3502 Research Article

IntroductionInduced pluripotent stem cells (iPSCs) have been generated fromthe fibroblasts of several species, including mouse, rat, rhesusmonkey and human, following ectopic expression of thetranscription factors Oct4 and Sox2, combined with either Klf4 andc-Myc or Lin28 and Nanog (Hockemeyer et al., 2008; Liao et al.,2008; Liu et al., 2008; Maherali et al., 2007; Okita et al., 2007;Park et al., 2008b; Takahashi and Yamanaka, 2006; Takahashi etal., 2007; Yu et al., 2007; Wernig et al., 2007; Hochedlinger andPlath, 2009; Li et al., 2008).

iPSCs acquire most of the features of embryonic stem cells (ESCs)including immortal growth and pluripotency, as measured by theirability to differentiate into multiple cell types in teratomas and theircontribution to germline-competent chimeras in mice (Maherali etal., 2007). Patient-derived iPSCs might be an ideal source forstudying complex diseases in vitro and potentially for treatingdisorders in the clinic. Indeed, iPSCs have been shown to alleviatethe symptoms in mouse models of Parkinson’s disease and sicklecell anemia (Hanna et al., 2007; Wernig et al., 2008a). Moreover,iPSCs have already been derived from human individuals sufferingfrom diseases such as Parkinson’s disease, diabetes or amyotrophiclateral sclerosis (ALS), thus enabling disease modeling and drugscreening approaches (Park et al., 2008a; Dimos et al., 2008).

Since the initial report of reprogramming fibroblasts into iPSCs,several additional cell types, including stomach cells (Aoi et al.,

2008), liver cells (Aoi et al., 2008; Stadtfeld et al., 2008b), pancreaticβ cells (Stadtfeld et al., 2008c), lymphocytes (Hanna et al., 2008)and neural progenitor cells (NPCs) (Eminli et al., 2008; Kim et al.,2008; Silva et al., 2008), have been successfully converted into iPSCsin mouse. By contrast, the derivation of human iPSCs is still limitedto fibroblasts and keratinocytes (Park et al., 2008b; Takahashi et al.,2007; Yu et al., 2007; Aasen et al., 2008; Maherali et al., 2008; Lowryet al., 2008). Interestingly, NPCs do not require ectopic Sox2expression for reprogramming (Eminli et al., 2008; Kim et al., 2008;Silva et al., 2008) due to their high endogenous Sox2 levels (Elliset al., 2004). Considering future clinical applications of the iPSCtechnology, however, NPCs are an undesirable cell type due to theirdifficult accessibility. We therefore evaluated whether melanocytescould be reprogrammed into iPSCs; melanocytes are, like NPCs, ofneuroectodermal origin and hence might require fewer factors forconversion into iPSCs, thus potentially facilitating replacement offactors with chemicals or non-integrating approaches (Stadtfeld etal., 2008; Okita et al., 2008). Moreover, melanocytes are easilyaccessible from the skin and can be marked genetically using Cre-lox technology in mice to trace their origin.

In addition to using nuclear reprogramming as a means ofderiving patient-specific iPSCs, induced pluripotency could be apowerful tool for distinguishing between epigenetic and geneticalterations occurring during development and tumorigenesis. Wehave previously shown that nuclear transfer can reprogram a

Induced pluripotent stem cells (iPSCs) have been derived at lowfrequencies from different cell types through ectopic expressionof the transcription factors Oct4 and Sox2, combined with eitherKlf4 and c-Myc or Lin28 and Nanog. In order to generate iPSCsmore effectively, it will be crucial to identify somatic cells thatare easily accessible and possibly require fewer factors forconversion into iPSCs. Here, we show that both human andmouse melanocytes give rise to iPSCs at higher efficiencies thanfibroblasts. Moreover, we demonstrate that a mouse malignantmelanoma cell line, which has previously been reprogrammedinto embryonic stem cells by nuclear transfer, remains equallyamenable to reprogramming into iPSCs by these transcriptionfactors. In contrast to skin fibroblasts, melanocytes andmelanoma cells did not require ectopic Sox2 expression for

conversion into iPSCs. iPSC lines from melanocytic cellsexpressed pluripotency markers, formed teratomas andcontributed to viable chimeric mice with germ line transmission.Our results identify skin melanocytes as an alternative sourcefor deriving patient-specific iPSCs at increased efficiency andwith fewer genetic elements. In addition, our results suggest thatcancer cells remain susceptible to transcription factor-mediatedreprogramming, which should facilitate the study of epigeneticchanges in human cancer.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/122/19/3502/DC1

Key words: Reprogramming, iPS cell, Melanocyte, Sox2

Summary

Sox2 is dispensable for the reprogramming ofmelanocytes and melanoma cells into inducedpluripotent stem cellsJochen Utikal1,2,3, Nimet Maherali1,2,4, Warakorn Kulalert1,2 and Konrad Hochedlinger1,2,*1Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Massachusetts General Hospital Center for RegenerativeMedicine, 185 Cambridge Street, Boston, MA 02114, USA2Massachusetts General Hospital Cancer Center, 185 Cambridge Street, Boston, MA 02114, USA3Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl-University of Heidelberg,Theodor-Kutzer-Ufer 1-3, 68135 Mannheim, Germany4Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA*Author for correspondence (khochedlinger@helix.mgh.harvard.edu)

Accepted 16 July 2009Journal of Cell Science 122, 3502-3510 Published by The Company of Biologists 2009doi:10.1242/jcs.054783

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malignant melanoma genome into a pluripotent state, demonstratingthat the epigenetic state of this particular tumor cell was reversibleupon exposure of its nucleus to oocyte cytoplasm (Hochedlinger etal., 2004). In order to assess whether transcription-factor-mediatedreprogramming is equally suitable for reprogramming tumor cellsinto a pluripotent state, in this study we attempted to convert thepreviously used melanoma cell line (Chin et al., 1999) into iPSCs.

ResultsReprogramming of genetically marked primary mousemelanocytes into iPSCsTo test whether cells of the melanocytic lineage can bereprogrammed into iPSCs, we isolated primary melanocytes fromthe epidermis of neonatal mice using previously published protocols(Yang et al., 2001). In order to rule out the presence of contaminatingfibroblasts in our cultures, we used animals that expressed Crerecombinase from promoter elements of the Wnt1 gene (Danielianet al., 1998), as well as a conditional (enhanced yellow fluorescenceprotein) EYFP reporter gene driven off the ROSA26 promoter

(Srinivas et al., 2001), leading to specific and irreversible labelingof neural-crest-derived cells, including melanocytes. After 5 daysin culture in melanocyte growth medium, EYFP-positive cells weresorted by fluorescence activated cell sorting (FACS) to separatethem from the remaining keratinocytes, and were cultured inmelanocyte growth medium. Resultant melanocyte cultures werehomogeneously positive for EYFP and contained melanosomes,which are melanin-containing organelles characteristic formelanocytes.

Primary melanocytes were infected on days 1, 2 and 3 withdoxycycline-inducible lentiviral vectors expressing Oct4, Sox2, c-Myc and Klf4 (Stadtfeld et al., 2008a) (Fig. 1A), giving rise toESC-like colonies at day 9. These colonies continued to grow afterdoxycycline withdrawal on day 19, indicating that colony growthwas independent of transgene expression. At day 24, ESC-likecolonies were picked and expanded into stable iPSC lines (Fig. 1B).Interestingly, the efficiency of reprogramming melanocytes wasmore than three times higher than that of fibroblasts (0.19% versus0.056%, see also Table 1).

To rule out the possibility that the Wnt1-Cretransgene became spuriously activated duringthe reprogramming process, we reprogrammedtail-tip fibroblasts from Wnt1-Cre/ROSA26-EYFP mice. No EYFP colonies were observed(0/45), confirming specificity of the lineagetracing system (Fig. 1C).

iPSCs derived from melanocytes expressedthe pluripotency markers Nanog and Oct4 (Fig.1D; data not shown), lost expression of themelanocyte markers tyrosinase and dopachrometautomerase, and attenuated melanin production(Fig. 1E,F; and data not shown). Demethylationof the Oct4 promoter region in iPSCs, which is

Fig. 1. Generation of iPSCs from genetically markedprimary mouse melanocytes in the presence or absenceof viral Sox2. (A) Experimental scheme for generatingiPSCs from primary melanocytes. (B) Derivation ofiPSCs from primary melanocytes of the lineage tracingmouse model Wnt1-Cre/ROSA26R-EYFP; shown areprimary melanocytes (left: brightfield and fluorescentimage) and one picked iPSC colony grown on feedercells (right: brightfield and fluorescent image) afterdoxycycline withdrawal. Note that high melanincontent of melanocytes results in quenching of theEYFP signal (lower left image). (C) EYFP-positiveiPSC colonies develop only from EYFP-positivemelanocytes. The reprogramming of EYFP-negativetail fibroblasts from Wnt1-Cre/ROSA26R-EYFP micedoes not give rise to EYFP-positive iPSC colonies.(D) Brightfield and fluorescent image of primarymelanocyte-derived iPSCs stained for the pluripotencymarker Nanog. (E) Primary melanocytes (PM) containthe pigment melanin whereas iPSCs derived frommelanocytes lost melanin. (F) Quantitative RT-PCRanalysis of marker gene expression in primarymelanocytes (PM), two iPSC lines derived with fourfactors (4F) or three factors (3F) from primarymelanocytes, and V6.5 control ESCs. Values representmeans ± s.e.m. (G) Bisulfite sequencing of the Oct4and Nanog promoter regions in primary melanocytes,iPSCs produced with three or four factors fromprimary melanocytes and ESCs. White circlesrepresent unmethylated CpGs; black circles denotemethylated CpGs.

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heavily methylated in primary melanocytes, demonstrated faithfulepigenetic reprogramming of iPSCs (Fig. 1G). In contrast to othermouse cell types that have been reprogrammed previously, such asfibroblasts, NPCs and hepatic cells (Maherali et al., 2007; Stadtfeldet al., 2008c; Stadtfeld et al., 2008b; Eminli et al., 2008), primarymouse melanocytes were devoid of methylation at the Nanogpromoter. Melanocyte-derived iPSCs differentiated in vitro intoembryoid bodies (data not shown) and into mesodermal, ectodermaland endodermal derivatives in the context of teratomas (Fig. 2A).This shows that melanocytes remain amenable to reprogramminginto pluripotent cells.

Sox2 is dispensable for the reprogramming of murinemelanocytes into iPSCsIt has been previously shown that cells with endogenous Sox2 levelscan be reprogrammed in the absence of ectopic Sox2 expression(Eminli et al., 2008; Kim et al., 2008; Silva et al., 2008). Giventhat melanocytes, like NPCs, are of neuroectodermal origin, weassessed Sox2 expression and indeed detected Sox2 transcripts,albeit at lower levels than in NPCs (Fig. 1F; supplementary materialFig. S1A,B). Interestingly, the expression of Sox2 was higher inlow passage (passage 1) melanocyte cultures compared with highpassage (passage 3+) cells. We therefore reasoned that Oct4, Klf4and c-Myc alone might be sufficient to induce pluripotency inprimary melanocytes. Indeed, Wnt1-EYFP-positive melanocytesthat were infected with lentiviruses expressing Oct4, c-Myc andKlf4 gave rise to ESC-like cells that grew into stable iPSC linesupon discontinuation of doxycycline (data not shown), indicatingthat ectopic Sox2 expression is dispensable for reprogrammingmelanocytes into iPSCs (supplementary material Fig. 1C). Inagreement with the progressive decline in Sox2 expression duringserial passaging, we were able to obtain iPSCs only at low passagesfrom Wnt1-EYFP-positive melanocytes using three factors. Athigher passages, the cells had to be supplemented by viral Sox2expression to generate iPSCs.

The efficiency of reprogramming melanocytes in the absence ofectopic Sox2 expression was lower than that with all four factors(0.19% with four factors versus 0.03% with three factors) (Table1). Three-factor iPSCs expressed pluripotency markers, asdemonstrated by quantitative PCR (qPCR) (Fig. 1F). They showeddemethylation of the Oct4 and Nanog promoter regions (Fig. 1G),and could differentiate both into embryoid bodies in vitro (data notshown) and into mesodermal, ectodermal and endodermalderivatives in teratomas (Fig. 2A). Further, melanocyte-derivediPSCs gave rise to chimeric mice competent of germlinetransmission (Fig. 2B-D).

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Because fibroblasts can be reprogrammed in the absence of viralc-Myc expression, we infected primary melanocytes with induciblelentiviruses expressing Klf4, Oct4 and Sox2 alone to assess theirpotential to produce iPSCs. We observed ESC-like colonies after 3weeks that could be expanded into stable, doxycycline-independentiPSC lines. The efficiency of iPSC formation was 0.02%, which isapproximately tenfold lower than with reprogramming melanocytesin the presence of c-Myc (see Table 1) and similar to observations

Table 1. Efficiency of reprogramming mouse and human melanocytes into iPS cells

Cell type Reprogramming factors Efficiency (%) ESC marker expression Teratoma formation Postnatal chimeras

Mouse tail-tip fibroblasts KOSM 0.056 Yes Yes n.d.

Primary mouse melanocytes KOSM 0.19 Yes Yes YesKOS 0.02 Yes Yes n.d.KOM 0.03 Yes Yes Yes

Primary human melanocytes KOSM 0.05 Yes Yes –KOS 0.01 Yes n.d. –KOM 0.01 Yes Yes –

R545 melanoma cells KOM n.d. Yes Yes Yes

K, Klf4; O, Oct4; S, Sox2; M, c-Myc; n.d., not determined.

Fig. 2. Differentiation potential of iPSCs obtained from primary mousemelanocytes with three (3F) or four (4F) reprogramming factors.(A) Hematoxylin and eosin stainings of teratomas derived from iPSCs showdifferentiation into cell types from all three germ layers [endoderm: epithelialstructures (left images); ectoderm: keratinized epithelium (center images) andmesoderm: muscle fibers (right images)]. (B) Viable newborn chimeras fromiPSCs derived from primary melanocytes. Chimeric pup (right) and non-chimeric littermate (left) are shown under regular light (left image) and UVlight (right image). (C) Adult chimera derived from three-factor female iPSCsshows obvious coat color chimerism. (D) Germline contribution of three-factormelanocyte iPSCs. Images of pups derived from matings between BDF1 wild-type males and female iPSC chimeras. The presence of agouti coat colorindicates germline transmission (arrow).

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in fibroblasts (Nakagawa et al., 2008; Wernig et al., 2008b). Three-factor iPSCs (without c-Myc) also differentiated in vitro intoembryoid bodies and into mesodermal, ectodermal and endodermalderivatives in the context of teratomas (data not shown). BecauseSox2 and c-Myc were individually not required for reprogrammingmelanocytes into iPSCs, we attempted to derive iPSCs with Oct4and Klf4 expressing doxycycline-inducible viruses alone. Althoughwe observed morphological changes in cells after doxycyclineaddition, we were unable to derive stable doxycycline-independentiPSC lines, despite repeated attempts.

Reprogramming of human melanocytes into iPSCsWe next tested whether human melanocytes are equally amenableto reprogramming into iPSCs as mouse melanocytes. To this end,we infected primary human melanocytes (obtained from Promocell,Heidelberg, Germany) with lentiviruses containing a reversetetracycline transactivator (rtTA) and the four reprogrammingfactors (Oct4, Sox2, c-Myc and Klf4) as previously described (Fig.

3A) (Maherali et al., 2008). After infection of melanocytes withviral supernatant for 3 consecutive days, cells were plated ontomouse embryonic fibroblasts (MEFs) in melanocyte growthconditions and induced with doxycycline. Medium changes with50% melanocyte and 50% human ESC medium were performedevery other day until human iPSCs developed. Human non-ESC-like colonies emerged at day 5 and contained only viral integrationsfor Oct4 and c-Myc (data not shown), as previously observed duringthe reprogramming of human fibroblasts (Takahashi et al., 2007).We were unable to expand these colonies after doxycyclinewithdrawal. After 10 days, however, colonies resembling humanESCs were observed. These colonies showed well-defined phase-bright borders with surrounding sheets of feeder cells and highnucleus-to-cytoplasm ratios with prominent nucleoli (Fig. 3C). Thecolonies all expressed the pluripotency marker Oct4 (Fig. 3D) andcould be expanded in the absence of doxycycline.

The reprogramming efficiency was calculated as 0.05% (Table1). All human iPSC lines lacked expression of the melanin-

Fig. 3. Generation and characterization ofhuman iPSCs derived from primary humanmelanocytes. (A) Experimental scheme for thegeneration of human iPSCs. Primary humanmelanocytes were infected with separatelentiviral vectors expressing a constitutivelyactive rtTA and doxycycline-inducible copiesof Oct4, Klf4, c-Myc and, optionally, Sox2.After infection, cells were seeded on mouseembryonic fibroblasts (MEFs), anddoxycycline was applied for 9 days. HumaniPSC clones were picked on the basis of humanESC-like morphology and doxycycline-independent growth. (B) Quantitative PCRexpression analysis of Sox2 in primary humanmelanocytes (PM), human iPSCs derived fromprimary human melanocytes produced with(4F) or without (3F) viral Sox2, fibroblastsdifferentiated from human iPSCs, and resultantsecondary human iPSCs (sec. hiPSCs).(C) Morphology of human iPSCs derived fromprimary human melanocytes in the presence(4F) or absence (3F) of viral Sox2.(D) Doxycycline-independent melanocyte-derived human iPSCs express Oct4 protein.(E) Bisulfite sequencing of the Nanog and Oct4promoter regions in primary melanocytes,human iPSCs derived from primarymelanocytes by three (3F) or four (4F) factors,and WA09 human ESCs. Promoter regionscontaining differentially methylated CpGs areshown. White circles represent unmethylatedCpGs; black circles denote methylated CpGs.(F) Hematoxylin and eosin staining ofteratomas generated from primary melanocyte-derived human iPSCs. Differentiated structuresfrom all three germ layers were present[endoderm: epithelial structures (left images);mesoderm: cartilage and muscle fibers (centerimages) and ectoderm: neural tissue andkeratinized epithelium (right images)].

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synthesizing enzymes tyrosinase and dopachrome tautomerase, incontrast to melanocytes that expressed these enzymes strongly (Fig.4B). In addition, all iPSC lines expressed pluripotency genes fromthe endogenous loci and lacked expression of the viral transgenes(Fig. 4B and supplementary material Fig. S2B). In accordance withthese observations, the Nanog and Oct4 promoter regions in iPSCswere demethylated to a similar extent as in human ESCs, whereasmelanocytes showed highly methylated promoter regions (Fig. 3E),demonstrating epigenetic reprogramming of key pluripotency genesin human iPSCs.

To test whether melanocyte-derived human iPSCs could formteratomas in vivo, around 5�104 human iPSCs were injected eitherunder one kidney or testis capsule of immunodeficient SCID mice.Teratomas developed after 7-10 weeks and contained structuresrepresentative of the three embryonic germ layers, includingcartilage, skeletal muscle, keratinized epithelium and mucousepithelium (Fig. 3F). This shows that human iPSCs derived fromhuman primary melanocytes resemble human ESCs and fulfil thecriteria for pluripotency (Maherali and Hochedlinger, 2008).

Ectopic Sox2 expression is dispensable for the reprogrammingof human melanocytesBecause mouse melanocytes were amenable to reprogramming intoiPSCs in the absence of exogenous Sox2 expression and becausehuman melanocyte cultures were also found to express Sox2 (Fig.3B), we reasoned that Oct4, Klf4 and c-Myc alone might besufficient to reprogram human melanocytes into iPSCs. Indeed,human melanocytes infected with viruses expressing Oct4, c-Mycand Klf4 alone gave rise to human ESC-like cells and grew intostable human iPSC lines upon discontinuation of doxycycline (Fig.3C), indicating that human melanocytes can be reprogrammed inthe absence of viral Sox2 expression. The efficiency ofreprogramming human melanocytes in the absence of Sox2 viruswas lower than that using all four factors (0.05% with four factorsversus 0.01% with three factors; see Table 1). iPSCs derived in theabsence of Sox2 virus expressed pluripotency markers (Fig. 3D andsupplementary material Fig. S2), showed demethylation of the Oct4and Nanog promoter regions (Fig. 3E), differentiated into embryoidbodies (data not shown) in vitro and into mesodermal, ectodermaland endodermal structures in teratomas (Fig. 3F).

To test whether human melanocytes can be reprogrammed intoiPSCs in the absence of c-Myc, we infected melanocytes with viralvectors expressing Oct4, Sox2 and Klf4 and detected a delayed onsetof colony formation after 4 weeks and obtained doxycycline-independent, stable iPSC lines only after 5 weeks. The efficiencyof iPSC derivation without c-Myc was ~fivefold lower than withc-Myc (0.01% versus 0.05%; Table 1). iPSCs that were derived inthe absence of ectopic c-Myc expression also activated pluripotencymarkers and differentiated in vitro into embryoid bodies (data notshown).

We next asked whether Oct4 and Klf4 alone might be sufficientfor reprogramming human melanocytes into iPSCs. Consistent withobservations in mouse, infection of melanocytes with viral vectorsexpressing Oct4 and Klf4 induced morphological changes in cultures,but no stable iPSC lines were recovered after withdrawal ofdoxycycline. This suggested that Oct4 and Klf4 together areinsufficient to induce pluripotency in human melanocytes. Treatmentof human fibroblasts with the histone deacetylase inhibitor valproicacid has recently been shown to replace ectopic Klf4 and c-Mycexpression during the reprogramming of human fibroblasts intoiPSCs (Huangfu et al., 2008; Maherali and Hochedlinger, 2008).

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However, when applied to human melanocytes infected with Oct4and Sox2 alone, we did not observe iPSC formation (data not shown).

Secondary cells derived from primary human iPSCsPrimary iPSCs produced with doxycycline-inducible lentivirusescan be differentiated in vitro into fibroblast-like cells, generatingso-called ‘secondary cells’, which express the four factors morehomogeneously and thus reprogram into secondary iPSCs at higherefficiency upon re-exposure to doxycycline (Hockemeyer et al.,2008; Maherali et al., 2008; Wernig et al., 2008c). To test whetherhuman melanocyte iPSCs can be converted into secondary cells,we differentiated several melanocyte-derived human iPSC lines invitro into fibroblast-like cells (Fig. 4A). Promoter methylationanalysis of the Oct4 and Nanog genes revealed a methylation patternakin to fibroblasts (Fig. 4A), confirming that the cells had acquireda somatic epigenetic pattern. Further, quantitative RT-PCR analysisconfirmed the lack of pluripotency gene expression in this cellpopulation (Fig. 4B).

For the generation of secondary human iPSCs, fibroblasts wereplated on mouse embryonal feeder layers and cultured underhuman ESC conditions in the presence of doxycycline. Fibroblast-derived secondary human iPSCs indeed gave rise to iPSCs in thepresence of doxycycline, whereas no colonies developed in theabsence of doxycycline (data not shown). The frequency ofconversion into human iPSCs was 0.24% and thus nearly five timeshigher than reprogramming using direct viral infection. However,iPSCs took longer to develop than melanocytes (21 versus 10 days).Moreover, we were only able to establish secondary iPSCs fromprimary iPSC clones produced with four factors, not from thoseproduced with three factors lacking Sox2, consistent with the notionthat fibroblasts require ectopic Sox2 expression for reprogramminginto iPSCs. Secondary human iPSCs were molecularly andfunctionally similar to primary human iPSCs. They showed a similarmethylation pattern of the Oct4 and Nanog promoter regions toESCs (Fig. 4A), expressed the pluripotency markers Oct4 andNanog (Fig. 4B), and formed teratomas after injection under thekidney capsule of SCID mice (data not shown).

Reprogramming of a malignant melanoma cell line into iPSCsin the absence of exogenous Sox2 expressionWe wanted to assess whether the malignant transformation ofmelanocytes would make them refractory to reprogramming bytranscription factors. To test this, we took advantage of the malignantR545 melanoma cell line (Chin et al., 1999), which we havepreviously shown to support the development of cloned embryosand the derivation of pluripotent ESCs (Hochedlinger et al., 2004).R545 cells carry a deletion of the ink4a/arf locus, and conditionallyexpress H-Ras from a melanocyte-specific promoter. Moreover,these cells were found to be trisomic for chromosomes 8 and 11(Hochedlinger et al., 2004).

Because R545 cells were of melanocyte origin, we first attemptedinfecting them with retroviruses expressing Oct4 and Klf4 alone,which also conferred resistance to puromycin and blasticidine,respectively (Fig. 5A). In the presence of both drugs, however, noESC-like colonies emerged despite the appearance of double-resistant cells (data not shown). We therefore superinfected resistantcells with doxycycline-dependent lentiviruses expressing Oct4, Klf4and c-Myc (Fig. 5A). ESC-like colonies appeared after 2 weeks inculture and were picked for further analysis. Melanoma-derivediPSC lines showed, like melanocyte-derived iPSCs, silencing of viralgene expression (supplementary material Fig. S3C) and

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demethylation of the Oct4 and Nanog promoters. They also formedteratomas and gave rise to chimeras after labeling with a lentivirusconstitutively expressing GFP and subsequent blastocyst injection(Fig. 5C-F). At five months of age and in the absence of doxycyclinetreatment, these mice were free of obvious tumors. Virus-specificPCR analysis confirmed that iPSCs were devoid of retroviral andlentiviral Sox2 expression (supplementary material Fig. S3A,B),demonstrating that melanoma cells, like primary melanocytes, donot require ectopic Sox2 expression for conversion into iPSCs.Together, these results indicate that the ectopic expression oftranscription factors can reverse the epigenetic state of primarymelanocytes and certain malignant cells of the melanocyte lineage.

DiscussionOur data allow four major conclusions: First, we demonstrate thatmouse and human melanocytes can be reprogrammed into iPSCsby the same combination of transcription factors previously usedto reprogram fibroblasts and keratinocytes. These melanocyte-derived iPSCs expressed markers similar to those expressed byESCs and gave rise to teratomas. In addition, mouse iPSCscontributed to adult chimeras that showed germline transmissionand hence fulfill all criteria of pluripotency.

Second, mouse melanocytes undergo reprogramming at roughlyfourfold higher efficiency than mouse fibroblasts (~0.19% versus~0.05%). Interestingly, primary mouse melanocytes were devoidof methylation at the Nanog promoter region, which is in contrastto human melanocytes and other cultured primary mouse cell typesthat have been recently reprogrammed (Eminli et al., 2008; Maheraliet al., 2007; Stadtfeld et al., 2008c) and might explain why mousemelanocytes are reprogrammed more efficiently. Consistent withthis notion is the previous observation that hypomethylatedfibroblasts, which lack methylation at the Nanog promoter, are moreefficiently reprogrammed to pluripotency by nuclear transfer(Blelloch et al., 2006). It remains unclear, however, whether

demethylation of the Nanog promoter in murine melanocytes waspre-existing in vivo or arose upon explantation of cells in vitro. Inaddition to mouse melanocytes, human melanocytes gave rise toiPSCs faster than did fibroblasts, showing colony formation after10 days of factor expression. These kinetics are similar tokeratinocytes infected with four factors, which yield iPSCs fasterand at higher efficiency than fibroblasts (Aasen et al., 2008;Maherali et al., 2008). Keratinocytes and melanocytes are both ofectodermal origin whereas fibroblasts are of mesodermal origin.Because ESCs are embryonic ectodermal cells, it might be fasterand more efficient to convert an ectodermal rather than amesodermal somatic cell type into iPSCs.

Third, we provide evidence that an aneuploid melanoma cell lineremains amenable to reprogramming into iPSCs that supported thedevelopment of chimeras, suggesting that certain cancer cells arenot refractory to reprogramming by transcription factors. Thisobservation should be useful for studying the relative contributionof reversible epigenetic and irreversible genetic changes to cancer.In contrast to nuclear transfer, however, iPSC formation does notrequire the generation of cloned embryos, which might beincompatible with some genetically aberrant cancer cells(Hochedlinger et al., 2004). Moreover, it should be possible toreprogram human cancer cells into iPSCs to assess their epigeneticstate.

Fourth, we show that fewer factors are required for the generationof iPSCs from mouse and human melanocytes as well as frommelanoma cells than previously used for reprogramming mouse andhuman fibroblasts and keratinocytes. To date, melanocytes andNPCs are the only cell types that can be reprogrammed intopluripotent cells in the absence of ectopic Sox2 expression. iPSCsproduced with three factors were molecularly and functionallysimilar to iPSCs produced with four factors, including their abilityto generate high-degree chimeric mice competent of germlinetransmission.

Fig. 4. Generation of secondary human iPSCs.(A) Experimental scheme depicting the generationof secondary human iPSCs. Primary human iPSCsderived from melanocytes were differentiated invitro as embryoid bodies for 7 days, then platedunder adherent conditions. Fibroblast-like colonieswere picked and expanded for at least two furtherpassages prior to re-inducing with doxycycline,resulting in secondary human iPSCs. Shown beloware the methylation states of the Nanog and Oct4promoter regions in primary human melanocytes,human iPSCs derived from melanocytes with fourfactors, fibroblasts differentiated from humaniPSCs and secondary human iPSCs derived fromfibroblasts. Promoter regions containingdifferentially methylated CpGs are shown. Whitecircles represent unmethylated CpGs; black circlesdenote methylated CpGs. (B) Quantitative PCRanalysis of Nanog and Oct4 pluripotency markerexpression as well as of the melanin-synthesizingenzymes tyrosinase (TYR) and dopachrometautomerase (DCT) in primary human melanocytes(PM), human iPSCs derived from primary humanmelanocytes generated in the presence (4F) orabsence (3F) of viral Sox2, differentiatedfibroblasts produced from four-factor human iPSCsand iPSCs obtained from secondary fibroblast-likecells (sec. hiPSCs). Values represent means ± s.e.m.

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The efficiency of reprogramming mouse and human primarymelanocytes in the absence of ectopic Sox2 expression is lowerthan that using all four factors. This is in contrast to NPCs, whosereprogramming efficiency with four factors was in our hands lowerthan that of three factors in the absence of Sox2 (Eminli et al., 2008).This suggests that cells with abundant levels of endogenous Sox2,such as NPCs, are more sensitive to ectopic Sox2 expression,resulting in toxicicity or the induction of differentiation as seenpreviously for ESCs (Kopp et al., 2008). In mouse and human earlypassage melanocyte cultures, however, which show low total levelsof Sox2 expression, forced expression of viral Sox2 does not appearto have an adverse effect on reprogramming, but rather enhancesefficiency.

We were able to detect higher Sox2 expression levels at earlypassages of mouse and human melanocyte cultures than at latepassages. In agreement, we show that primary melanocytes can bereprogrammed in the absence of exogenous Sox2 expression onlyat lower passages. This suggests that cultured cells might changequalitatively over time or as they reach senescence, thus resultingin a gradual loss of Sox2 expression. Alternatively, there may be arare Sox2-positive subpopulation in the culture, possibly

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progenitors, that is lost during passaging through differentiation.We also cannot rule out the possibility that other Sox familymembers expressed in melanocytes functionally replace the role ofSox2 during reprogramming.

Our findings imply that melanocyte cultures that express one ofthe reprogramming factors endogenously are more amenable toreprogramming than fibroblasts or keratinocytes and thus might bean appropriate source of cells for attempts to replace viral genedelivery systems with transient expression approaches such asplasmids (Okita et al., 2008), adenoviruses (Stadtfeld et al., 2008b)or small compounds (Huangfu et al., 2008; Shi et al., 2008). Lastly,melanocytes can be easily obtained by skin punch biopsies, whichmake them an accessible cell type for clinical applications.

Materials and MethodsLentiviral productionThe generation and structure of replication-defective doxycycline-inducible lentiviralvectors, a lentiviral vector constitutively expressing rtTA, and retroviral vectors havebeen described in detail elsewhere (Stadtfeld et al., 2008a). To produce infectiouslentiviral particles, 293T cells cultured on 10-cm dishes were transfected with theLV-tetO vectors (11 μg) together with the packaging plasmids VSV-G (5.5 μg) and�8.9 (8.25 μg) using Fugene (Roche). Viral supernatants were harvested on three

Fig. 5. Reprogramming of a malignant melanomacell into iPSCs. (A) Experimental outline forgenerating iPSCs from R545 melanoma cells.(B) Shown are R545 cells (left image) and R545-derived iPSC colonies on feeder cells at day 50(right image). (C) Brightfield and fluorescent imageof R545-derived iPSCs stained for the pluripotencymarker Nanog. (D) Bisulfite sequencing of the Oct4and Nanog promotor regions in R545 cells, R545derived iPSCs and ESCs. White circles representunmethylated CpGs; black circles denotemethylated CpGs. (E) Hematoxylin and eosinstaining of a teratoma derived from a R545-iPSCshows differentiation into cell types from all threegerm layers: keratinized epithelium (left image),epithelium and glandular structures (center image)and cartilage (right image). (F) Viable newbornchimera from iPSCs derived from R545 cells.Chimeric pup (lower image) and non-chimericlittermate (upper image) are shown under UV light(left image). Adult chimera derived from three-factor R545-iPSCs shows coat color chimerism(right image).

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3509Reprogramming of melanocytic cells into iPSCs

consecutive days starting 24 hours after transfection, yielding a total of ~30 ml ofsupernatant per viral vector. Viral supernatant was concentrated approximately 100-fold by ultracentrifugation at 50,000 g for 1.5 hours at 4°C and resuspended in 300μl phosphate-buffered saline (PBS), and stored at –80°C. Infections were carried outin 1 ml medium using 5 μl of each viral concentrate per 35-mm plate. Comparisonsof different cell types were performed with the same batch and concentration of viralparticles.

Mouse cell culture and generation of iPSCsNewborn pups of the lineage tracing mouse model Wnt1-Cre � Rosa26R-EYFP(mixed background of C57Bl/6 and 129SvJae) (Stock no. 003829, The JacksonLaboratory, Bar Harbor, ME) were sacrificed by CO2 asphyxiation. The mice weresterilized by soaking several times in 1:200 diluted Wescodyne Solution (SterisCorporation) and washed three times in 1� PBS containing penicillin andstreptomycin. The skin was carefully peeled off and placed dermis side down in aPetri dish with 5 ml of 0.2% trypsin in Hank’s buffered salt solution (HBSS) containingpenicillin and streptomycin. The Petri dish was placed in the refrigerator at 4°Covernight. The next day, the trypsinized skin parts were carefully pulled away fromthe thin sheet of epidermis leaving the dermis. The epidermis sheets were rinsed inPBS and cut into small pieces. The tissue pieces were transferred into a 75-ml flaskwith 6 ml of melanocyte growth medium (Cascade Biologicals), and individual cellswere released by pipetting up and down (Yang et al., 2001). After 3-4 days of culture,cells were treated with 0.25% trypsin-EDTA for 1-2 minutes and EYFP-positive cellswere isolated by FACS and further cultured in melanocyte growth medium.

Lentiviral vector infections were carried out in mouse melanocytes in six-well platesat a density of 100,000 cells/well on 3 subsequent days. Medium changes wereperformed 12-24 hours after infection. One day after the last infection, ESC mediumcontaining 1ug/ml doxycycline was added. Fresh ESC medium with doxycycline wasadded every other day until iPSC colonies developed. Five days later, cell cultureconditions were switched to ESC medium without doxycycline. iPSC colonies werepicked into 96-well plates containing PBS without magnesium and calcium using a10-ul pipette. Trypsin was added to each well and incubated for 5 minutes. Single-cell suspensions were transferred into 24-well dishes containing MEFs. Picked iPSCswere grown on MEFs under ESC conditions. Prior to blastocyst injections, iPSCswere marked with a FUGW lentiviral vector constitutively expressing GFP.

The infection efficiency of primary melanocytes and tail-tip fibroblasts wascalculated after three subsequent infections with tetO-GFP lentiviral vector in thepresence of the rtTA-expressing lentiviral vector (24% and 87%, respectively). Thefraction of cells receiving all viral vectors in the various combinations was determinedby multiplying the infection rate of GFP-positive cells, as described previously(Stadfeld et al., 2008a). The final percentage of efficiency was determined by dividingthe number of GFP positive colonies by the fraction of cells calculated to expresseither three or four viral transgenes.

R545 mouse melanoma cells were cultured in RPMI supplemented with 10% FCSas recently reported (Hochedlinger et al., 2004) and viral infections were performedas described in detail elsewhere (Stadtfeld et al., 2008a).

Human cell culture and generation of iPSCsHuman primary melanocytes were obtained from Promocell, Heidelberg, Germany(NHEM.f-c M2, Lot 6022001 and Lot 7062002.1) and cultured in human melanocytegrowth medium (M2, Promocell). Lentiviral vector infections were carried out withhuman melanocytes in six-well plates at a density of 100,000 cells/well on 3subsequent days. The infection efficiency of primary human melanocytes after threesubsequent infections with tetO-GFP lentiviral vector in the presence of the rtTA-expressing lentiviral vector was 30.1%. Medium changes were performed 12-24 hoursafter infection. One day after the last infection, cells were transferred on MEFs. Mediacontaining 50% melanocyte M2 medium and 50% human ESC medium containing0.5 ug/ml doxycycline was added. Medium changes were performed every other dayin the presence of 0.5 ug/ml doxycycline until iPSC colonies developed. Uponappearance of human ESC-like colonies, medium was switched to human ESC cultureconditions as previously described (Cowan et al., 2004).

ImmunofluorescenceMouse and human iPSCs were cultured on MEFs that were seeded the day beforeon pretreated coverslips. Cells were fixed with 4% paraformaldehyde, andpermeabilized with 0.5% Triton X-100. The cells were then stained with primaryantibodies against mOct4 (sc-8628, Santa Cruz Biotechnology) and mNanog(ab21603, Abcam). Respective secondary antibodies were conjugated to Alexa Fluor546 (Invitrogen). Nuclei were counterstained with DAPI (Invitrogen). Cells wereimaged with a Leica DMI4000B inverted fluorescence microscope equipped with aLeica DFC350FX camera. Images were processed and analyzed with AdobePhotoshop software.

Quantitative PCRRNA was isolated from cells with the TRIzol reagent (Invitrogen). For melanocytes,an additional phenol-chloroform step was performed before RNA clean up with theRNeasy Minikit (Qiagen) to remove melanin. cDNA was produced with theSuperscript III First-Strand synthesis system (Invitrogen) using oligo-dT primers. Real-

time quantitative PCR (qPCR) reactions were set up in triplicates with the BrilliantII SYBR Green qPCR Master Mix (Stratagene) and run on a Mx3000P qPCR System(Stratagene). Primer sequences are listed in supplementary material Table S1.

Bisulfite sequencingBisulfite treatment of DNA was performed with the EpiTect Bisulfite Kit (Qiagen)according to manufacturer’s instructions. Oct4 and Nanog promoter sequences wereas previously described for mouse (Eminli et al., 2008) and human (Maherali et al.,2008). Amplified products were purified using gel filtration columns, then clonedinto the pCR4-TOPO vector (Invitrogen) and sequenced with M13 forward and reverseprimers. Analysis shows the promoter regions containing the methylated andunmethylated dinucleotide CpG.

Generation of mouse teratomas and chimerasFor teratoma induction, 2�106 cells of each iPSC line were injected subcutaneouslyinto the dorsal flank of isoflurane-anesthetized SCID mice. Teratomas were recovered3-5 weeks post-injection, fixed overnight in 10% formalin, paraffin embedded andprocessed with hematoxylin and eosin.

For chimera production, female BDF1 mice were superovulated with PMS(pregnant mare serum) and hCG (human chorion gonadotropin) and mated to BDF1stud males. Zygotes were isolated from plugged females 24 hours after hCG injection.After 3 days of in vitro culture in potassium simplex optimized medium (KSOM),blastocysts were injected with iPSCs and transferred into d2.5 pseudopregnantrecipient females. C-sections were performed 17 days later and the pups fosteredwith lactating Swiss females.

Differentiation of human iPSCsFor in vitro differentiation, human iPSC colonies were picked and placed insuspension culture with fibroblast growth media. After 10 days, embryoid bodieswere plated under adherent conditions on gelatin-coated plates and further culturedwith fibroblast growth media. For teratoma formation around 50,000 human iPSCswere pelleted and injected as a suspension into SCID mice underneath one kidneyor both testis capsules of anaesthetized mice. Tumors developed after 8-12 weeksand were processed for histological analysis.

We thank Matthias Stadtfeld for critical review of the manuscript.We also thank Laura Prickett, Kathryn E. Folz-Donahue for help withFACS analysis and Patricia Follett for performing blastocyst injections.J.U. was supported by the Dr Mildred Scheel Foundation for CancerResearch. N.M. was supported by a graduate scholarship from theNatural Sciences and Engineering Research Council of Canada and aSir James Lougheed Award from Alberta Scholarships. K.H. wassupported by the NIH Director’s Innovator Award, the Harvard StemCell Institute, the V Foundation and the Kimmel Foundation. This studyhas been approved by the MGH animal and biosafety committees.Deposited in PMC for release after 12 months.

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