EGFR marks a subpopulation of dermal mesenchymal cells highly expressing IGF1 which enhances hair follicle regeneration.

The skin harbours transcriptionally and functionally heterogeneous mesenchymal cells that participate in various physiological activities by secreting biochemical cues. In this study, we aimed to identify a new subpopulation of dermal mesenchymal cells that enhance hair follicle regeneration through a paracrine mechanism. Integrated single-cell RNA sequencing (scRNA-seq) data analysis revealed epidermal growth factor receptor (EGFR) as a marker of distinct fibroblast subpopulation in the neonatal murine dermis. Immunofluorescence staining and fluorescence-activated cell sorting (FACS) were used to validate the existence of the cell population in Krt14-rtTA-H2BGFP mouse. The difference of gene expression between separated cell subpopulation was examined by real-time PCR. Potential effect of the designated factor on hair follicle regeneration was observed after the application on excisional wounds in Krt14-rtTA-H2BGFP mouse. Immunofluorescence staining demonstrated the existence of dermal EGFR+ cells in neonatal and adult mouse dermis. The EGFR+ mesenchymal population, sorted by FACS, displayed a higher expression level of Igf1 (insulin-like growth factor 1). Co-localisation of IGF1 with EGFR in the mouse dermis and upregulated numbers of hair follicles in healed wounds following the application of exogenous IGF1 illustrated the contribution of EGFR+ cells in promoting wound-induced hair follicle neogenesis. Our results indicate that EGFR identifies a subpopulation of dermal fibroblasts that contribute to IGF1 promotion of hair follicle neogenesis. It broadens the understanding of heterogeneity and the mesenchymal cell function in skin and may facilitate the potential translational application of these cells.

An Engineered Dermal Substitute with Mesenchymal Stem Cells Enhances Cutaneous Wound Healing.

The treatment of refractory cutaneous wounds remains to be a clinical challenge. There is growing evidence to show that mesenchymal stem cells (MSCs) have great potential in promoting wound healing. However, the therapeutic effects of MSCs are greatly dampened by their poor survival and engraftment in the wounds. To address this limitation, in this study, MSCs were grown into a collagen-glycosaminoglycan (C-GAG) matrix to form a dermis-like tissue sheet, named engineered dermal substitute (EDS). When seeded on C-GAG matrix, MSCs adhered rapidly, migrated into the pores, and proliferated readily. When applied onto excisional wounds in healthy and diabetic mice, the EDS survived well, and accelerated wound closure, compared with C-GAG matrix alone or MSCs in collagen hydrogel. Histological analysis revealed that EDS prolonged the retention of MSCs in the wounds, associated with increased macrophage infiltration and enhanced angiogenesis. RNA-Seq analysis of EDS-treated wounds uncovered the expression of abundant human chemokines and proangiogenic factors and their corresponding murine receptors, suggesting a mechanism of ligand/receptor-mediated signals in wound healing. Thus, our results indicate that EDS prolongs the survival and retention of MSCs in the wounds and enhances wound healing.

Mesenchymal stem cell aggregation mediated by integrin α4/VCAM-1 after intrathecal transplantation in MCAO rats.

BACKGROUND: Mesenchymal stem cells (MSCs) have shown immense therapeutic potential for various brain diseases. Intrathecal administration of MSCs may enhance their recruitment to lesions in the central nervous system, but any impact on cerebrospinal fluid (CSF) flow remains unclear. METHODS: Rats with or without middle cerebral artery occlusion (MCAO) received intrathecal injections of 2D cultured MSCs, 3D cultured MSCs or an equal volume of artificial cerebrospinal fluid (ACSF). Ventricle volume was assessed by MRI on Days 2 and 14 post-MCAO surgery. A beam walking test was used to assess fine motor coordination and balance. Aggregation of MSCs was evaluated in CSF and frozen brain tissue. Differential expression of cell adhesion molecules was evaluated by RNA-Seq, flow cytometry and immunofluorescence analyses. The influence of VCAM-1 blockade in mediating the aggregation of 2D MSCs was investigated in vitro by counting cells that passed through a strainer and in vivo by evaluating ventricular dilation. RESULTS: MSC expanded in 2D culture formed aggregates in the CSF and caused ventricular enlargement in both MCAO and normal rats. Aggregates were associated with impaired motor function. 2D MSCs expressed higher levels of integrin α4 and VCAM-1 than 3D MSCs. Blockade of VCAM-1 in 2D MSCs reduced their aggregation in vitro and reduced lateral ventricular enlargement after intrathecal infusion. 3D MSCs exhibited lower cell aggregation and reduced cerebral ventricular dilation after intrathecal transplantation CONCLUSIONS: The aggregation of 2D MSCs, mediated by the interaction of integrin α4 and VCAM-1, is a potential risk for obstruction of CSF flow after intrathecal transplantation.

Three-dimensional cultured mesenchymal stem cells enhance repair of ischemic stroke through inhibition of microglia.

BACKGROUND: We show previously that three-dimensional (3D) spheroid cultured mesenchymal stem cells (MSCs) exhibit reduced cell size thus devoid of lung entrapment following intravenous (IV) infusion. In this study, we determined the therapeutic effect of 3D-cultured MSCs on ischemic stroke and investigated the mechanisms involved. METHODS: Rats underwent middle cerebral artery occlusion (MCAO) and reperfusion. 1 × 106 of 3D- or 2D-cultured MSCs, which were pre-labeled with GFP, were injected through the tail vain three and seven days after MCAO. Two days after infusion, MSC engraftment into the ischemic brain tissues was assessed by histological analysis for GFP-expressing cells, and infarct volume was determined by MRI. Microglia in the lesion were sorted and subjected to gene expressional analysis by RNA-seq. RESULTS: We found that infusion of 3D-cultured MSCs significantly reduced the infarct volume of the brain with increased engraftment of the cells into the ischemic tissue, compared to 2D-cultured MSCs. Accordingly, in the brain lesion of 3D MSC-treated animals, there were significantly reduced numbers of amoeboid microglia and decreased levels of proinflammatory cytokines, indicating attenuated activation of the microglia. RNA-seq of microglia derived from the lesions suggested that 3D-cultured MSCs decreased the response of microglia to the ischemic insult. Interestingly, we observed a decreased expression of mincle, a damage-associated molecular patterns (DAMPs) receptor, which induces the production of proinflammatory cytokines, suggestive of a potential mechanism in 3D MSC-mediated enhanced repair to ischemic stroke. CONCLUSIONS: Our data indicate that 3D-cultured MSCs exhibit enhanced repair to ischemic stroke, probably through a suppression to ischemia-induced microglial activation.

Engineered Skin Substitute Regenerates the Skin with Hair Follicle Formation.

Currently, engineered skin substitutes (ESS) are unable to regenerate cutaneous appendages. Recent studies have shown that skin-derived precursors (SKPs), which are extensively available, have the potential to induce hair follicle neogenesis. Here, we demonstrate that ESS consisting of culture-expanded SKPs and epidermal stem cells (Epi-SCs) reconstitute the skin with hair follicle regeneration after grafting into nude mice. SKPs seeded in a C-GAG matrix proliferated and expressed higher levels of hair induction signature genes-such as Akp2, Sox2, CD133 and Bmp6-compared to dermal fibroblasts. Moreover, when ESS prepared by seeding a mixture of culture-expanded murine SKPs and human adult Epi-SCs into a C-GAG matrix was grafted into full-thickness skin wounds in nude mice, black hairs were generated within 3 weeks. Immunofluorescence analysis showed that the SKPs were localized to the dermal papillae of the newly-formed hair follicle. Our results indicate that SKPs can serve as the hair-inductive cells in ESS to furnish it with hair genesis potential.