Generation of human melanocytes from induced pluripotent stem cells - PubMed (original) (raw)
Generation of human melanocytes from induced pluripotent stem cells
Shigeki Ohta et al. PLoS One. 2011.
Abstract
Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC). These iPS cell lines were subsequently used to form embryoid bodies (EBs) and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Induction of human 3F- and 4F-iPS cells from adult dermal fibroblasts and characterization of their pluripotency.
(A) Morphology of human dermal fibroblasts. (B) Representative image of an established iPS cell colony cultured on mitomycin C-treated STO feeders. (C, D) Alkaline phosphatase (AP) staining in a 3F- (C) and 4F- (D) iPS cell colony. (E–I′) Expression of pluripotency markers in established 3F- and 4F-iPS colonies. 3F-iPS (E–I) and 4F-iPS (E′–I′) colonies express markers common to pluripotent cells including OCT3/4, NANOG, SSEA-4, TRA-1-60, and TRA-1-81. DAPI (4, 6-diamidino-2-phenylindole) staining indicates the total cell content per field. Scale bars, 20 µm (A), 100 µm (B, C, and D), 50 µm (E–I′). (J) Expression of pluripotency genes. Endogenous gene expression levels of OCT3/4, SOX2, KLF4, c-MYC, NANOG, and REX1 were determined by quantitative PCR in parental human dermal fibroblasts (HDF), 3F- and 4F-iPS cells, 201B7 (4F-iPS cells) and ES cells (KhES-1). The graphs show the average of two independent experiments. Error bar indicates mean±S.E.M. (K) iPS cells are demethylated at the OCT3/4 promoter relative to their parental fibroblasts. Bisulfite sequencing analysis of the OCT3/4 promoter in parental HDFs, 3F-, and 4F-iPS cells. Each horizontal row of circles represents an individual sequencing reaction for a given amplification. White circles represent unmethylated CpG dinucleotides; black circles represent methylated CpG dinucleotides. (L–N′) Images of differentiated cells at day 12. 3F- (L–N) and 4F-iPS (L′–N′) cells were cultured in suspension to form EBs for three weeks and then transferred to gelatin-coated plates and cultivated for another 12 days. Immunocytochemical analyses showed positive cells in spontaneously differentiated iPS cell colonies for β-III tubulin (ectoderm, L and L′), α-smooth muscle actin (α-SMA, mesoderm, M and M′), and α-fetoprotein (AFP, endoderm, N and N′). Scale bar, 50 µm. (O–Q′) Generation of teratoma-like masses in testis by xenograft of 3F- and 4F-iPS. Paraffin-embedded sections were stained with hematoxylin and eosin. Resulting teratomas display features of ectoderm (O and O′), mesoderm (P and P′) and endoderm (Q and Q′). Scale bar, 100 µm.
Figure 2. Generation of melanocytes from iPS cells.
Efficient differentiation of human iPS cells into pigmented melanocytes in defined conditions. (A) A representative image of a 3F-iPS cell colony grown on a Mitomycin-C-treated STO feeder layer. (B) Embryoid bodies (EBs) formed in suspension under feeder-free conditions. (C) After one week in differentiation medium, cells were observed migrating out from EBs on a fibronectin–coated plate. (D) Morphology of putative melanocytes differentiated from human EBs 7 weeks after differentiation. Cells display melanocytic morphology and pigmentation. (E) Pigmented cell pellet of iPS cell-derived melanocytes 9 weeks after differentiation. Scale bar, 100 µm (A, B, C), 40 µm (D).
Figure 3. Marker expression characteristic of the melanocyte lineage in human iPS cell-derived melanocytes observed 8 weeks after differentiation.
Either 3F- (3F-iPS MEL, A–E) or 4F- (4F-iPS MEL, F–J) iPS cells derived melanocytes were positive for an array of melanocyte markers: SILV (silver protein), TYRP1 (tyrosinase-related protein-1), TYR (tyrosinase), MITF (microphthalmia-associated transcription factor) , and S100. Normal human epidermal melanocytes (NHEM) were also stained with the same antibodies (K–O). Transmission electron microscopy images of either 3F (P), 4F (Q)-iPS cells derived melanocytes, or NHEM (R) showed that those melanocytes have many melanosomes in the cytoplasm. Scale bar, 20 µm (A–O), 0.5 µm (P, Q, and R). (S) Quantitative real-time PCR analysis of MITF-M and c-Kit in 3F-iPS, 3F-iPS MEL, and NHEM. The data are normalized to GAPDH and represented as fold change relative to RNA levels in 3F-iPS. The graphs show the average of two independent experiments. Error bar indicates mean±S.E.M. (T) Quantitation of SILV-positive cells induced from 3F and 4F-iPS cells. Percentage of SILV positive cells upon total differentiation of EBs cells 7 weeks after differentiation under melanocyte differentiation medium. Error bar indicates mean±S.E.M (3F-iPS, n = 3; 4F-iPS, n = 2). (U) The global gene-expression patterns were compared between human 3F-iPS cells and 3F-iPS derived-MEL (3F-iPS MEL), and between NHEM and 3F-iPS MEL. The lines indicate the linear equivalent and 5-fold differences on either side in gene expression levels between the two samples.
Figure 4. 3F- and 4F-iPS cells can be differentiated into NCSC and MELSCs.
(A) Gene expression analysis of a neural crest stem cell marker (Slug), a melanocyte stem cell marker (PAX3), a melanocytic marker (MITF), pluripotency marker (NANOG) in 3F-iPS cells, differentiated cells derived from EBs on fibronectin-coated plates in melanocyte differentiation medium at 7 days after differentiation (EB-DIFF), and 3F-iPS derived melanocytes (3F-iPS MEL). Transcript levels were normalized to GAPDH. The graphs show the average of two independent experiments. Error bar indicates mean±S.E.M. (B) Representative flow cytometry results for HNK-1 and p75 staining in 3F-iPS EB-derived cells cultured in melanocyte differentiation medium for one week. Dead cells were excluded using PI staining. (C) Immunocytochemistry revealing cells positive for NCSC markers (p75, HNK-1, and AP2α) in both 3F- and 4F-iPS cells derived EB-differentiated cells 7 days after differentiation. Scale bar, 50 µm. (D) Images show cells positive for neural crest cell marker SOX10, as well as possible melanocyte stem cell marker, PAX3 in both 3F- and 4F-iPS cell-derived EB-differentiated cells 7 days after differentiation. Scale bar, 50 µm and 10 µm (insert images).
References
- Alkhateeb A, Fain PR, Thody A, Bennett DC, Spritz RA. Epidemiology of vitiligo and associated autoimmune diseases in Caucasian probands and their families. Pigment Cell Res. 2003;16:208–214. - PubMed
- Czajkowski R, Placek W, Drewa T, Kowaliszyn B, Sir J, et al. Autologous cultured melanocytes in vitiligo treatment. Dermatol Surg. 2007;33:1027–1036; discussion 1035–1026. - PubMed
- van Geel N, Ongenae K, Naeyaert JM. Surgical techniques for vitiligo: a review. Dermatology. 2001;202:162–166. - PubMed
- Grichnik JM. Melanoma, nevogenesis, and stem cell biology. J Invest Dermatol. 2008;128:2365–2380. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials