Large-scale isolation of functional dermal papilla cells using novel surface marker LEPTIN Receptor.

Dermal papilla (DP) cells regulate hair follicle epithelial cells and melanocytes by secreting functional factors, playing a key role in hair follicle morphogenesis and hair growth. DP cells can reconstitute new hair follicles and induce hair regeneration, providing a potential therapeutic strategy for treating hair loss. However, current methods for isolating DP cells are either inefficient (physical microdissection) or only applied to genetically labeled mice. We systematically screened for the surface proteins specifically expressed in skin DP using mRNA expression databases. We identified two antibodies against receptors LEPTIN Receptor (LEPR ) and Scavenger Receptor Class A Member 5 (SCARA5) which could specifically label and isolate DP cells by flow cytometry from mice back skin at the growth phase. The sorted LEPR+ cells maintained the DP characteristics after culturing in vitro, expressing DP marker alkaline phosphatase and functional factors including RSPO1/2 and EDN3, the three major DP secretory factors that regulate hair follicle epithelial cells and melanocytes. Furthermore, the low-passage LEPR+ DP cells could reconstitute hair follicles on nude mice using chamber graft assay when combined with epithelial stem cells. The method of isolating functional DP cells we established here lays a solid foundation for developing DP cell-based therapy.

EGFL6 expression in hair follicle central isthmus is dependent on the presence of terminal Schwann cells.

Hair follicle central isthmus is surrounded by dense nerve endings and terminal Schwann cells (TSCs), forming a specialized sensory structure called lanceolate complexes. Extracellular matrix protein EGFL6 expressed from epidermis has been found closely associated with lanceolate complexes and important for proper alignment of nerve fibres and TSCs processes, and for proper response to light touch. However, how EGFL6 itself is specifically induced/deposited/maintained at the central isthmus remains to be elucidated. Previous reports and our results showed that nerve endings and TSCs docking at the central isthmus during hair follicle development occur before the specific depositing of EGFL6 protein. Furthermore, we found nude mice rarely maintain the lanceolate complex, and EGFL6 is lost in their aberrant hair follicle. Instead, reconstituted hair follicle in nude mice by stem cells chamber grafting assay expresses EGFL6 at the central isthmus area after hair follicle innervation. At last, long-term but not short-term cutaneous denervation leads to degeneration of TSCs and loss of EGFL6 expression. Together, our results demonstrate that EGFL6 expression in the central isthmus is dependent on the presence of TSCs, proposing that the interplay of epidermis and neuronal components is important for maintaining functional structure of lanceolate complexes.

Calcitonin gene-related peptide receptor Calcrl is enriched in hair follicles stem cells and differentially expressed in interfollicular epidermis in murine skin.

Calcitonin gene-related peptide (CGRP) and adrenomedullin are structurally similar neuropeptides acting as potent vasodilators of blood pressure and mediator of inflammation in skin. Revealing the expression pattern of their common receptor-Calcitonin gene-related peptide receptor (Calcrl) in their targeted cells is important to explain the functions of CGRP and adrenomedullin in skin. Our immunostaining results showed that Calcrl is enriched in hair follicles bulge stem cells and differentially expressed in basal stem cells of interfollicular epidermis. In addition, Calcrl expression in interfollicular epidermis is dependent on presence of nerve fibers. Long-term ablation of the murine cutaneous nerve leads to loss of Calcrl expression in interfollicular epidermis but not in hair follicle bulge stem cells. Our results demonstrate a tight interaction between neuronal components and epidermis, and indicates potential roles of Calcrl in epidermal stem cells.

Spatial association of SEMA3C with nerve endings/terminal Schwann cells in hair follicle isthmus region.

Nerve endings and terminal Schwann cells (TSCs) specifically and densely surround hair follicle at isthmus area, forming a neuromuscular-junction-like structure called lanceolate complex. The interplay between neuronal components and epidermis in this specialized structure enables hair to properly sense complex stimuli from environments. However, how nerves precisely attach to and innervate this specific region during development remains to be elucidated. Here, we demonstrate that SEMA3C, a secreted protein member of semaphorin family responsible for axonal guidance, is localized right below sebaceous gland and in close approximation with nerve endings and TSCs processes all through the entire hair cycle. SEMA3C protein is deposited outside of epithelial cells and its expression is independent on the presence of nerve endings/TSCs. SEMA3C is also found in portions of dermal papilla at growth phase. The tight spatial association of SEMA3C with lanceolate complex suggests that it might play roles in establishment and/or maintenance of the lanceolate complex in hair follicle.