Work

Melanocytes Derived from Transgene-Free Human Induced Pluripotent Stem Cells

Year: 2013

Type: article

Abstract: human induced pluripotent stem cell human primary melanocyte α-melanocyte-stimulating hormone microphthalmia-associated transcription factor pluripotent stem cell single-nucleotide polymorphism Defects in melanocytes have been implicated in the etiology of a variety of human skin diseases and disorders (Lin and Fisher, 2007Lin J.Y. Fisher D.E. Melanocyte biology and skin pigmentation.Nature. 2007; 445: 843-850Crossref PubMed Scopus (851) Google Scholar; Fistarol and Itin, 2010Fistarol S.K. Itin P.H. Disorders of pigmentation.J Dtsch Dermatol Ges. 2010; 8: 187-201Crossref PubMed Google Scholar; Rees, 2011Rees J.L. The genetics of human pigmentary disorders.J Invest Dermatol. 2011; 131: E12-E13Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). There is long-standing interest in studying the development and dysfunction of human melanocytes, but there has not been a reliable and accessible system to study early events in human melanocyte differentiation. An in vitro system that reliably and efficiently produces normal human melanocytes from embryonic stage cells would allow us to better dissect the physiological and pathological development of melanocytes. Recent advances in stem cell biology have led to the establishment of human induced pluripotent stem cell (hiPSC) techniques that enable researchers to reprogram somatic cells to the pluripotent state (Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14962) Google Scholar). Differentiation of human and mouse pluripotent stem cells (PSCs) toward the melanocyte lineage has been reported (Yamane et al., 1999Yamane T. Hayashi S. Mizoguchi M. et al.Derivation of melanocytes from embryonic stem cells in culture.Dev Dyn. 1999; 216: 450-458Crossref PubMed Google Scholar; Pla et al., 2005Pla P. Alberti C. Solov'eva O. et al.Ednrb2 orients cell migration towards the dorsolateral neural crest pathway and promotes melanocyte differentiation.Pigment Cell Res. 2005; 18: 181-187Crossref PubMed Scopus (52) Google Scholar; Fang et al., 2006Fang D. Leishear K. Nguyen T.K. et al.Defining the conditions for the generation of melanocytes from human embryonic stem cells.Stem Cells. 2006; 24: 1668-1677Crossref PubMed Scopus (95) Google Scholar; Nissan et al., 2011Nissan X. Larribere L. Saidani M. et al.Functional melanocytes derived from human pluripotent stem cells engraft into pluristratified epidermis.Proc Natl Acad Sci USA. 2011; 108: 14861-14866Crossref PubMed Scopus (54) Google Scholar; Ohta et al., 2011Ohta S. Imaizumi Y. Okada Y. et al.Generation of human melanocytes from induced pluripotent stem cells.PLoS One. 2011; 6: e16182Crossref PubMed Scopus (88) Google Scholar; Yang et al., 2011Yang R. Jiang M. Kumar S.M. et al.Generation of melanocytes from induced pluripotent stem cells.J Invest Dermatol. 2011; 131: 2458-2466Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), but existing protocols have shortcomings that may limit their research and clinical applications. For example, the use of embryonic stem cells could lead to allogeneic immunoincompatibility of differentiated melanocytes and transplant recipients. In addition, the use of hiPSCs generated by integrative reprogramming strategies raises concerns about reactivation of retained transgenes, some of which are oncogenes. In addition, the current methods for melanocyte differentiation from hiPSCs require optimization in order to reproducibly generate high-purity melanocytes from multiple hiPSC lines. We have established a strategy to produce human melanocytes in vitro for use as a platform for pigment cell research and the development of cell-based therapies. We first derived transgene-free hiPSCs from two distinct types of skin cells: human primary melanocytes (HMs) and human dermal fibroblasts (HDF51) (Figure 1a and Supplementary Figure S1a online). We used a nonintegrative reprogramming approach mediated by Sendai virus–based vectors independently encoding POU5F1, SOX2, KLF4, and MYC (Fusaki et al., 2009Fusaki N. Ban H. Nishiyama A. et al.Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.Proc Jpn Acad Ser B Phys Biol Sci. 2009; 85: 348-362Crossref PubMed Scopus (1001) Google Scholar; Macarthur et al., 2012Macarthur C.C. Fontes A. Ravinder N. et al.Generation of human-induced pluripotent stem cells by a nonintegrating RNA Sendai virus vector in feeder-free or xeno-free conditions.Stem Cells Int. 2012; 2012: 564612Crossref PubMed Scopus (67) Google Scholar). As shown in Figure 1b and Supplementary Figure S1b online, biomarkers of cellular pluripotency, including endogenous OCT4/POU5F1, NANOG, Tra-1-81, and UEA-I (Wang et al., 2011Wang Y.C. Nakagawa M. Garitaonandia I. et al.Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis.Cell Res. 2011; 21: 1551-1563Crossref PubMed Scopus (68) Google Scholar), were positive in HMi-506, HMi-503, and HDF51i-509 hiPSCs. Cells were also shown to be pluripotent using a gene expression diagnostic test (PluriTest; Muller et al., 2011Muller F.J. Schuldt B.M. Williams R. et al.A bioinformatic assay for pluripotency in human cells.Nat Methods. 2011; 8: 315-317Crossref PubMed Scopus (339) Google Scholar), by differentiation into cells that express biomarkers relevant to all three germ layers in vitro (Figure 1c and Supplementary Figure S1c, S1d and S1e online) and by generation of teratomas (Supplementary Figure S1d online). Download .pdf (2.03 MB) Help with pdf files Supplementary Information We newly developed two differentiation protocols based on previously reported methods. One protocol involves an aggregation-in-suspension step, whereas the other does not (Supplementary Figure S2 online). Both protocols generated cells displaying typical melanocyte morphology and pigmentation (Figure 1d) from hiPSCs after 30 days of directed differentiation, suggesting that the aggregation-in-suspension step is dispensable. The melanin granules that accumulated at the dendritic tips of differentiated cells were intensely stained by Fontana–Masson staining, indicating that the pigmentation of these cells was due to melanogenesis (Supplementary Figure S3 online). In addition, MITF (microphthalmia-associated transcription factor), a marker for melanocyte progenitors, was expressed in more than 90% of the differentiated derivatives after 30 days (Figure 1e and Supplementary Figure S4 online), which appears to be a higher differentiation efficiency than other reported protocols (Nissan et al., 2011Nissan X. Larribere L. Saidani M. et al.Functional melanocytes derived from human pluripotent stem cells engraft into pluristratified epidermis.Proc Natl Acad Sci USA. 2011; 108: 14861-14866Crossref PubMed Scopus (54) Google Scholar; Ohta et al., 2011Ohta S. Imaizumi Y. Okada Y. et al.Generation of human melanocytes from induced pluripotent stem cells.PLoS One. 2011; 6: e16182Crossref PubMed Scopus (88) Google Scholar). As expected, MITF was not detected in the undifferentiated hiPSCs, and was present in the primary melanocytes (Figure 1e). Notably, our protocols resulted in similarly high levels of melanocyte differentiation for all four independent hiPSC lines examined, highlighting their reproducibility. Other melanocytic biomarkers including TYR (tyrosinase), MLANA (melan-A), TYRP1 (tyrosinase-related protein 1), PMEL (premelanosome protein), PAX3 (paired box 3), and SOX10 (SRY-box 10) were highly expressed in the differentiated derivatives (similar to primary melanocytes, Figure 2a and b). The melanin content and cell signaling involved in melanin production in the differentiated derivatives was increased by treatment with α-melanocyte-stimulating hormone (α-MSH) in a dose-dependent manner (Figure 2c and d and Supplementary Figure S5 online). These findings indicate that the differentiated derivatives possess molecular features of bona fide melanocytes and accurately mimic their ability to respond to α-MSH, which is the factor that activates melanogenesis and enhances skin pigmentation during the tanning response (Thody, 1999Thody A.J. alpha-MSH and the regulation of melanocyte function.Ann NY Acad Sci. 1999; 885: 217-229Crossref PubMed Scopus (81) Google Scholar). Genome-wide gene expression profiling and unsupervised hierarchical clustering revealed that the melanocytes (HMi-506_Mel Diff_1 and HMi-506_Mel Diff_2) differentiated from the HMi-506 cells were closely clustered with HMs and were distinct from all undifferentiated hiPSC samples (Figure 2e). As genetic abnormalities may occur in hiPSC genomes during the reprogramming and differentiation processes, we tested the genomic stability of the cells by comparing the differentiated derivatives with the parental primary melanocytes using high-resolution single-nucleotide polymorphism (SNP) genotyping and copy number variation analysis. As shown in Figure 2f, the HMi-506_Mel Diff derivatives and parental cells showed highly similar genotyping profiles, showing that the cellular genome remained stable during reprogramming and differentiation. Similar to human melanocytes in vivo, the differentiated derivatives in semiautologous skin reconstructs were located at the dermis–epidermis interface and interspersed with keratinocytes (Supplementary Figure S6a, S6b, S6c and S6d online), indicating their ability to integrate with the skin tissue of transplant recipients. Similar to the autologous dermal fibroblasts used for generating transgene-free hiPSCs, the differentiated derivatives stimulated limited proliferation of peripheral blood mononuclear cells that were isolated from the blood of the same individual in a mixed lymphocyte reaction assay (Supplementary Figure S6e online). These results attest to the clinical advantages of melanocytes differentiated from hiPSCs using the reprogramming and differentiation approaches described here. In this study, we have demonstrated that genetically stable melanocytes can be efficiently differentiated from transgene-free hiPSCs generated from two different types of cutaneous cells. This differentiation protocol takes less time than previously reported melanocytic differentiation protocols, and we showed that it is equally effective for multiple independent hiPSC lines. We performed a thorough investigation of the differentiated cells, including genome-wide gene expression analysis and SNP genotyping in addition to functional assays. Our approach can serve as an unlimited source of custom human melanocytes that can be used for novel approaches for modeling human skin disease (e.g., melanoma and vitiligo) and to provide material for transplantation. JCJ was supported by the California Institute for Regenerative Medicine (CIRM) Bridges to Stem Cell Research Internship Program (TB1-01177-1). Y-CW was supported by the Marie Mayer Foundation Postdoctoral Fellowship. JFL and SEP were supported by grants from CIRM (RT1-01108, TR1-01250, and CL1-00502), NIH (R33MH087925), the Esther O’Keeffe Foundation, and the Millipore Foundation. MMP was supported by CIRM (RN2-00931) and NIH (1R01HD071100). YL was supported by CIRM (RT1-011071), the Memorial Hermann Foundation (Staman Ogilvie Fund), and Bentsen Stroke Center. LCL was supported by NIH/NICHD Career Development Award (K12 HD001259). We thank Ms Nastaran Afari and Ms Kellen Na at University of California, San Diego, for their assistance in teratoma analysis. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

Source: Journal of Investigative Dermatology

DOI: 10.1038/jid.2013.139