Abstract
Melanocytes are pigment-producing cells of neural crest (NC) origin that are responsible for protecting the skin against UV irradiation. Pluripotent stem cell (PSC) technology offers a promising approach for studying human melanocyte development and disease. Here, we report that timed exposure to activators of WNT, BMP, and EDN3 signaling triggers the sequential induction of NC and melanocyte precursor fates under dual-SMAD-inhibition conditions. Using a SOX10::GFP human embryonic stem cell (hESC) reporter line, we demonstrate that the temporal onset of WNT activation is particularly critical for human NC induction. Subsequent maturation of hESC-derived melanocytes yields pure populations that match the molecular and functional properties of adult melanocytes. Melanocytes from Hermansky-Pudlak syndrome and Chediak-Higashi syndrome patient-specific induced PSCs (iPSCs) faithfully reproduce the ultrastructural features of disease-associated pigmentation defects. Our data define a highly specific requirement for WNT signaling during NC induction and enable the generation of pure populations of human iPSC-derived melanocytes for faithful modeling of pigmentation disorders.
Highlights
Epidermal melanocytes are pigment-producing cells found at the basement membrane of the skin where they establish a photo-protective barrier against UV-irradiation
To recapitulate the progressive differentiation that occurs during normal development we established a stepwise differentiation protocol in which human pluripotent stem cells are first differentiated to the multipotent neural crest (NC) stage before becoming melanoblast precursors capable of maturing into terminally-differentiated melanocytes
In the dual SMAD inhibition (DSi) protocol human embryonic stem cells (hESCs) are treated for 11 days with two small molecules that inhibit the separate branches of SMAD signaling; LDN-193189 (LDN) which inhibits BMP signaling and SB431542 (SB) which inhibits TGF-β, Activin, and Nodal signaling (Figure 1A)
Summary
Epidermal melanocytes are pigment-producing cells found at the basement membrane of the skin where they establish a photo-protective barrier against UV-irradiation. While the developmental biology of melanocytes has been well studied in avian and murine models, the processes underlying melanocyte development in humans remain poorly understood. The derivation of melanocytes from human embryonic stem cells (hESCs) provides a valuable tool for studying human melanocyte development and for modeling disease biology. Previous work on the derivation of melanocytes from murine (Yamane et al, 1999) and human (Fang et al, 2006; Nissan et al, 2011) ESCs relied on stromal co-culture or embryoid body formation in combination with conditioned media from a WNT3a producing stromal cell line to trigger melanocytic differentiation. The lack of a defined culture system has complicated efforts to gain better mechanistic insights into early melanocyte development and maturation
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