Induced pluripotent stem cell‐derived melanocyte precursor cells undergoing differentiation into melanocytes

Induced pluripotent stem cell (iPSC) technology offers a novel approach for conversion of human primary fibroblasts into melanocytes. During attempts to explore various protocols for differentiation of iPSCs into melanocytes, we found a distinct and self‐renewing cell lineage that could differentiate into melanocytes, named as melanocyte precursor cells (MPCs). The MPCs exhibited a morphology distinctive from that of melanocytes, in lacking either the melanosomal structure or the melanocyte‐specific marker genes MITF, TYR, and SOX10. In addition, gene expression studies in the MPCs showed high‐level expression of WNT5A, ROR2, which are non‐canonical WNT pathway markers, and its related receptor TGFβR2. In contrast, MPC differentiation into melanocytes was achieved by activating the canonical WNT pathway using the GSK3β inhibitor. Our data demonstrated the distinct characteristic of MPCs' ability to differentiate into melanocytes, and the underlying mechanism of interfacing between canonical WNT signaling pathway and non‐canonical WNT signaling pathway.     

Inhibition of adipocytogenesis by canonical WNT signaling in human mesenchymal stem cells

The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study, we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPARγ2, lipoprotein lipase, and adipsin increased during adipocytogenesis of hMSCs. Simultaneously, the expression of canonical WNT2, 10B, 13, and 14 decreased, whereas non-canonical WNT4 and 11 increased, and WNT5A was unchanged. A small molecule WNT mimetic, SB-216763, increased accumulation of β-catenin protein, inhibited induction of WNT4 and 11 and inhibited adipocytogenesis. In contrast, knockdown of β-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells.     

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.     

Coupling of the radiosensitivity of melanocyte stem cells to their dormancy during the hair cycle

Current studies have revealed that stem cells are more radiosensitive than mature cells. As somatic stem cells are mostly kept in a quiescent state, this conflicts with Bergonié and Tribondeau's law that actively mitotic cells are the most radiosensitive. In this study, we focused on hair graying to understand the stress‐resistance of melanocyte stem cells (McSCs). We used Dct‐H2B‐GFP transgenic mice which enables the stable visualization of McSCs and an anti‐Kit monoclonal antibody which selectively eradicates amplifying McSCs. The results demonstrate that quiescent McSCs are rather radiosensitive, but the coexistence of non‐quiescent McSCs provides the stem cell pool with radioresistance. The irradiated quiescent McSCs prematurely differentiate in the niche upon their activation without sufficiently renewing themselves for cyclic hair pigmentation. These data indicate that tissue radiosensitivity is largely dependent on the state of somatic stem cells under their local microenvironment.     

CXCR7 mediates SDF1‐induced melanocyte migration

Melanoblasts are derived from the neural crest and migrate to the dermal/epidermal border of skin and hair bulges. Although melanoblast migration during embryogenesis has been well investigated, there are only a few reports regarding the migration of mature melanocytes. Here, we demonstrate that a chemokine, stromal‐derived factor‐1 (SDF1, also known as CXCL12), and one of its receptor CXCR7 regulate normal human epidermal melanocyte (NHEM) migration. We found that SDF1 induces the directional migration of NHEMs. Interestingly, although both CXCR4 and CXCR7 are expressed in NHEMs, blockade of CXCR4 using a CXCR4‐specific neutralizing antibody did not exert any influence on the SDF1‐induced migration of NHEMs, whereas blockade of CXCR7 using a CXCR7‐specific neutralizing antibody did influence migration. Furthermore, SDF1‐induced NHEMs migration exhibited the early hallmark events of CXCR7 signaling associated with MAP kinase activation. It is known that the phosphorylation of ERK through CXCR7 signaling is mediated by β‐arrestins. The treatment of NHEMs with SDF1 resulted in the phosphorylation of ERK in a β‐arrestin 2‐dependent manner. These results suggest that melanocytes may have a unique mechanism of migration via SDF1/CXCR7 signaling that is different from that of other cell types.     

Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

Epithelial–mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin–vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.     

Histidase function in human epidermal cells

The activity of histidase was studied in (1) epidermal tissue scraped from human infant foreskin, (2) fibroblast-like cells in monolayer serial culture from human foreskin, and (3) epithelial-like (epidermal) outgrowth from foreskin primary explants. Foreskin epidermal tissue without in vitro culture and epidermal outgrowth in primary culture from explants of foreskin showed equivalent mean levels of histidase activity, 5.22 × 10^?3 and 5.01 × 10^?3 μMoles urocanic acid produced per milligram protein per minute. Under the same assay conditions, there was no measurable histidase activity in cultured fibroblast-like cells from foreskin at various times after subculture. The K_m for enzyme from human foreskin epidermal tissue ranged between 2 and 5 × 10^?3 M histidine. Ability to demonstrate the presence or absence of this tissue-specific enzyme function in cultured cells suggests a useful means for studying differentiation, as well as a more precise way to identify epidermal origin of cultured cell types than morphological characteristics alone would permit.     

Epigenetic loss of putative tumor suppressor SFRP3 correlates with poor prognosis of lung adenocarcinoma patients

Secreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing of SFRP3 in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate SFRP3 expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, SFRP3 mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of SFRP3 in LUAD and LUSC patients. Moreover, DNA hypermethylation of SFRP3 was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of SFRP3 expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and in vitro demethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of CyclinD1 expression in vitro. Our results indicate that SFRP3 acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.     

BMP4 is required for the initial expression of MITF in melanocyte precursor differentiation from embryonic stem cells

Although the differentiation of melanoblasts to melanocytes is known to depend on many distinct factors, it is still poorly understood which factors lead to the induction of melanoblasts. To determine which factors might induce melanoblasts, we examined a set of candidate factors for their ability to induce expression of MITF, a master regulator of melanoblast development, in an ES cell-based melanocyte differentiation system. It appears that BMP4 is capable of inducing MITF expression in stem cells. In contrast, a number of other factors normally implicated in the development of the melanocyte lineage, including WNT1, WNT3a, SCF, EDN3, IGF1, PDGF, and RA, cannot induce MITF expression. Nevertheless, BMP4 alone does not allow MITF-expressing precursors to become differentiated melanocytes, but the addition of EDN3 further promotes differentiation of the precursors into mature melanocytes. Our results support a model in which BMP4 induces MITF expression in pluripotent stem cells and EDN3 subsequently promotes differentiation of these MITF expressing cells along the melanocyte lineage.     

Sodium-N-butyrate induces cytoskeletal rearrangements and formation of cornified envelopes in cultured adult human keratinocytes

Abstract The technique developed in our laboratory allows us to culture multilayered, stratified sheets of human keratinocytes, which can be used to cover the burn wounds of patients. Organization of cells in these cultures resembles stratum germinativum and stratum spinosum but there are only a few fully keratinized cells and the stratum corneum is not developed. Since the fully differentiated sheets may offer additional advantages as epidermal transplants, attempts were made to enhance the degree of differentiation in vitro. In the present study sodium-N-butyrate (NaB) was used as a differentiating agent and its effect on the cell cycle and cytoarchitecture of epidermal cells was investigated. Incubation of keratinocytes in the presence of 2.5 mM NaB induced the appearance of enucleated cornified envelopes, covering approximately 70–80% of the surface of the cultures. Their appearance correlated with a decrease in expression of keratin K13, previously shown to be inhibited during terminal differentiation of human keratinocytes. An increase in transglutaminase transferase activity was also observed. The induction of cornified layers also correlated with an increase in the amount of microfilament (MF)-associated actin. NaB also induced changes in the cell cycle distribution of the keratinocyte cultures. A decrease in the proportion of S and G_1B phase cells was paralleled by an increase in G_1A cells, maximally expressed 30–48 h following addition of the inducer. Interestingly, NaB also induced a cell arrest in G₂ phase. These cell cycle perturbations preceded the onset of keratinocyte differentiation. The results indicate that the enhanced differentiation of human keratinocytes in the presence of NaB may serve as a means to produce epidermal sheets with improved properties for transplantation in a clinical setting. It also serves as an in vitro model system to study the interrelationships between biochemical events and cell cycle changes accompanying differentiation.     

Cellular and molecular mechanisms controlling the migration of melanocytes and melanoma cells

During embryonic development in vertebrates, the neural crest‐derived melanoblasts migrate along the dorsolateral axis and cross the basal membrane separating the dermis from the epidermis to reach their final location in the interfollicular epidermis and epidermal hair follicles. Neoplastic transformation converts melanocytes into highly invasive and metastatic melanoma cells. In vitro, these cells extend various types of protrusions and adopt two interconvertible modes of migration, mesenchymal and amoeboid, driven by different signalling molecules. In this review, we describe the major contributions of natural mouse mutants, mouse models generated by genetic engineering and in vitro culture systems, to identification of the genes, signalling pathways and mechanisms regulating the migration of normal and pathological cells of the melanocyte lineage, at both the cellular and molecular levels.     

Effect of keratinocytes on regulation of melanogenesis in culture of melanocytes

Melanocytes are the melanin-producing cells by melanogenesis, and the pigment melanin is primarily responsible for the color of skin. These cells contain dendrites that are in close contact with neighboring keratinocytes. Keratinocytes produce and secrete factors that regulate the proliferation and melanogenesis of melanocytes in vitro. Therefore, adopting only melanocyte pure culture may not clearly reflect the skin physiology in vivo. In this study, we applied a two-culture model using melanocytes and keratinocytes from human skin, such as melanocyte pure culture and melanocyte co-culture with keratinocyte. And then, there was compared the responses of melanocytes under different culture conditions (treatment with arbutin, MSH-α and UV-B irradiation). The results show that there was no significant difference in melanocyte proliferation and melanogenesis between arbutin and MSH-α treatment. However, the co-culture model was more stable than the pure culture model in terms of melanocyte proliferation and melanogenesis upon UV-B irradiation. Therefore, the co-culture model was superior to the pure culture as a useful method for the study of melanocytes and epidermal melanin unit.     

Secreted Frizzled related protein-4 (sFRP4) promotes epidermal differentiation and apoptosis

The skin provides vital protection from infection and dehydration. Maintenance of the skin is through a constant program of proliferation, differentiation and apoptosis of epidermal cells, whereby proliferating cells in the basal layer differentiating to form the keratinized, anucleated stratum corneum. The WNT signalling pathway is known to be important in the skin. WNT signalling has been shown to be important both in epidermal development and in the maintenance and cycling of hair follicles and epidermal stem cells. However, the precise role for this pathway in epidermal differentiation remains unknown.We investigated the role of the WNT signalling inhibitor sFRP4 in epidermal differentiation. sFRP4 is expressed in both normal skin and keratinocytes in culture. Expression of sFRP4 mRNA and protein increases with keratinocyte differentiation and apoptosis, whilst exposure of keratinocytes to exogenous sFRP4 promotes apoptosis and expression of the terminal differentiation marker Involucrin.These data suggest sFRP4 promotes epidermal differentiation.