Lgr6 marks epidermal stem cells with a nerve-dependent role in wound re-epithelialization

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Hair follicle and interfollicular epidermal stem cells make varying contributions to wound regeneration

Upon wounding, multiple stem cell populations in the hair follicle (HF) and interfollicular epidermis (IFE) converge at the site of injury. Although these cells can contribute permanently to the regenerating epithelium, it remains unclear whether these contributions vary among cells originating from diverse compartments in the skin. By comparing the fates of several keratinocyte lineages, we observed here an initial decrease in both HF- and IFE-derived cells within the transient acanthotic layers of the regenerating epithelium. At the same time, the relative abundance of early-arriving IFE-derived cells specifically in the wound basal layer declined as later-arriving HF-derived cells entered the site of injury. Although laggard bulge-derived cells were typically constrained at the regenerative periphery, these cells persisted in the wound basal layer. Finally, suppressing Notch enabled IFE-derived cells to out-compete HF-derived cells. Taken together, these findings indicate that IFE-, HF- and bulge-derived cells make distinct contributions to regeneration over time. Furthermore, we speculate that extrinsic, non-genetic factors such as spatial constraint, distance from the wound, and basal versus suprabasal position may largely determine whether a cell ultimately persists.     

Activity of mesenchymal stem cells in therapies for chronic skin wound healing

Chronic or non-healing skin wounds present an ongoing challenge in advanced wound care, particularly as the number of patients increases while technology aimed at stimulating wound healing in these cases remains inefficient. Mesenchymal stem cells (MSCs) have proved to be an attractive cell type for various cell therapies due to their ability to differentiate into various cell lineages, multiple donor tissue types, and relative resilience in ex-vivo expansion, as well as immunomodulatory effects during transplants. More recently, these cells have been targeted for use in strategies to improve chronic wound healing in patients with diabetic ulcers or other stasis wounds. Here, we outline several mechanisms by which MSCs can improve healing outcomes in these cases, including reducing tissue inflammation, inducing angiogenesis in the wound bed, and reducing scarring following the repair process. Approaches to extend MSC life span in implant sites are also examined.     

The use of adipose-derived stem cells as sheets for wound healing

Cellular therapies have shown immense promise in the treatment of nonhealing wounds. Cell sheets are an emerging strategy in tissue engineering, and these cell sheets are promising as a delivery method of mesenchymal stem cells to the wound bed. Cell sheet technology utilizes temperature dependent polymers to allow for lifting of cultured cells and extracellular matrix without the use of digestive enzymes. While mesenchymal stem cells (MSCs) have shown success in cell sheets for myocardial repair, examination of cell sheets in the field of wound healing has been limited. We previously developed a novel cell sheet composed of human adipose-derived stem cells (ASCs). Both single and triple layer cell sheets were examined in a full-thickness murine wound model. The treatment cell sheets were compared with untreated controls and analyzed at timepoints of 7, 14, 18 and 21 d. The ASC cell sheets showed increased healing at 7, 14 and 18 d, and this effect was increased in the triple layer cell sheet group. Future development of these cell sheets will focus on increasing angiogenesis in the wound bed, utilizing multiple cell types, and examining allogeneic cell sheets. Here we review our experiment, expand upon our future directions and discuss the potential of an off-the-shelf cell sheet. In the field of wound healing, such a cell sheet is both clinically and scientifically exciting.     

Role of caveolin-1 in epidermal stem cells during burn wound healing in rats

Local transplantation of stem cells has therapeutic effects on skin damage but cannot provide satisfactory wound healing. Studies on the mechanisms underlying the therapeutic effects of stem cells on skin wound healing will be needed. Hence, in the present study, we explored the role of Caveolin-1 in epidermal stem cells (EpiSCs) in the modulation of wound healing. We first isolated EpiSCs from mouse skin tissues and established stable EpiSCs with overexpression of Caveolin-1 using a lentiviral construct. We then evaluated the epidermal growth factor (EGF)-induced cell proliferation ability using cell counting Kit-8 (CCK-8) assay and assessed EpiSC pluripotency by examining Nanog mRNA levels in EpiSCs. Furthermore, we treated mice with skin burn injury using EpiSCs with overexpression of Caveolin-1. Histological examinations were conducted to evaluate re-epithelialization, wound scores, cell proliferation and capillary density in wounds. We found that overexpression of Caveolin-1 in EpiSCs promoted EGF-induced cell proliferation ability and increased wound closure in a mouse model of skin burn injury. Histological evaluation demonstrated that overexpression of Caveolin-1 in EpiSCs promoted re-epithelialization in wounds, enhanced cellularity, and increased vasculature, as well as increased wound scores. Taken together, our results suggested that Caveolin-1 expression in the EpiSCs play a critical role in the regulation of EpiSC proliferation ability and alteration of EpiSC proliferation ability may be an effective approach in promoting EpiSC-based therapy in skin wound healing.     

Adipose-derived stem cells for wound healing

Wound healing is a complex but a fine-tuned biological process in which human skin has the ability to regenerate itself following damage. However, in particular conditions such as deep burn or diabetes the process of wound healing is compromised. Despite investigations on the potency of a wide variety of stem cells for wound healing, adipose-derived stem cells (ASCs) seem to possess the least limitations for clinical applications, and literature showed that ASCs can improve the process of wound healing very likely by promoting angiogenesis and/or vascularisation, modulating immune response, and inducing epithelialization in the wound. In the present review, advantages and disadvantages of various stem cells which can be used for promoting wound healing are discussed. In addition, potential mechanisms of action by which ASCs may accelerate wound healing are summarised. Finally, clinical studies applying ASCs for wound healing and the associated limitations are reviewed.     

Human hair follicle-derived mesenchymal stem cells: Isolation,expansion, and differentiation

Hair follicles are easily accessible skin appendages that protect against cold and potential injuries. Hair follicles contain various pools of stem cells, such as epithelial, melanocyte, and mesenchymal stem cells (MSCs) that continuously self-renew, differentiate, regulate hair growth, and maintain skin homeostasis. Recently, MSCs derived from the dermal papilla or dermal sheath of the human hair follicle have received attention because of their accessibility and broad differentiation potential. In this review, we describe the applications of human hair follicle-derived MSCs (hHF-MSCs) in tissue engineering and regenerative medicine. We have described protocols for isolating hHF-MSCs from human hair follicles and their culture condition in detail. We also summarize strategies for maintaining hHF-MSCs in a highly proliferative but undifferentiated state after repeated in vitro passages, including supplementation of growth factors, 3D suspension culture technology, and 3D aggregates of MSCs. In addition, we report the potential of hHF-MSCs in obtaining induced smooth muscle cells and tissue-engineered blood vessels, regenerated hair follicles, induced red blood cells, and induced pluripotent stem cells. In summary, the abundance, convenient accessibility, and broad differentiation potential make hHF-MSCs an ideal seed cell source of regenerative medical and cell therapy.     

Elimination of foreign material by epidermal malpighian cells during wound healing in fish skin

Cultured epidermal malpighian cells and experimental wounds of Atlantic salmon Salmo salar were challenged with a variety of particulate materials. Latex beads and the bacteria Carnobacterium piscicola, Pseudomonas fluorescens and Aeromonas salmonicida salmonicida were engulfed by cultured cells, whereas Staphylococcus intermedius were not. The cells engulfed bacteria that proliferated in culture medium devoid of antibiotics and melanin granules and other cellular debris. Cells at wound margins engulfed latex beads and C. piscicola, P. fluorescens and A. s. salmonicida , but not S. intermedius. Malpighian cells thus appear to be both phagocytic and discriminatory. The results support the hypothesis that malpighian cells remove foreign material from fish skin by sloughing after becoming laden with engulfed material.     

Effects of porcine acellular dermal matrix treatment on wound healing and scar formation: Role of Jag1 expression in epidermal stem cells

Skin wound healing involves Notch/Jagged1 signaling. However, little is known how Jag1 expression level in epidermal stem cells (ESCs) contributes to wound healing and scar formation. We applied multiple cellular and molecular techniques to examine how Jag1 expression in ESCs modulates ESCs differentiation to myofibroblasts (MFB) in vitro, interpret how Jag1 expression in ESCs is involved in wound healing and scar formation in mice, and evaluate the effects of porcine acellular dermal matrix (ADM) treatment on wound healing and scar formation. We found that Jag1, Notch1 and Hes1 expression was up-regulated in the wound tissue during the period of wound healing. Furthermore, Jag1 expression level in the ESCs was positively associated with the level of differentiation to MFB. ESC-specific knockout of Jag1 delayed wound healing and promoted scar formation in vivo. In addition, we reported that porcine ADM treatment after skin incision could accelerate wound closure and reduce scar formation in vivo. This effect was associated with decreased expression of MFB markers, including α-SMA Col-1 and Col-III in wound tissues. Finally, we confirmed that porcine ADM treatment could increase Jag1, Notch1 and Hesl expression in wound tissues. Taken together, our results suggested that ESC-specific Jag1 expression levels are critical for wound healing and scar formation, and porcine ADM treatment would be beneficial in promoting wound healing and preventing scar formation by enhancing Notch/Jagged1 signaling pathway in ESCs.     

Aspirin promotes tenogenic differentiation of tendon stem cells and facilitates tendinopathy healing through regulating the GDF7/Smad1/5 signaling pathway

Tendinopathy is a common musculoskeletal system disorder in sports medicine, but regeneration ability of injury tendon is limited. Tendon stem cells (TSCs) have shown the definitive treatment evidence for tendinopathy and tendon injuries due to their tenogenesis capacity. Aspirin, as the representative of nonsteroidal anti-inflammatory drugs for its anti-inflammatory and analgestic actions, has been commonly used in treating tendinopathy in clinical, but the effect of aspirin on tenogenesis of TSCs is unclear. We hypothesized that aspirin could promote injury tendon healing through inducing TSCs tenogenesis. The aim of the present study is to make clear the effect of aspirin on TSC tenogenesis and tendon healing in tendinopathy, and thus provide new treatment evidence and strategy of aspirin for clinical practice. First, TSCs were treated with aspirin under tenogenic medium for 3, 7, and 14 days. Sirius Red staining was performed to observe the TSC differentiation. Furthermore, RNA sequencing was utilized to screen out different genes between the induction group and aspirin treatment group. Then, we identified the filtrated molecules and compared their effect on tenogenesis and related signaling pathway. At last, we constructed the tendinopathy model and compared biomechanical changes after aspirin intake. From the results, we found that aspirin promoted tenogenesis of TSCs. RNA sequencing showed that growth differentiation factor 6 (GDF6), GDF7, and GDF11 were upregulated in induction medium with the aspirin group compared with the induction medium group. GDF7 increased tenogenesis and activated Smad1/5 signaling. In addition, aspirin increased the expression of TNC, TNMD, and Scx and biomechanical properties of the injured tendon. In conclusion, aspirin promoted TSC tenogenesis and tendinopathy healing through GDF7/Smad1/5 signaling, and this provided new treatment evidence of aspirin for tendinopathy and tendon injuries.     

Early-age-dependent selective decrease of differentiation potential of hair-follicle-associated pluripotent (HAP) stem cells to beating cardiac-muscle cells

We have previously discovered nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells and have shown that they can differentiate to neurons, glia, and many other cell types. HAP stem cells can be used for nerve and spinal cord repair. We have recently shown the HAP stem cells can differentiate to beating heart-muscle cells and tissue sheets of beating heart-muscle cells. In the present study, we determined the efficiency of HAP stem cells from mouse vibrissa hair follicles of various ages to differentiate to beating heart-muscle cells. We observed that the whiskers located near the ear were more efficient to differentiate to cardiac-muscle cells compared to whiskers located near the nose. Differentiation to cardiac-muscle cells from HAP stem cells in cultured whiskers in 4-week-old mice was significantly greater than in 10-, 20-, and 40-week-old mice. There was a strong decrease in differentiation potential of HAP stem cells to cardiac-muscle cells by 10 weeks of age. In contrast, the differentiation potential of HAP stem cells to other cell types did not decrease with age. The possibility of rejuvenation of HAP stem cells to differentiate at high efficiency to cardiac-muscle cells is discussed.     

Hair follicle stem cell cultures reveal self‐organizing plasticity of stem cells and their progeny

Understanding how complex tissues are formed, maintained, and regenerated through local growth, differentiation, and remodeling requires knowledge on how single‐cell behaviors are coordinated on the population level. The self‐renewing hair follicle, maintained by a distinct stem cell population, represents an excellent paradigm to address this question. A major obstacle in mechanistic understanding of hair follicle stem cell (HFSC) regulation has been the lack of a culture system that recapitulates HFSC behavior while allowing their precise monitoring and manipulation. Here, we establish an in vitro culture system based on a 3D extracellular matrix environment and defined soluble factors, which for the first time allows expansion and long‐term maintenance of murine multipotent HFSCs in the absence of heterologous cell types. Strikingly, this scheme promotes de novo generation of HFSCs from non‐HFSCs and vice versa in a dynamic self‐organizing process. This bidirectional interconversion of HFSCs and their progeny drives the system into a population equilibrium state. Our study uncovers regulatory dynamics by which phenotypic plasticity of cells drives population‐level homeostasis within a niche, and provides a discovery tool for studies on adult stem cell fate.     

Mesenchymal stem cell-conditioned media: A novel alternative of stem cell therapy for quality wound healing

Mesenchymal stem cells-conditioned media (MSCs-CM) contains several growth factors and cytokines, thus may be used as a better alternative to stem cell therapy, which needs to be elucidated. The present study was conducted to evaluate the therapeutic potential of caprine, canine, and guinea pig bone marrow-derived MSCs-CM in excision wound healing in a guinea pig model. MSCs were obtained from bone marrow, expanded ex vivo and characterized as per ISCT criteria. CM was collected assayed by western blot to ascertain the presence of important secretory biomolecules. Quantitative estimation by enzyme-linked immunosorbent assay was done for a vascular epidermal growth factor (VEGF) and interleukin-6 (IL-6) in caprine MSCs-CM and optimum time for collection of CM was decided as 72 hr. CM from all the species was lyophilized by freeze-drying method. Full-thickness (2 × 2 cm²) excision skin wounds were created in guinea pigs (six animals in each group) and respective lyophilized CM mixed with laminin gel was applied topically at weekly interval. On Day 28, histopathological examinations of healed skin were done by hemotoxylin and eosin staining. MSCs were found to secrete important growth factors and cytokines (i.e., VEGF, transforming growth factor-β1, fibroblast growth factor-2, insulin-like growth factor-1, stem cell factor, and IL-6) as demonstrated by immunohistochemistry and western blot assay. It was found that allogenic and xenogenic application of CM significantly improved quality wound healing with minimal scar formation. Thus, MSCs-CM can be used allogenically as well as xenogenically for quality wound healing.     

Biochemistry of epidermal stem cells

Background

The epidermis is an important protective barrier that is essential for maintenance of life. Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processes must be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locations in the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment of this tissue.

Scope of review

A great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifying stem cell niche locations, and in understanding how stem cell populations are related. We discuss these studies as they apply to understanding normal epidermal homeostasis and skin cancer.

Major conclusions

An assortment of stem cell markers have been identified that permit assignment of stem cells to specific regions of the epidermis, and progress has been made in understanding the role of these cells in normal epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations exist in epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair in damaged epidermis.

General significance

Understanding epidermal stem cell biology is likely to lead to important therapies for treating skin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

表皮干细胞鉴别方法研究进展

表皮干细胞不仅在组织稳态和皮肤损伤修复中发挥重要作用,而且对研究肿瘤发生机制和皮肤疾病的基因治疗具有重大意义,多年来,人们一直致力寻找干细胞特异性分子标坊物以及体内干细胞的精确度定位研究,并且发现,根据细胞动力学特点以及细胞内和细胞膜表面的一些特异性标记物可以鉴别出表皮干细胞,本综述了近年来国内外学在表皮干细胞鉴定方面取得的一些进展。     

鹿茸角柄断面伤口无疤痕愈合过程中骨形态蛋白的表达及功能分析

为研究骨形态蛋白（bone morphogenetic proteins ,BMP）在鹿茸再生早期角柄断面伤口无疤痕愈合过程中的功能,本研究通过免疫组化技术对比分析了BMP 2 和BMP 4在正常皮肤及茸皮中的表达及分布差异,同时利用添加外源性蛋白分析了BMP 4对鹿真皮成纤维细胞和毛乳头细胞的影响。结果显示：(1)原代培养的真皮成纤维细胞表达波形蛋白阳性率几乎为100%;(2) BMP 2和BMP 4强烈表达于茸皮中新生毛囊的毛基质细胞中;(3) BMP 4可促进鹿真皮成纤维细胞向脂肪细胞转分化;(4) BMP 4可促进鹿真皮毛乳头细胞成团。以此推测BMP在鹿毛囊形成及伤口愈合过程中发挥重要作用。