPS} cells within six and four passages after the receipt. {BJ} fibroblasts from neonatal foreskin and {NTERA-2} clone D1 human embryonic carcinoma cells were obtained from American Type Culture Collection. Human fibroblasts, {NTERA-2,} {PLAT-E,} and {PLAT-A} cells were maintained in Dulbecco's modified eagle medium {(DMEM,} Nacalai Tesque, Japan) containing 10\% fetal bovine serum {(FBS,} Japan Serum) and 0.5\% penicillin and streptomycin {(Invitrogen).} {293FT} cells were maintained in {DMEM} containing 10\% {FBS,} 2 {mM} L-glutamine {(Invitrogen),} 1 × 10−4 M nonessential amino acids {(Invitrogen),} 1 {mM} sodium pyruvate {(Sigma)} and 0.5\% penicillin and streptomycin. {PA6} stroma cells {(RIKEN} Bioresource Center, Japan) were maintained in {α-MEM} containing 10\% {FBS} and 0.5\% penicillin and streptomycin. {iPS} cells were generated and maintained in Primate {ES} medium {(ReproCELL,} Japan) supplemented with 4 ng/ml recombinant human basic fibroblast growth factor {(bFGF,} {WAKO,} Japan). For passaging, human {iPS} cells were washed once with {PBS} and then incubated with {DMEM/F12} containing 1 mg/ml collagenase {IV} {(Invitrogen)} at {37°C.} When colonies at the edge of the dish started dissociating from the bottom, {DMEF/F12/collangenase} was removed and washed with Primate {ES} cell medium. Cells were scraped and collected into 15 ml conical tube. An appropriate volume of the medium was added, and the contents were transferred to a new dish on {SNL} feeder cells. The split ratio was routinely 1:3. For feeder-free culture of {iPS} cells, the plate was coated with 0.3 mg/ml Matrigel (growth-factor reduced, {BD} Biosciences) at {4°C} overnight. The plate was warmed to room temperature before use. Unbound Matrigel was aspirated off and washed out with {DMEM/F12.} {iPS} cells were seeded on Matrigel-coated plate in {MEF-conditioned} or nonconditioned primate {ES} cell medium, both supplemented with 4 ng/ml {bFGF.} The medium was changed daily. For preparation of {MEF-conditioned} medium, {MEFs} derived from embryonic day 13.5 embryo pool of {ICR} mice were plated at 1 × 106 cells per 100 mm dish and incubated overnight. Next day, the cells were washed once with {PBS} and cultured in 10 ml of primate {ES} cell medium. After 24 hr incubation, the supernatant of {MEF} culture was collected, filtered through a 0.22 μm pore-size filter, and stored at {−20°C} until use. The open reading frame of human {OCT3/4} was amplified by {RT-PCR} and cloned into {pCR2.1-TOPO.} An {EcoRI} fragment of {pCR2.1-hOCT3/4} was introduced into the {EcoRI} site of {pMXs} retroviral vector. To discriminate each experiment, we introduced a 20 bp random sequence, which we designated N20 barcode, into the {NotI/SalI} site of Oct3/4 expression vector. We used a unique barcode sequence in each experiment to avoid interexperimental contamination. The open reading frames of human {SOX2,} {KLF4,} and {c-MYC} were also amplified by {RT-PCR} and subcloned into {pENTR-D-TOPO} {(Invitrogen).} All of the genes subcloned into {pENTR-D-TOPO} were transferred to {pMXs} by using the Gateway cloning system {(Invitrogen),} according to the manufacturer's instructions. Mouse Slc7a1 {ORF} was also amplified, subcloned into {pENTR-D-TOPO,} and transferred to {pLenti6/UbC/V5-DEST} {(Invitrogen)} by the Gateway system. The regulatory regions of the human {OCT3/4} gene and the {REX1} gene were amplified by {PCR} and subcloned into {pCRXL-TOPO} {(Invitrogen).} For {phOCT4-Luc} and {phREX1-Luc,} the fragments were removed by {KpnI/BglII} digestion from {pCRXL} vector and subcloned into the {KpnI/BglII} site of {pGV-BM2.} For {pPolII-Luc,} an {AatII} {(blunted)/NheI} fragment of {pQBI-polII} was inserted into the {KpnI} {(blunted)/NheI} site of {pGV-BM2.} All of the fragments were verified by sequencing. Primer sequences are shown in Table S12. {293FT} cells {(Invitrogen)} were plated at 6 × 106 cells per 100 mm dish and incubated overnight. Cells were transfected with 3 μg of {pLenti6/UbC-Slc7a1} along with 9 μg of Virapower packaging mix by Lipofectamine 2000 {(Invitrogen),} according to the manufacturer's instructions. Forty-eight hours after transfection, the supernatant of transfectant was collected and filtered through a 0.45 μm pore-size cellulose acetate filter {(Whatman).} Human fibroblasts were seeded at 8 × 105 cells per 100 mm dish 1 day before transduction. The medium was replaced with virus-containing supernatant supplemented with 4 μg/ml polybrene {(Nacalai} Tesque), and incubated for 24 hr. {PLAT-E} packaging cells were plated at 8 × 106 cells per 100 mm dish and incubated overnight. Next day, the cells were transfected with {pMXs} vectors with Fugene 6 transfection reagent {(Roche).} Twenty-four hours after transfection, the medium was collected as the first virus-containing supernatant and replaced with a new medium, which was collected after twenty-four hours as the second virus-containing supernatant. Human fibroblasts expressing mouse Slc7a1 gene were seeded at 8 × 105 cells per 100 mm dish 1 day before transduction. The virus-containing supernatants were filtered through a 0.45 μm pore-size filter and supplemented with 4 μg/ml polybrene. Equal amounts of supernatants containing each of the four retroviruses were mixed, transferred to the fibroblast dish, and incubated overnight. Twenty-four hours after transduction, the virus-containing medium was replaced with the second supernatant. Six days after transduction, fibroblasts were harvested by trypsinization and replated at 5 × 104 cells per 100 mm dish on an {SNL} feeder layer. Next day, the medium was replaced with Primate {ES} cell medium supplemented with 4 ng/ml {bFGF.} The medium was changed every other day. Thirty days after transduction, colonies were picked up and transferred into 0.2 ml of Primate {ES} cell medium. The colonies were mechanically dissociated to small clamps by pipeting up and down. The cell suspension was transferred on {SNL} feeder in 24-well plates. We defined this stage as passage 1. Total {RNA} was purified with Trizol reagent {(Invitrogen)} and treated with Turbo {DNA-free} kit {(Ambion)} to remove genomic {DNA} contamination. One microgram of total {RNA} was used for reverse transcription reaction with {ReverTraAce-α} {(Toyobo,} Japan) and {dT20} primer, according to the manufacturer's instructions. {PCR} was performed with {ExTaq} {(Takara,} Japan). Quantitative {PCR} was performed with Platinum {SYBR} Green {qPCR} Supermix {UDG} {(Invitrogen)} and analyzed with the 7300 real-time {PCR} system {(Applied} Biosystems). Primer sequences are shown in Table S12. Alkaline phosphatase staining was performed using the Leukocyte Alkaline Phosphatase kit {(Sigma).} For immunocytochemistry, cells were fixed with {PBS} containing 4\% paraformaldehyde for 10 min at room temperature. After washing with {PBS,} the cells were treated 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 min at room temperature. Primary antibodies included {SSEA1} (1:100, Developmental Studies Hybridoma Bank), {SSEA3} (1:10, a kind gift from Dr. Peter W. Andrews), {SSEA4} (1:100, Developmental Studies Hybridoma Bank), {TRA-2-49/6E} (1:20, Developmental Studies Hybridoma Bank), {TRA-1-60} (1:50, a kind gift from Dr. Peter W. Andrews), {TRA-1-81} (1:50, a kind gift from Dr. Peter W. Andrews), Nanog (1:20, {AF1997,} {R\&D} Systems), {βIII-tubulin} (1:100, {CB412,} Chemicon), glial fibrillary acidic protein (1:500, Z0334, {DAKO),} α-smooth muscle actin (pre-diluted, N1584, {DAKO),} desmin (1:100, {RB-9014,} Lab Vision), vimentin (1:100, {SC-6260,} Santa Cruz), α-fetoprotein (1:100, {MAB1368,} {R\&D} Systems), tyrosine hydroxylase (1:100, {AB152,} Chemicon). Secondary antibodies used were cyanine 3 {(Cy3)} -conjugated goat anti-rat {IgM} (1:500, Jackson Immunoresearch), Alexa546-conjugated goat anti-mouse {IgM} (1:500, Invitrogen), Alexa488-conjugated goat anti-rabbit {IgG} (1:500, Invitrogen), Alexa488-conjugated donkey anti-goat {IgG} (1:500, Invitrogen), Cy3-conjugated goat anti-mouse {IgG} (1:500, Chemicon), and Alexa488-conjugated goat anti-mouse {IgG} (1:500, Invitrogen). Nucleuses were stained with 1 μg/ml Hoechst 33342 {(Invitrogen).} For {EB} formation, human {iPS} cells were harvested by treating with collagenase {IV.} The clumps of the cells were transferred to poly (2-hydroxyrthyl methacrylate)-coated dish in {DMEM/F12} containing 20\% knockout serum replacement {(KSR,} Invitrogen), 2 {mM} L-glutamine, 1 × 10−4 M nonessential amino acids, 1 × 10−4 M 2-mercaptoethanol {(Invitrogen),} and 0.5\% penicillin and streptomycin. The medium was changed every other day. After 8 days as a floating culture, {EBs} were transferred to gelatin-coated plate and cultured in the same medium for another 8 days. Coculture with {PA6} was used for differentiation into dopaminergic neurons. {PA6} cells were plated on gelatin-coated 6-well plates and incubated for 4 days to reach confluence. Small clumps of {iPS} cells were plated on {PA6-feeder} layer in Glasgow minimum essential medium {(Invitrogen)} containing 10\% {KSR} {(Invitrogen),} 1 × 10−4 M nonessential amino acids, 1 × 10−4 M 2-mercaptoethanol {(Invitrogen),} and 0.5\% penicillin and streptomycin. For cardiomyocyte differentiation, {iPS} cells were maintained on Matrigel-coated plate in {MEF-CM} supplemented with 4 ng/ml {bFGF} for 6 days. The medium was then replaced with {RPMI1640} {(Invitrogen)} plus B27 supplement {(Invitrogen)} medium {(RPMI/B27),} supplemented with 100 ng/ml human recombinant activin A {(R} \& D Systems) for 24 hr, followed by 10 ng/ml human recombinant bone morphologenic protein 4 {(BMP4,} {R\&D} Systems) for 4 days. After cytokine stimulation, the cells were maintained in {RPMI/B27} without any cytokines. The medium was changed every other day. Genomic {DNA} (1 μg) was treated with {CpGenome} {DNA} modification kit {(Chemicon),} according to the manufacturer's recommendations. Treated {DNA} was purified with {QIAquick} column {(QIAGEN).} The promoter regions of the human Oct3/4, Nanog, and Rex1 genes were amplified by {PCR.} The {PCR} products were subcloned into {pCR2.1-TOPO.} Ten clones of each sample were verified by sequencing with the M13 universal primer. Primer sequences used for {PCR} amplification were provided in Table S12. Each reporter plasmid (1 μg) containing the firefly luciferase gene was introduced into human {iPS} cells or {HDF} with 50 ng of {pRL-TK} {(Promega).} Forty-eight hours after transfection, the cells were lysed with {1X} passive lysis buffer {(Promega)} and incubated for 15 min at room temperature. Luciferase activities were measured with a {Dual-Luciferase} reporter assay system {(Promega)} and Centro {LB} 960 detection system {(BERTHOLD),} according to the manufacturer's protocol. The cells were harvested by collagenase {IV} treatment, collected into tubes, and centrifuged, and the pellets were suspended in {DMEM/F12.} One quarter of the cells from a confluent 100 mm dish was injected subcutaneously to dorsal flank of a {SCID} mouse {(CREA,} Japan). Nine weeks after injection, tumors were dissected, weighted, and fixed with {PBS} containing 4\% paraformaldehyde. Paraffin-embedded tissue was sliced and stained with hematoxylin and eosin. The cells at semiconfluent state were lysed with {RIPA} buffer (50 {mM} {Tris-HCl,} {pH} 8.0, 150 {mM} {NaCl,} 1\% Nonidet P-40 {(NP-40),} 1\% sodium deoxycholate, and 0.1\% {SDS),} supplemented with protease inhibitor cocktail {(Roche).} The cell lysate of {MEL-1} {hES} cell line was purchased from Abcam. Cell lysates (20 μg) were separated by electrophoresis on 8\% or 12\% {SDS-polyacrylamide} gel and transferred to a polyvinylidine difluoride membrane {(Millipore).} The blot was blocked with {TBST} (20 {mM} {Tris-HCl,} {pH} 7.6, 136 {mM} {NaCl,} and 0.1\% Tween-20) containing 1\% skim milk and then incubated with primary antibody solution at {4°C} overnight. After washing with {TBST,} the membrane was incubated with horseradish peroxidase {(HRP)-conjugated} secondary antibody for 1 hr at room temperature. Signals were detected with Immobilon Western chemiluminescent {HRP} substrate {(Millipore)} and {LAS3000} imaging system {(FUJIFILM,} Japan). Antibodies used for western blotting were {anti-Oct3/4} (1:600, {SC-5279,} Santa Cruz), {anti-Sox2} (1:2000, {AB5603,} Chemicon), {anti-Nanog} (1:200, {R\&D} Systems), {anti-Klf4} (1:200, {SC-20691,} Santa Cruz), {anti-c-Myc} (1:200, {SC-764,} Santa Cruz), {anti-E-cadherin} (1:1000, 610182, {BD} Biosciences), {anti-Dppa4} (1:500, ab31648, Abcam), {anti-FoxD3} (1:200, {AB5687,} Chemicon), anti-telomerase (1:1000, ab23699, Abcam), {anti-Sall4} (1:400, ab29112, Abcam), {anti-LIN-28} (1:500, {AF3757,} {R\&D} systems), anti-β-actin (1:5000, A5441, Sigma), anti-mouse {IgG-HRP} (1:3000, \#7076, Cell Signaling), anti-rabbit {IgG-HRP} (1:2000, \#7074, Cell Signaling), and anti-goat {IgG-HRP} (1:3000, {SC-2056,} Santa Cruz). Genomic {DNA} (5 μg) was digested with {BglII,} {EcoRI,} and {NcoI} overnight. Digested {DNA} fragments were separated on 0.8\% agarose gel and transferred to a nylon membrane {(Amersham).} The membrane was incubated with digoxigenin {(DIG)-labeled} {DNA} probe in {DIG} Easy Hyb buffer {(Roche)} at {42°C} overnight with constant agitation. After washing, alkaline phosphatase-conjugated {anti-DIG} antibody (1:10,000, Roche) was added to a membrane. Signals were raised by {CDP-star} {(Roche)} and detected by {LAS3000} imaging system. The genomic {DNA} was used for {PCR} with Powerplex 16 system {(Promega)} and analyzed by {ABI} {PRISM} 3100 Genetic analyzer and Gene Mapper v3.5 {(Applied} Biosystems). Chromosomal G-band analyses were performed at the Nihon Gene Research Laboratories, Japan. Telomerase activity was detected with a {TRAPEZE} telomerase detection kit {(Chemicon),} according to the manufacturer's instructions. The samples were separated by {TBE-based} 10\% acrylamide nondenaturing gel electrophoresis. The gel was stained with {SYBR} Gold (1:10,000, Invitrogen). Approximately 1 × 107 cells were crosslinked with 1\% formaldehyde for 5 min at room temperature and quenched by addition of glycine. The cell lysate was sonicated to share {chromatin-DNA} complex. Immunoprecipitation was performed with Dynabeads Protein G {(Invitrogen)} -linked anti-trimethyl Lys 4 histone H3 (07-473, Upstate), anti-trimethyl Lys 27 histone H3 (07-449, Upstate) or normal rabbit {IgG} antibody. Eluates were used for quantitative {PCR} as templates. Total {RNA} from {HDF} and {hiPS} cells (clone {201B)} was labeled with Cy3. Samples were hybridized with Whole Human Genome Microarray 4 × {44K} {(G4112F,} Agilent). Each sample was hybridized once with the one color protocol. Arrays were scanned with a {G2565BA} Microarray Scanner System {(Agilent).} Data analyzed by using {GeneSpring} {GX7.3.1} software {(Agilent).} Two normalization procedures were applied; first, signal intensities less than 0.01 were set to 0.01. Then each chip was normalized to the 50th percentile of the measurements taken from that chip. The microarray data of {hES} H9 cells {(Tesar} et al., 2007) were retrieved from {GEO} {DataSets} {(GSM194390,} {http://www.ncbi.nlm.nih.gov/sites/entrez?db=gds\&cmd=search\&term=GSE7902).} Genes with “present” flag value in all three samples were used for analyses (32,266 genes). We have deposited the microarray data of {HDF} and {hiPS} cells to {GEO} {DataSets} with the accession number {GSE9561.} We thank Dr. Deepak Srivastava for critical reading of the manuscript; Gary Howard and Stephen Ordway for editorial review; Drs. Masato Nakagawa, Keisuke Okita, and Takashi Aoi and other members of our laboratory for scientific comment and valuable discussion; Dr. Peter. W. Andrews for {SSEA-3,} {TRA-1-60,} and {TRA-1-81} antibodies; and Dr. Toshio Kitamura for retroviral system. We are also grateful to Aki Okada for technical support and Rie Kato and Ryoko Iyama for administrative supports. This study was supported in part by a grant from the Program for Promotion of Fundamental Studies in Health Sciences of {NIBIO,} a grant from the Leading Project of {MEXT,} a grant from Uehara Memorial Foundation, and {Grants-in-Aid} for Scientific Research of {JSPS} and {MEXT.}   },
	number = {5},
	journal = {Cell},
	author = {Kazutoshi Takahashi and Koji Tanabe and Mari Ohnuki and Megumi Narita and Tomoko Ichisaka and Kiichiro Tomoda and Shinya Yamanaka},
	month = nov,
	year = {2007},
	pages = {861--872}
},

@article{nakagawa_generation_2008,
	title = {Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts},
	volume = {26},
	issn = {1087-0156},
	url = {http://dx.doi.org/10.1038/nbt1374},
	doi = {10.1038/nbt1374},
	number = {1},
	journal = {Nat Biotech},
	author = {Masato Nakagawa and Michiyo Koyanagi and Koji Tanabe and Kazutoshi Takahashi and Tomoko Ichisaka and Takashi Aoi and Keisuke Okita and Yuji Mochiduki and Nanako Takizawa and Shinya Yamanaka},
	year = {2008},
	pages = {101--106}
},

@article{okita_generation_2007,
	title = {Generation of germline-competent induced pluripotent stem cells},
	volume = {448},
	issn = {0028-0836},
	url = {http://dx.doi.org/10.1038/nature05934},
	doi = {10.1038/nature05934},
	number = {7151},
	journal = {Nature},
	author = {Keisuke Okita and Tomoko Ichisaka and Shinya Yamanaka},
	month = jul,
	year = {2007},
	pages = {313--317}
},

@article{huangfu_induction_2008-1,
	title = {Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2},
	volume = {26},
	issn = {1087-0156},
	url = {http://dx.doi.org/10.1038/nbt.1502},
	doi = {10.1038/nbt.1502},
	number = {11},
	journal = {Nat Biotech},
	author = {Danwei Huangfu and Kenji Osafune and Rene Maehr and Wenjun Guo and Astrid Eijkelenboom and Shuibing Chen and Whitney Muhlestein and Douglas A Melton},
	month = nov,
	year = {2008},
	pages = {1269--1275}
},

@article{wernig_in_2007,
	title = {In vitro reprogramming of fibroblasts into a pluripotent {ES-cell-like} state},
	volume = {448},
	issn = {0028-0836},
	url = {http://dx.doi.org/10.1038/nature05944},
	doi = {10.1038/nature05944},
	number = {7151},
	journal = {Nature},
	author = {Marius Wernig and Alexander Meissner and Ruth Foreman and Tobias Brambrink and Manching Ku and Konrad Hochedlinger and Bradley E. Bernstein and Rudolf Jaenisch},
	month = jul,
	year = {2007},
	pages = {318--324}
},

@article{yu_induced_2007,
	title = {Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells},
	volume = {318},
	url = {http://www.sciencemag.org/cgi/content/abstract/318/5858/1917},
	doi = {10.1126/science.1151526},
	abstract = {Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors {(OCT4,} {SOX2,} {NANOG,} and {LIN28)} are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem {(ES)} cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human {ES} cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.
},
	number = {5858},
	journal = {Science},
	author = {Junying Yu and Maxim A. Vodyanik and Kim {Smuga-Otto} and Jessica {Antosiewicz-Bourget} and Jennifer L. Frane and Shulan Tian and Jeff Nie and Gudrun A. Jonsdottir and Victor Ruotti and Ron Stewart and Igor I. Slukvin and James A. Thomson},
	month = dec,
	year = {2007},
	pages = {1917--1920}
},

@article{judson_embryonic_2009,
	title = {Embryonic stem cell-specific {microRNAs} promote induced pluripotency},
	volume = {advanced online publication},
	issn = {1546-1696},
	url = {http://dx.doi.org/10.1038/nbt.1535},
	doi = {10.1038/nbt.1535},
	journal = {Nat Biotech},
	author = {Robert L Judson and Joshua E Babiarz and Monica Venere and Robert Blelloch},
	month = apr,
	year = {2009}
}