Enrichment of putative human epidermal stem cells based on cell size and collagen type IV adhesiveness

The enrichment and identification of human epidermal stem cells （EpSCs） are of paramount importance for both basic research and clinical application. Although several approaches for the enrichment of EpSCs have been established, enriching a pure population of viable EpSCs is still a challenging task. An improved approach is worth developing to enhance the purity and viability of EpSCs. Here we report that cell size combined with collagen type IV adhesiveness can be used in an improved approach to enrich pure and viable human EpSCs. We separated the rap- idly adherent keratinocytes into three populations that range in size from 5-7 μm （population A）, to 7-9 μm （population B）, to ≥9μm （population C） in diameter, and found that human putative EpSCs could be further enriched in population A with the smallest size. Among the three populations, population A displayed the highest density of plintegrin receptor, contained the highest percentage of cells in G0/G1 phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony formation efficiency （CFE）. When injected into murine blastocysts, these cells participated in multi-tissue formation. More significantly, compared with a previous approach that sorted putative EpSCs according to pl-integrin antibody staining, the viability of the EpSCs enriched by the improved approach was significantly enhanced. Our results provide a putative strategy for the enrichment of human EpSCs, and encourage further study into the role of cell size in stem cell biology.

Substance P combined with epidermal stem cells promotes wound healing and nerve regeneration in diabetes mellitus

Exogenous substance P accelerates wound healing in diabetes,but the mechanism remains poorly understood.Here,we established a rat model by intraperitoneally injecting streptozotocin.Four wounds（1.8 cm diameter） were drilled using a self-made punch onto the back,bilateral to the vertebral column,and then treated using amniotic membrane with epidermal stem cells and/or substance P around and in the middle of the wounds.With the combined treatment the wound-healing rate was 100% at 14 days.With prolonged time,type I collagen content gradually increased,yet type III collagen content gradually diminished.Abundant protein gene product 9.5-and substance P-immunoreactive nerve fibers regenerated.Partial nerve fiber endings extended to the epidermis.The therapeutic effects of combined substance P and epidermal stem cells were better than with amniotic membrane and either factor alone.Our results suggest that the combination of substance P and epidermal stem cells effectively contributes to nerve regeneration and wound healing in diabetic rats.

Calcium silicate accelerates cutaneous wound healing with enhanced re-epithelialization through EGF/EGFR/ERK-mediated promotion of epidermal stem cell functions

Background:Human epidermal stem cells(hESCs)play an important role in re-epithelialization and thereby in facilitating wound healing,while an effective way to activate hESCs remains to be explored.Calcium silicate(CS)is a form of bioceramic that can alter cell behavior and promote tissue regeneration.Here,we have observed the effect of CS on hESCs and investigated its possible mechanism.Methods:Using a mouse full-thickness skin excision model,we explored the therapeutic effect of CS on wound healing and re-epithelialization.In vitro,hESCs were cultured with diluted CS ion extracts(CSIEs),and the proliferation,migration ability and stemness of hESCs were evaluated.The effects of CS on the epidermal growth factor(EGF),epidermal growth factor receptor(EGFR)and extracellular signal-related kinase(ERK)signaling pathway were also explored.Results:In vivo,CS accelerated wound healing and re-epithelialization.Immunohistochemistry demonstrated that CS upregulated cytokeratin 19 and integrinβ1 expression,indicating that CS improved hESCs stemness.In vitro studies confirmed that CS improved the biological function of hESCs.And the possible mechanism could be due to the activation of the EGF/EGFR/ERK signaling pathway.Conclusion:CS can promote re-epithelialization and improve the biological functions of hESCs via activating the EGF/EGFR/ERK signaling pathway.

Epithelial stem cell islands in the regenerated epidermis

Objective: The effects of growth factors on wound healing have been studied extensively, however, their molecular and genetic mechanisms that regulate epidermal regeneration are not fully understood. In this study, we explore the cell reversion characteristics and epithelial stem cell distribution in human regenerated epidermis treated with recombinant human epidermal growth factor (rhEGF). Methods:Tissue biospies from 8 regenerated skins treated with rhEGF were used to evaluate the cell reversion.

Genetic reporter analysis reveals an expandable reservoir of OCT4+cells in adult skin

The transcription factor Oct4(Pou5f1)is a critical regulator of pluripotency in embryonic and induced pluripotent stem cells.Therefore,Oct4 expression might identify somatic stem cell populations with inherent multipotent potential or a propensity for facilitated reprogramming.However,analysis of Oct4 expression is confounded by Oct4 pseudogenes or non-pluripotency-related isoforms.Systematic analysis of a transgenic Oct4-EGFP reporter mouse identified testis and skin as two principle sources of Oct4^(+)cells in postnatal mice.While the prevalence of GFP^(+)cells in testis rapidly declined with age,the skin-resident GFP^(+)population expanded in a cyclical fashion.These cells were identified as epidermal stem cells dwelling in the stem cell niche of the hair follicle,which endogenously expressed all principle reprogramming factors at low levels.Interestingly,skin wounding or non-traumatic hair removal robustly expanded the GFP^(+)epidermal cell pool not only locally,but also in uninjured skin areas,demonstrating the existence of a systemic response.Thus,the epithelial stem cell niche of the hair follicle harbors an expandable pool of Oct4^(+)stem cells,which might be useful for therapeutic cell transfer or facilitated reprogramming.

Platelet-rich plasma accelerates skin wound healing by promoting re-epithelialization

Background:Autologous platelet-rich plasma(PRP)has been suggested to be effective for wound healing.However,evidence for its use in patients with acute and chronic wounds remains insufficient.The aims of this study were to comprehensively examine the effectiveness,synergy and possible mechanism of PRP-mediated improvement of acute skin wound repair.Methods:Full-thickness wounds were made on the back of C57/BL6 mice.PRP or saline solution as a control was administered to the wound area.Wound healing rate,local inflammation,angiogenesis,re-epithelialization and collagen deposition were measured at days 3,5,7 and 14 after skin injury.The biological character of epidermal stem cells(ESCs),which reflect the potential for re-epithelialization,was further evaluated in vitro and in vivo.Results:PRP strongly improved skin wound healing,which was associated with regulation of local inflammation,enhancement of angiogenesis and re-epithelialization.PRP treatment significantly reduced the production of inflammatory cytokines interleukin-17A and interleukin-1β.An increase in the local vessel intensity and enhancement of re-epithelialization were also observed in animals with PRP administration and were associated with enhanced secretion of growth factors such as vascular endothelial growth factor and insulin-like growth factor-1.Moreover,PRP treatment ameliorated the survival and activated the migration and proliferation of primary cultured ESCs,and these effects were accompanied by the differentiation of ESCs into adult cells following the changes of CD49f and keratin 10 and keratin 14.Conclusion:PRP improved skin wound healing by modulating inflammation and increasing angiogenesis and re-epithelialization.However,the underlying regulatory mechanism needs to be investigated in the future.Our data provide a preliminary theoretical foundation for the clinical administration of PRP in wound healing and skin regeneration.

Photodynamic therapy accelerates skin wound healing through promoting re-epithelialization

Background:Epidermal stem cells(EpSCs)that reside in cutaneous hair follicles and the basal layer of the epidermis are indispensable for wound healing and skin homeostasis.Little is known about the effects of photochemical activation on EpSC differentiation,proliferation and migration during wound healing.The present study aimed to determine the effects of photodynamic therapy(PDT)on wound healing in vivo and in vitro.Methods:We created mouse full-thickness skin resection models and applied 5-aminolevulinic acid(ALA)for PDT to the wound beds.Wound healing was analysed by gross evaluation and haematoxylin–eosin staining in vivo.In cultured EpSCs,protein expression was measured using flow cytometry and immunohistochemistry.Cell migration was examined using a scratch model;apoptosis and differentiation were measured using flow cytometry.Results:PDT accelerated wound closure by enhancing EpSC differentiation,proliferation and migration,thereby promoting re-epithelialization and angiogenesis.PDT inhibited inflammatory infiltration and expression of proinflammatory cytokines,whereas the secretion of growth factors was greater than in other groups.The proportion of transient amplifying cells was significantly greater in vivo and in vitro in the PDT groups.EpSC migration was markedly enhanced after ALAinduced PDT.Conclusions:Topical ALA-induced PDT stimulates wound healing by enhancing re-epithelialization,promoting angiogenesis as well as modulating skin homeostasis.This work provides a preliminary theoretical foundation for the clinical administration of topical ALA-induced PDT in skin wound healing.