| Budget Amount *help |
¥3,950,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2007: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2006: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
Several putative biomarkers have been suggested for identifying murine follicular stem cells; however, human hair follicles have distinct biomarker expression, and follicular stem cell isolation methods have not been established. To characterize human follicular bulge cells and their progeny, we examined the expression of stem cell-associated (K15, CD200, CD34, CD271) and other biomarkers (K1, K14, CD29 and CD49f) in immunohistological sections of the human epidermis and follicular outer root sheath (ORS). We also examined freshly-isolated and cultured epidermal or follicular cells with single-and multi-color flow cytometry or immuncytochemistry. After sorting cells by CD200, CD34, and forward scatter (FSC) values (cell size), colony-forming assays were performed. We found that biomarkers were differentially expressed in the epidermis and ORS. Basal bulge cells were mainly K15+CD200+CD34-CD271-, and suprabasal cells were K15+CD200+CD34-CD271-. We categorized follicular cells into nine
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subpopulations according to biomarker expression profiles. The CD200+CD34-bulge cells had much higher colony-forming abilities than the CD34+population, and was divided into two subpopulations: a CD200+CD34-FSChigh (K15-rich, basal) and a CD200+CD34-FSClow (K15-poor, suprabasal) population. The former formed fewer but larger-sized colonies than the latter. Follicular epithelial cell cultivation resulted in loss of K15, CD200, CD34, and CD271 expression, but maintenance of K14, CD29, and CD49f expression. We found that the bulge contained two populations with different localizations, cell sizes, and colony forming abilities. We showed that K15, CD200, CD34, and CD271 were useful biomarkers for characterizing freshly isolated human follicular epithelial cells in diverse stages of differentiation.
Dermal papilla cells (DPCs) in the mammalian hair follicle have been shown to develop hair follicles through epithelial-mesenchymal interactions. A cell therapy to regenerate human hair is theoretically possible by expanding autologous human DPCs and transplanting them into bald skin, though much remains to be overcome before clinical success. In this study, we compared gene signatures of human DPCs at different passages and human dermal fibroblasts, and found TGF-β2 to be highly expressed in cultured human DPCs. Keratinocyte conditioned medium, which is known to help preserve the hair inducing capacity of hDPCs, upregulated TGF-β2 expression of human DPCs and also enhanced their alkaline phosphatase activity, a known index for hair inductive capacity. Through screening of components secreted from keratinocytes, the vitamin D3 analogue was found to promote TGF-β2 and Wnt10b expression and alkaline phosphatase activity of human DPCs. In animal hair folliculogenesis models using rat epidermis and expanded human DPCs, inhibition of TGF-β2 signaling at the ligand or receptor level significantly impaired hair folliculogenesis and maturation. These results suggest an important role for TGF-β2 in hair follicle morphogenesis and provide insights into the establishment of future cell therapies for hair regrowth by transplanting expanded DPCs. Less
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