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The hair follicle is a complex of mesenchymal and epithelial cells acquiring different properties and characteristics responsible for fulfilling its inductive and regenerative role. The epidermal and dermal crosstalk induces morphogenesis and maintains hair follicle cycling properties. The hair follicle is enriched with pluripotent stem cells, where dermal papilla (DP) cells and dermal sheath (DS) cells constitute the dermal compartment and the epithelial stem cells existing in the bulge region exert their regenerative role by mediating the epithelial-mesenchymal interaction (EMI). Many studies have developed and focused on various methods to optimize the EMI through in vivo and in vitro approaches for hair regeneration. The culturing of human hair mesenchymal cells resulted in the loss of trichogenicity and inductive properties of DP cells, limiting their potential application in de novo hair follicle generation in vivo. Epithelial stem cells derived from human hair follicles are challenging to isolate and culture, making it difficult to obtain enough cells for hair regeneration purposes. Mesenchymal stem cells and epithelial stem cells derived from human hair follicles lose their ability to form hair follicles during culture, limiting the study of hair follicle formation in vivo. Therefore, many attempts and methods have been developed to overcome these limitations. Here, we review the possible and necessary cell methods and techniques used for human hair follicle regeneration and the restoration of hair follicle cell inductivity in culture.

Abstract Background Hair follicle (HF) formation and growth are sustained by epithelial-mesenchymal interaction via growth factors and cytokines. Pivotal roles of FGFs on HF regeneration and neogenesis have been reported mainly in rodent models. FGF expression is regulated by upstream pathways, represented by canonical WNT signaling; however, how FGFs influence on human folliculogenesis remains elusive. The aim of this study is to assess if human scalp-derived fibroblasts (sFBs) are able to modulate their FGF expression profile in response to WNT activation and to evaluate the influence of WNT-activated or suppressed FGFs on folliculogenesis. Methods Dermal papilla cells (DPCs), dermal sheath cells (DSCs), and sFBs were isolated from the human scalp and cultured independently. The gene expression profile of FGFs in DPCs, DSCs, and sFBs and the influence of WNT activator, CHIR99021, on FGF expression pattern in sFBs were evaluated by reverse transcription polymerase chain reaction, which were confirmed at protein level by western blotting analysis. The changes in the expression of DPC or keratinocyte (KC) biomarkers under the presence of FGF7 or 9 were examined in both single and co-culture assay of DPCs and/or KCs. The influence of FGF 7 and FGF 9 on hair morphogenesis and growth was analyzed in vivo using mouse chamber assay. Results In single culture, sFBs were distinguished from DPCs and DSCs by relatively high expression of FGF5 and FGF 18, potential inducers of hair cycle retardation or catagen phase. In WNT-activated state, sFBs downregulated FGF7 while upregulating FGF9, a positive regulator of HF morphogenesis, FGF16 and FGF 20 belonging to the same FGF subfamily. In addition, CHIR99021, a WNT activator, dose-dependently modulated FGF7 and 9 expression to be folliculogenic. Altered expressions of FGF7 and FGF9 by CHIR99021 were confirmed at protein level. Supplementation of FGF9 to cultured DPCs resulted in upregulation of representative DP biomarkers and this tendency was sustained, when DPCs were co-cultured with KCs. In mouse chamber assay, FGF9 increased both the number and the diameter of newly formed HFs, while FGF7 decreased HF diameter. Conclusion The results implied that sFBs support HF formation by modulating regional FGF expression profile responding to WNT activation.

The dermal sheath (DS) is a population of mesenchyme-derived skin cells with emerging importance for skin homeostasis. The DS includes hair follicle dermal stem cells, which exhibit self-renewal and serve as bipotent progenitors of dermal papilla (DP) cells and DS cells. Upon aging, stem cells exhibit deficiencies in self-renewal and their number is reduced. While the DS of mice has been examined in considerable detail, our knowledge of the human DS, the pathways contributing to its self-renewal and differentiation capacity and potential paracrine effects important for tissue regeneration and aging is very limited. Using single-cell RNA sequencing of human skin biopsies from donors of different ages we have now analyzed the transcriptome of 72,048 cells, including 50,149 fibroblasts. Our results show that DS cells that exhibit stem cell characteristics were lost upon aging. We further show that HES1, COL11A1, MYL4 and CTNNB1 regulate DS stem cell characteristics. Finally, the DS secreted protein Activin A showed paracrine effects on keratinocytes and dermal fibroblasts, promoting proliferation, epidermal thickness and pro-collagen production. Our work provides a detailed description of human DS identity on the single-cell level, its loss upon aging, its stem cell characteristics and its contribution to a juvenile skin phenotype.

The dermal sheath (DS) is a population of mesenchyme-derived skin cells with emerging importance for skin homeostasis. The DS includes hair follicle dermal stem cells, which exhibit self-renewal and serve as bipotent progenitors of dermal papilla (DP) cells and DS cells. Upon aging, stem cells exhibit deficiencies in self-renewal and their number is reduced. While the DS of mice has been examined in considerable detail, our knowledge of the human DS, the pathways contributing to its self-renewal and differentiation capacity and potential paracrine effects important for tissue regeneration and aging is very limited. Using single-cell RNA sequencing of human skin biopsies from donors of different ages we have now analyzed the transcriptome of 72,048 cells, including 50,149 fibroblasts. Our results show that DS cells that exhibit stem cell characteristics were lost upon aging. We further show that HES1 , COL11A1 , MYL4 and CTNNB1 regulate DS stem cell characteristics. Finally, the DS secreted protein Activin A showed paracrine effects on keratinocytes and dermal fibroblasts, promoting proliferation, epidermal thickness and pro-collagen production. Our work provides a detailed description of human DS identity on the single-cell level, its loss upon aging, its stem cell characteristics and its contribution to a juvenile skin phenotype. [ABSTRACT FROM AUTHOR]

BACKGROUND: Noggin is an inhibitory molecule of bone morphogenetic protein (BMP) signaling, which can bind to BMP2/4 to form a complex, thereby blocking BMP signaling and affecting normal development. Some studies have suggested that dermal sheath cells are a kind of dermal stem cells that can self-renew for a long time, and participate in the formation of dermal papilla and dermal sheath during hair follicle regeneration. During the development of hair follicles, Noggin is involved in the induction of hair follicle regeneration, and its loss leads to a decrease in hair follicle number and slows hair follicle growth. However, little is known about the Noggin in the study of hair follicle dermal sheaths. OBJECTIVE: To study the biological function of Noggin protein in the dermal sheath of hair follicles. METHODS: The dermal sheath specific α-SMA-Cre ERT2 tool mice were used to specifically overexpress Noggin protein in the dermal sheath. At 8 and 9 days after birth, αSMA-CreER; pMES-Noggin mice in the experimental group and αSMA-CreER mice in the control group were injected with 4-hydroxytamoxifen. At 21, 23, and 28 days after birth, mouse skin tissues were obtained. Hematoxylin-eosin staining was used to observe hair follicle growth and subcutaneous fat layer thickness, and immunohistochemical staining was used to analyze cell phenotype. RESULTS AND CONCLUSION: Based on the findings of hematoxylin-eosin staining, we found that the growth of hair follicle was not affected, but the growth and development of subcutaneous adipose tissue of hair follicles were seriously impacted, presenting with the subcutaneous fat layer becoming thinned. The immunohistochemical results indicated that the decrease of BMP signaling may result in the thinning of the hair follicle fat layer. This discovery expands the understanding of dermal sheath cells and Noggin protein, and provides an important basis for more accurate understanding of hair follicle regeneration and tissue growth mechanisms. [ABSTRACT FROM AUTHOR]

Objective: To explore the research progress of hair follicle and related stem cells in scar-free skin healing in recent years., Methods: The literature related to hair follicle and related stem cells, wound healing, and scar formation in recent years was extensively reviewed and summarized from the aspects of cell function and molecular mechanism., Results: Scar tissue after wound healing treated with hair follicle transplantation and related stem cell therapy is more mild or even without scar formation. The cell types and molecular mechanisms of the above phenomena are complex, and the bone morphogenetic protein signal transduction pathway and Wnt signal transduction pathway are strongly correlated., Conclusion: The research of hair follicle and related stem cells in scar-free skin healing is at the initial stage at present. Strengthening the mechanism research may provide new ideas for the treatment of wound and scar.

We investigated the effect of oxygen tension on the proliferation and hair-inductive capacity of human dermal papilla cells (DPCs) and dermal sheath cells (DSCs). DPCs and DSCs were separately obtained from human hair follicles and each cultured under atmospheric/hyperoxic (20% O 2 ), physiological/normoxic (6% O 2 ), or hypoxic (1% O 2 ) conditions. Proliferation of DPCs and DSCs was highest under normoxia. Compared with hyperoxia, hypoxia inhibited proliferation of DPCs, but enhanced that of DSCs. In DPCs, hypoxia downregulated the expression of hair-inductive capacity-related genes, including BMP4 , LEF1 , SOX2 , and VCAN . In DSCs, both normoxia and hypoxia upregulated SOX2 expression, whereas hypoxia downregulated BMP4 expression. Microarray analysis revealed that normoxia increased the expression of pluripotency-related genes, including SPRY , NR0B1 , MSX2 , IFITM1 , and DAZL , compared with hyperoxia. In an in vivo hair follicle reconstitution assay, cultured DPCs and DSCs were transplanted with newborn mouse epidermal keratinocytes into nude mice using a chamber method. In this experiment, normoxia resulted in the most efficient induction of DPC hair follicles, whereas hypoxia caused the most efficient induction and maturation of DSC hair follicles. These results suggest that application of physiological/hypoxic oxygen tension to cultured human DSCs enhances proliferation and maintenance of hair inductivity for skin engineering and clinical applications. Impact statement Dermal sheath cells (DSCs) and dermal papilla cells (DPCs) are useful cell sources for cell-based regenerative therapy. This is the first report to describe that low-oxygen conditions are better for DSCs. Normoxic and hypoxic culture of DSCs is beneficial for expanding these hair follicular cells and advancing development of cell-based therapy for both wound healing and hair regeneration. The current study supports that optimized oxygen tension can be applied to use expanded human DPCs and DSCs for skin engineering and clinical applications.

We developed a nude rat model for determining the capacity of trichogenous cells to restore in vivo-damaged hair follicles (HFs). A surgical scalpel was inserted into the rat's dermis to generate the in vivo-damaged pelage HFs, the HFs whose lower parts were lost, but the upper parts containing sebaceous and bulge regions remained intact. Dermal papilla cells (DPCs) and dermal sheath cells (DSCs) from EGFP transgenic rat vibrissae were propagated in culture, and each alone (DPC or DSC) or a mixture (DPC/DSC) was transplanted into the intradermal path made by a scalpel. It was found that the in vivo-damaged HFs had hair self-restoration ability, and the transplanted trichogenic dermal cells prominently enhanced this ability, DPC/DSC transplants being more effective in enhancement than DPC or DSC alone. The restored bulbs contained EGFP-positive cells, shed their original straight shafts, generated new shafts, and further developed into hairs with a sebaceous gland and bulge structures by ~6 weeks post-transplantation. Compared to the preceding animal models, this model is less invasive, requires fewer donor cells and allows repeated operations with higher reproducibility and accuracy. The present study suggests that conditions causing in situ-damaged HFs, such as androgenic alopecia, in which HFs are damaged and miniaturized, can be restored by functional trichogenous dermal cell transplantation therapy. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)