Defining the Conditions for the Generation of Melanocytes from Human Embryonic Stem Cells

Abstract

Because of their undifferentiated nature, human embryonic stem cells (hESCs) are an ideal model system for studying both normal human development and the processes that underlie disease. In the current study, we describe an efficient method for differentiating hESCs into a melanocyte population within 4 -6 weeks using three growth factors: Wnt3a, endothelin-3, and stem cell factor. The hESC-derived melanocytes expressed melanocyte markers (such as microphthalmia-associated transcription factor and tyrosinase), developed melanosomes, and produced melanin. They retained the melanocyte phenotype during long-term cell culture (>90 days) and, when incorporated into human reconstructed skin, homed to the appropriate location along the basement membrane in the same manner as epidermisderived melanocytes. They maintained a stable phenotype even after grafting of the reconstructs to immunodeficient mice. Over time in culture, the hESC-derived melanocytes lost expression of telomerase and underwent senescence. In summary, we have shown for the first time the differentiation of hESCs into melanocytes. This method provides a novel in vitro system for studying the development biology of human melanocytes.

Generation of Human Melanocytes from Induced Pluripotent Stem Cells

“…To induce melanocytic differentiation, EBs were derived as described above, however, after three weeks, EBs were plated on a fibronectin-coated dish in DMEM and MCDB 201 based differentiation medium containing 50 ng/ml Wnt3a (R&D Systems, Minneapolis, MN), 50 ng/ml stem cell factor (SCF, R&D Systems), 100 mM Endothelin-3 (ET-3, American Peptide Company, Sunnyvale, CA), 20 pM cholera toxin (Toyobo, Osaka, Japan), 50 nM TPA (12-tetra decanoyl-phorbol 13-acetate, SigmaAldrich, St Louis, MO), 4 ng/ml FGF-2 (WAKO), 100 mM Lascorbic acid (Sigma), 0.05 mM dexamethasone (Sigma), 1 mg/ml linoleic acid-bovine serum albumin (Sigma), and 1X insulintransferrin-selenium (Sigma) following the method described by Fang et al [14]. Before attaining confluence, cells were dissociated using TrypLE Select (Invitrogen) and replated onto a fibronectin (BD Biosciences, San Jose, CA)-coated dish in differentiation medium until the appearance of pigmented cells and continued to be cultured for few months.…”

“…Thus, it is desirable to develop a new in vitro system for generating human melanocytes through MELSCs, which mimics in vivo differentiation processes to better understand human melanocyte development. Melanocyte generation from embryonic stem (ES) cells has been previously reported [14,15]. Alternatively, induced pluripotent stem (iPS) cells have specific advantages compared to ES cells.…”

In vitro and in vivo differentiation of human embryonic stem cells into retina‐like organs and comparison with that from mouse pluripotent epiblast stem cells

“…In some cases, very complex culture strategies have been used, including the sequential addition and withdrawal of factors and purification of specific cell populations during the culture period (Fang et al, 2006;Wang et al, 2005). As for the induction of ocular tissues, lens and several retinal cell types were reported to have been differentiated from ES cells (Gong et al, 2008;Lamba et al, 2006;Osakada et al, 2008).…”

Concise Review: Epidermal Grafting: The Case for Pluripotent Stem Cells

“…This issue may eventually be much better addressed by pluripotent stem cell derivatives. At this point, in addition to basal keratinocytes, protocols are available for differentiation into melanocytes (unpublished data and [33]) and fibroblasts [34], mesenchymal stem cells [35], and adipocytes [36] that can be introduced into the final cell therapy product to improve its overall quality.…”