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Generation of human melanocytes from induced pluripotent stem cells.

Ohta S, Imaizumi Y, Okada Y, Akamatsu W, Kuwahara R, Ohyama M, Amagai M, Matsuzaki Y, Yamanaka S, Okano H, Kawakami Y - PLoS ONE (2011)

Bottom Line: Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan.

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.

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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) [20] 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.
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pone-0016182-g001: 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) [20] 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.

Mentions: We established two human iPS cell lines following the methods established by Takahashi et al. [20] from dermal fibroblasts using all four retrovirally expressed Yamanaka factors (4F) (SOX2, OCT3/4, KLF4, c-MYC) or using three factors (3F) without c-MYC (Figure 1A and 1B). To examine the expression of pluripotency markers in both 3F-iPS cells and 4F-iPS cells, we performed alkaline phosphatase staining (Figure 1C and 1D) and immunocytochemical analyses on OCT3/4, NANOG, SSEA4, TRA-1-60, and TRA-1-81 in 3F and 4F-iPS cell colonies (Figure 1E–I′). In addition, the gene expression profiles of pluripotent stem cell markers including the endogenous Yamanaka factors, NANOG and REX1 were analyzed in 3F and 4F-iPS cells by quantitative PCR analyses and compared to 4F-iPS cells' parental human dermal fibroblasts and previously characterized 4F-iPS cells, 201B7 [20]. The generated 3F- and 4F-iPS cells expressed endogenous pluripotency markers similarly to 201B7 iPS cells and ES cells (Figure 1J). Transgene expression was also examined using quantitative PCR analyses, indicating a similarly low gene expression level in the both established 3F- and 4F-iPS cells as compared to 201B7 iPS cells, apart from a higher expression of retroviral KLF4 in 3F-iPS cells (Supplemental Figure S1). Evaluation of the methylation status of cytosine guanine dinucleotides (CpG) in the promoter regions of OCT3/4 was analyzed by bisulfite sequencing and compared to 4F-iPS parental dermal fibroblasts. 3F- and 4F-iPS cells were highly unmethylated in comparison (Figure 1K). Furthermore, the differentiation ability of both 3F- and 4F-iPS cells was examined in vitro. Floating embryoid bodies (EBs) were cultivated for 3 weeks from each iPS cell set, and then spontaneously differentiated onto gelatin coated dishes for 12 days. Attached cells were immunopositive for three germ cell layers' markers (β-III-tubulin, ectoderm; α-smooth muscle actin, mesoderm; α-fetoprotein, endoderm) (Figure 1L-1N′). Finally, to confirm the pluripotency of 4F-iPS cells in vivo, we transplanted 3F- and 4F-iPS cells into the testis of immunodeficient mice (NOD-SCID) to observe teratoma formation. Tumors formed eight weeks after injection and contained various tissues representing all three germ layers (ectoderm, mesoderm, and endoderm; Figure 1O–1Q′) and various tissues (Supplemental Figure S2). Collectively, these results confirmed the establishment of de novo iPS cells from human dermal fibroblasts.


Generation of human melanocytes from induced pluripotent stem cells.

Ohta S, Imaizumi Y, Okada Y, Akamatsu W, Kuwahara R, Ohyama M, Amagai M, Matsuzaki Y, Yamanaka S, Okano H, Kawakami Y - PLoS ONE (2011)

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) [20] 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.
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pone-0016182-g001: 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) [20] 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.
Mentions: We established two human iPS cell lines following the methods established by Takahashi et al. [20] from dermal fibroblasts using all four retrovirally expressed Yamanaka factors (4F) (SOX2, OCT3/4, KLF4, c-MYC) or using three factors (3F) without c-MYC (Figure 1A and 1B). To examine the expression of pluripotency markers in both 3F-iPS cells and 4F-iPS cells, we performed alkaline phosphatase staining (Figure 1C and 1D) and immunocytochemical analyses on OCT3/4, NANOG, SSEA4, TRA-1-60, and TRA-1-81 in 3F and 4F-iPS cell colonies (Figure 1E–I′). In addition, the gene expression profiles of pluripotent stem cell markers including the endogenous Yamanaka factors, NANOG and REX1 were analyzed in 3F and 4F-iPS cells by quantitative PCR analyses and compared to 4F-iPS cells' parental human dermal fibroblasts and previously characterized 4F-iPS cells, 201B7 [20]. The generated 3F- and 4F-iPS cells expressed endogenous pluripotency markers similarly to 201B7 iPS cells and ES cells (Figure 1J). Transgene expression was also examined using quantitative PCR analyses, indicating a similarly low gene expression level in the both established 3F- and 4F-iPS cells as compared to 201B7 iPS cells, apart from a higher expression of retroviral KLF4 in 3F-iPS cells (Supplemental Figure S1). Evaluation of the methylation status of cytosine guanine dinucleotides (CpG) in the promoter regions of OCT3/4 was analyzed by bisulfite sequencing and compared to 4F-iPS parental dermal fibroblasts. 3F- and 4F-iPS cells were highly unmethylated in comparison (Figure 1K). Furthermore, the differentiation ability of both 3F- and 4F-iPS cells was examined in vitro. Floating embryoid bodies (EBs) were cultivated for 3 weeks from each iPS cell set, and then spontaneously differentiated onto gelatin coated dishes for 12 days. Attached cells were immunopositive for three germ cell layers' markers (β-III-tubulin, ectoderm; α-smooth muscle actin, mesoderm; α-fetoprotein, endoderm) (Figure 1L-1N′). Finally, to confirm the pluripotency of 4F-iPS cells in vivo, we transplanted 3F- and 4F-iPS cells into the testis of immunodeficient mice (NOD-SCID) to observe teratoma formation. Tumors formed eight weeks after injection and contained various tissues representing all three germ layers (ectoderm, mesoderm, and endoderm; Figure 1O–1Q′) and various tissues (Supplemental Figure S2). Collectively, these results confirmed the establishment of de novo iPS cells from human dermal fibroblasts.

Bottom Line: Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan.

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.

Show MeSH
Related in: MedlinePlus