Establishment of co‐culture of human induced pluripotent stem cell‐derived melanocytes and keratinocytes in vitro

Abstract

Dear Editor,We previously established human induced pluripotent stem (iPS) cell-derived melanocytes in large quantities within a short period and induced their differentiation and high melanogenic potency using original iPS cell-derived melanocyte medium (iDMM). 1 We used original iPS cell lines established from T cells in the blood of a healthy 40-year-old Japanese man and a Sendai virus vector. 1 In the present study, we established melanocytes derived from more general iPS cells (1383D6, HPS1006) obtained from the Japanese Center for iPS Cell Research and Application using advanced iDMM (Table S1). The iPS cells were maintained in StemFit medium (AHS, AK02N; Ajinomoto, Tokyo, Japan). We found that many pigmented cells developed from the border of human iPS cell nests within 10 days under advanced iDMM (Fig. 1a). The granules in them were strongly tyrosinase-positive as observed by confocal immunofluorescence laser microscopy, which suggested that they were mature melanocytes (Fig. 1b). Previous approaches to grow and differentiate iPS cells into melanocytes needed first to produce embryoid bodies and then to process them to differentiate into melanocytes, which took approximately 30 days. 2,3 The present approach further improves in production of large quantities within a short period and induces differentiation and high melanogenic potency in vitro. We examined the mRNA expression levels of NANOG, Pax3, MITF, TRP1 and tyrosinase in the human iPS cell-derived melanocytes using quantitative reverse transcription polymerase chain reaction (Table S2, Fig. 1c). Normal human adult epidermal melanocytes were purchased from Invitrogen Gibco (Carlsbad, CA, USA) as controls (Table S3).The mRNA expression level of the pluripotency marker NANOG in these iPS cell-derived melanocytes was significantly lower than in human iPS cells. In contrast, mRNA expression levels of four melanocyte-specific markers (Pax3, MITF, TRP1 and tyrosinase) in the iPS cell-derived melanocytes that we established from a line of human melanocytes were significantly higher than in human iPS cells. The melanocyte-specific marker mRNA expression levels in the iPS cell-derived melanocytes were similar to the levels found in normal human adult epidermal melanocytes (Table S4).

Surgical procedures and innovative approaches for vitiligo regenerative treatment and melanocytorrhagy

“…It has been demonstrated that induced pluripotent stem (iPS) cells have the ability to differentiate into multiple cell types in the body and have an unlimited growth potential. A number of approaches have been used to establish human iPS cell‐derived melanocytes 41–44 . We established human iPS cell‐derived melanocytes from T cells in the blood of healthy persons and vitiligo patients and a Sendai virus vector using original iPS cell‐derived melanocyte medium (advanced iDMM) (Figure 2).…”

“…We mechanically isolated human iPS cell‐derived melanocyte colonies from those dishes by picking them up gently with a pipette. These mechanically isolated colonies were then transferred to new gelatinous dishes coated with Matrigel and were cultured with advanced iDMM 43,44 . Previous approaches to grow and differentiate iPS cells into melanocytes needed first to produce embryoid bodies and then to process them to differentiate into melanocytes, 41,42 which took approximately 30 days.…”

“…To establish a melanocyte–keratinocyte co‐culture system, human iPS cell‐derived melanocytes produced by advanced iDMM and normal human adult keratinocytes were co‐cultured in mixed culture media (advanced iDMM + keratinocyte growth medium). Seeding ratio of melanocytes to keratinocytes is 1:10 and proper mix medium condition for a co‐culture system is a 1:2 ratio for melanocyte medium to keratinocyte medium 44 . The co‐culture model provides a more physiologically realistic condition to study the interaction between melanocytes and keratinocytes, which enables a reliable screening system for depigmenting compounds.…”

“…A number of approaches have been used to establish human iPS cell-derived melanocytes. [41][42][43][44] We established human iPS cell-derived melanocytes from T cells in the blood of healthy persons and vitiligo patients and a Sendai virus vector using original iPS cell-derived melanocyte medium (advanced iDMM) (Figure 2). The medium included stem cell factor (SCF), WNT3a, bone morphogenetic protein 4 (BMP4), endothelin 1, endothelin 3, α-melanocytestimulating hormone (αMSH), dibutyryl-cAMP (DBcAMP), Ltyrosine, transferrin, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and granulocyte macrophage colony-stimulating factor (GMCSF), and under fetal bovine serum-free culture conditions without supplementation by nonphysiologic agents.…”

“…These mechanically isolated colonies were then transferred to new gelatinous dishes coated with Matrigel and were cultured with advanced iDMM. 43,44 Previous approaches to grow differentiate iPS cells into melanocytes needed first to produce embryoid bodies and then to process them to differentiate into melanocytes, 41,42 which took approximately 30 days. In contrast, our new approach to utilize a Matrigel coating has shortened this differentiation process to 14 days in total.…”