Yu Inoue

Wnt/β-Catenin and Kit Signaling Sequentially Regulate Melanocyte Stem Cell Differentiation in UVB-Induced Epidermal Pigmentation

UV radiation is a well-known inducer of epidermal pigmentation that is utilized in therapy for vitiligo, one of the skin depigmentation disorders. Although it has been reported that melanocyte stem cells (McSCs) play essential roles in hair pigmentation, the relationship between McSCs and epidermal pigmentation remains unclear. Repetitive UVB irradiation on the dorsal skin of F1 mice of HR-1 × HR/De caused apparent epidermal pigmentation, and it was characterized by increase in the number of melanocytes. Interestingly, differentiation of McSCs into melanoblasts in hair follicles was followed by induction of epidermal melanocyte differentiation. Administration of a neutralizing antibody for Kit receptor that depletes resident melanoblasts could not suppress increased number of melanocytes. UVB irradiation also induced robust expression of Wnt7a as well as Kitl in epidermis, and β-catenin translocation into nucleus in McSCs. Intradermal injection of IWR-1 (inhibitor of Wnt response 1), a chemical inhibitor of β-catenin activation, and small interfering RNA (siRNA) against Wnt7a suppressed increase in the number of epidermal melanocytes. Taken altogether, it was demonstrated that Wnt7a triggered McSCs differentiation through β-catenin activation, and Kitl might induce following migration of melanoblasts to epidermis. These findings will help in developing therapeutic technologies for vitiligo and other pigmentary disorders.

ZIP2 Protein, a Zinc Transporter, Is Associated with Keratinocyte Differentiation

Background: Few studies have investigated the ZIP proteins specifically expressed in keratinocytes. Results: ZIP2 is highly expressed in differentiating keratinocytes, and their differentiation is inhibited by ZIP2 siRNA. Conclusion: ZIP2 is essential for the differentiation of keratinocytes. Significance: Understanding the regulation of keratinocyte differentiation by zinc and its transporters is crucial for developing new therapies against skin disease. Zinc is essential for the proper functioning of various enzymes and transcription factors, and its homeostasis is rigorously controlled by zinc transporters (SLC39/ZIP, importers; SLC30/ZnT, exporters). Skin disease is commonly caused by a zinc deficiency. Dietary and inherited zinc deficiencies are known to cause alopecia and the development of vesicular or pustular dermatitis. A previous study demonstrated that zinc played crucial roles in the survival of keratinocytes and their unique functions. High levels of zinc have been detected in the epidermis. Epidermal layers are considered to use a mechanism that preferentially takes in zinc, which is involved with the unique functions of keratinocytes. However, few studies have investigated the ZIP (Zrt- and Irt-like protein) proteins specifically expressed in keratinocytes and their functions. We explored the ZIP proteins specifically expressed in the epidermis and analyzed their functions. Gene expression analysis showed that the expression of ZIP2 was consistently higher in the epidermis than in the dermis. Immunohistochemistry analysis confirmed the expression of ZIP2 in differentiating keratinocytes. The expression of ZIP2 was found to be up-regulated by the differentiation induction of cultured keratinocytes. Intracellular zinc levels were decreased in keratinocytes when ZIP2 was knocked down by siRNA, and this subsequently inhibited the differentiation of keratinocytes. Moreover, we demonstrated that ZIP2 knockdown inhibited the normal formation of a three-dimensional cultured epidermis. Taken together, the results of this study suggest that ZIP2, a zinc transporter expressed specifically in the epidermis, and zinc taken up by ZIP2 are necessary for the differentiation of keratinocytes.

Age-related changes of p75 Neurotrophin receptor-positive adipose-derived stem cells

BACKGROUND The existence of multipotent stem cells in subcutaneous adipose tissue has been reported. We previously confirmed that p75 neurotrophin receptor (p75NTR; CD271)-positive cells in subcutaneous adipose tissue possessed multipotency, although changes of the characteristics in p75NTR-positive adipose-derived stem cells (ASCs) with aging remain unclear. OBJECTIVE To investigate the effect of aging on p75NTR-positive ASCs. METHODS The number of p75NTR-positive ASCs in subcutaneous adipose tissue of ICR mice aged 3-24 weeks was analyzed by immunostaining and flow cytometry. Subsequently, the cells were isolated and their ability to attach to the cell culture dish, proliferation rate (doubling time) and the expression of senescence-associated beta-galactosidase (SA-beta gal), a cellular senescence marker, were assessed. Age-related changes in the differentiation potential of p75NTR-positive cells in adipogenic, osteogenic, chondrogenic and myogenic lineage were also investigated. RESULTS The number of ASCs per unit of tissue weight in adipose tissue and the attachment rate of isolated cells decreased with aging. No difference in the cell proliferation rate and the percentage of SA-beta gal-positive cells was detected. Although the efficacy of differentiation into adipogenic and osteogenic lineages slightly decreased with aging, the differentiation potential into chondrogenic and myogenic lineages was not changed. CONCLUSION The number of ASCs per unit of tissue weight decreased in aged mice. However, the cells possessed proliferation and differentiation potentials almost equal to those of young mice even though the differentiation potentials showed a tendency of decrease. These results raise the possibility that stem cell functions, self-renewal and multipotency, are maintained regardless of aging.

Melanocyte stem cells express receptors for canonical Wnt-signaling pathway on their surface.

It has been reported that melanocytes play important roles in skin and hair pigmentation and are differentiated from melanocyte stem cells (MSCs) residing in the bulge area of hair follicles. Recently, interest has been growing in MSCs because regulation of the upstream of differentiated melanocytes is essential for the determination of skin and hair pigmentation; however, their precise characteristics remain to be elucidated. The aim of this study is to explore cell-surface markers expressed on MSCs in order to understand their characteristics. To explore genes specifically expressed in the bulge region, we classified a hair follicle into four areas, hair bulb, hair bulb to bulge (lower bulge), bulge, and epidermis to bulge (upper bulge), and collected these areas from back skin sections of C57BL/6 mice by laser microdissection. Real-time RT-PCR performed on these areas revealed that Frizzled (Fzd)-4, Fzd7, low density lipoprotein receptor-related protein 5 (Lrp5), and Lrp6, receptors for Wnt molecules, were expressed higher in the bulge area than other areas. Furthermore, FACS analysis showed that populations of Fzd4(+) cells and Fzd7(+) cells were different from those of Kit(+) cells (precursor of melanocytes: melanoblasts). Fzd4(+) and Fzd7(+) cells isolated by FACS required a longer culture period to differentiate into mature melanocytes than Kit(+) cells. Up-regulation of mRNA expressions of melanocyte markers (dopa chrometautomerase: Dct, tyrosinase: Tyr, tyrosinase-related protein 1: Tyrp1) was observed in Fzd4(+) and Fzd7(+) cells following Kit(+) cells during differentiation. These results suggested that Fzd4(+) and Fzd7(+) cells were more immature than melanoblasts, therefore raising the possibility that Fzd4(+) and Fzd7(+) cells are MSCs.

Accelerated differentiation of melanocyte stem cells contributes to the formation of hyperpigmented maculae.

It has been reported that the abnormal regulation of melanocyte stem cells (McSCs) causes hair greying; however, little is known about the role of McSCs in skin hyperpigmentation such as solar lentigines (SLs). To investigate the involvement of McSCs in SLs, the canonical Wnt signalling pathway that triggers the differentiation of McSCs was analysed in UVB‐induced delayed hyperpigmented maculae in mice and human SL lesions. After inducing hyperpigmented maculae on dorsal skin of F1 mice of HR‐1× HR/De, which was formed long after repeated UVB irradiation, the epidermal Wnt1 expression and the number of nuclear β‐catenin‐positive McSCs were increased as compared to non‐irradiated control mice. Furthermore, the expression of dopachrome tautomerase (Dct), a downstream target of β‐catenin, was significantly upregulated in McSCs of UVB‐irradiated mice. The Wnt1 expression and the number of nuclear β‐catenin‐positive McSCs were also higher in human SL lesions than in normal skin. Recombinant Wnt1 protein induced melanocyte‐related genes including Dct in early‐passage normal human melanocytes (NHEMs), an in vitro McSC model. These results demonstrate that the canonical Wnt signalling pathway is activated in SL lesions and strongly suggest that the accelerated differentiation of McSCs is involved in SL pathogenesis.

Bimodal effect of retinoic acid on melanocyte differentiation identified by time-dependent analysis.

Retinoic acid (RA) is considered to control melanocytes; however, its precise mechanism remains unclear because of a bimodal effect, which promotes or inhibits melanin synthesis depending on the cell type, culture condition of melanocytes and skin conditions. In this study, we examined the effects of RA throughout each stage of differentiation of melanocytes using a mouse embryonic stem cell culture system to induce melanocytes. The results showed that RA has significantly different effects depending on the stage of differentiation of melanocytes. More specifically, RA promoted differentiation in earlier stages, wherein embryonic stem cells became melanoblasts via neural crest cells, and inhibited differentiation in later stages, wherein melanoblasts became melanocytes. It was revealed for the first time that melanocytes show markedly different reactions to RA depending on the stage of differentiation.

Comprehensive analysis of melanogenesis and proliferation potential of melanocyte lineage in solar lentigines

BACKGROUND Solar lentigines (SLs) are characterized by hyperpigmented macules, commonly seen on sun-exposed areas of the skin. Although it has been reported that an increase in the number of melanocytes and epidermal melanin content was observed in the lesions, the following questions remain to be answered: (1) Is acceleration of melanogenesis in the epidermis caused by an increased number of melanocytes or the high melanogenic potential of each melanocyte? (2) Why does the number of melanocytes increase? OBJECTIVE To elucidate the pathogenic mechanism of SLs by investigating the number, melanogenic potential and proliferation status of the melanocyte lineage in healthy skin and SL lesions. METHODS Immunostaining for melanocyte lineage markers (tyrosinase, MART-1, MITF, and Frizzled-4) and a proliferation marker, Ki67, was performed on skin sections, and the obtained images were analyzed by image analysis software. RESULTS The expression level of tyrosinase to MART-1 of each melanocyte was significantly higher in SL lesions than healthy skin. The numbers of melanocytes in the epidermis, melanoblasts in the hair follicular infundibulum and melanocyte stem cells in the bulge region were increased in SL; however, no significant difference was observed in the Ki67-positive rate of these cells. CONCLUSION The melanogenic potential of each melanocyte was elevated in SL lesions. It was suggested that the increased number of melanocytes in the SL epidermis might be attributed to the abnormal increase of melanocyte stem cells in the bulge.

Major amino acids in collagen hydrolysate regulate the differentiation of mouse embryoid bodies.

To take advantage of the therapeutic potential of embryonic stem cells (ESCs), it is necessary to regulate their differentiation in response to defined factors. In this study, in order to explore novel molecules that regulate the differentiation of ESCs, we investigated whether collagen hydrolysate, collagen-characteristic amino acids, glycine (Gly), l-proline and trans-4-hydroxy-l-proline (l-Hyp); or dipeptides, proline-hydroxyproline and hydroxyproline-glycine regulate the differentiation of mouse embryoid bodies (EBs). We identified that treatment with collagen hydrolysate or Gly repressed the expression of the mesendodermal markers, Brachyury and Foxa2 in EBs and maintained the undifferentiated state of mESCs in a feeder-free monolayer culture. In contrast, l-Hyp promoted the expression of Brachyury, Mixl1, Gsc and Foxa2 in EBs. And the treatment with l-Hyp promoted cardiac differentiation within EBs, which was proven by the spontaneous contraction of cardiomyocytes and the expression of the cardiac markers, α-MHC, MLC-2v and Nkx2.5. Results suggest that l-Hyp is a promising new inducer for reproducible and efficient differentiation of mesendoderm lineages.

Aberrant Gene Expression and Sexually Incompatible Genomic Imprinting in Oocytes Derived from XY Mouse Embryonic Stem Cells In Vitro

Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) in vivo and in vitro. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis in vivo; however, in vitro differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development. To address this issue, a mouse ESC line, capable of producing follicle-like structures (FLSs) efficiently, was established to investigate their properties using conventional molecular biological methods. The results revealed that the ESC-derived FLSs were morphologically similar to ovarian primary-to-secondary follicles but never formed an antrum; instead, the FLSs eventually underwent abnormal development or cell death in culture, or formed teratomas when transplanted under the kidney capsule in mice. Gene expression analyses demonstrated that the FLSs lacked transcripts for genes essential to late folliculogenesis, including gonadotropin receptors and steroidogenic enzymes, whereas some other genes were overexpressed in FLSs compared to the adult ovary. The E-Cadherin protein, which is involved in cell-to-cell interactions, was also expressed ectopically. Remarkably, it was seen that oocyte-like cells in the FLSs exhibited androgenetic genomic imprinting, which is ordinarily indicative of male GCs. Although the FLSs did not express male GC marker genes, the DNA methyltransferase, Dnmt3L, was expressed at an abnormally high level. Furthermore, the expression of sex determination factors was ambiguous in FLSs as both male and female determinants were expressed weakly. These data suggest that the developmental dysfunction of the ESC-derived FLSs may be attributable to aberrant gene expression and genomic imprinting, possibly associated with uncertain sex determination in culture.

Lignin Induces ES Cells to Differentiate into Neuroectodermal Cells through Mediation of the Wnt Signaling Pathway

Embryonic stem cells (ES cells) are characterized by their pluripotency and infinite proliferation potential. Ever since ES cells were first established in 1981, there have been a growing number of studies aimed at clinical applications of ES cells. In recent years, various types of differentiation inducement systems using ES cells have been established. Further studies have been conducted to utilize differentiation inducement systems in the field of regenerative medicine. For cellular treatments using stem cells including ES cells, differentiation induction should be performed in a sufficient manner to obtain the intended cell lineages. Lignin is a high-molecular amorphous material that forms plants together with cellulose and hemicelluloses, in which phenylpropane fundamental units are complexly condensed. Lignin derivatives have been shown to have several bioactive functions. In spite of these findings, few studies have focused on the effects of lignin on stem cells. Our study aimed to develop a novel technology using lignin to effectively induce ES cells to differentiate into neuroectodermal cells including ocular cells and neural cells. Since lignin can be produced at a relatively low cost in large volumes, its utilization is expected for more convenient differentiation induction technologies and in the field of regenerative medicine in the future.