Shijie Tang

Three-dimensional culture and identification of human eccrine sweat glands in matrigel basement membrane matrix

Interactions between the extracellular matrix (ECM) and epithelial cells are necessary for the proper organization and function of the epithelium. In the present study, we show that human eccrine sweat gland epithelial cells cultured in matrigel, a representation of ECM components, constitute a good model for studying three-dimensional reconstruction, wound repair and regeneration and differentiation of the human eccrine sweat gland. In matrigel, epithelial cells from the human eccrine sweat gland form tubular-like structures and then the tubular-like structures coil into sphere-like shapes that structurally resemble human eccrine sweat glands in vivo. One sphere-like shape can be linked to another sphere-like shape or to a cell monolayer via tubular-like structures. Hematoxylin and eosin staining has revealed that the tubular-like structures have a single layer or stratified epithelial cells located peripherally and a lumen at the center, similar to the secretory part or duct part, respectively, of the eccrine sweat gland in sections of skin tissue. Immunohistochemical analysis of the cultures has demonstrated that the cells express CK7, CK19, epithelial membrane antigen and actin. Thus, matrigel promotes the organization and differentiation of epithelial cells from the human eccrine sweat gland into eccrine sweat gland tissues.

Matrigel basement membrane matrix induces eccrine sweat gland cells to reconstitute sweat gland-like structures in nude mice

BACKGROUND Severe burn results in irreversible damage to eccrine sweat glands, for which no effective treatment is available. Interaction between the extracellular matrix and epithelial cells is critical for proper three-dimensional organization and function of the epithelium. METHODS Matrigel-embedded eccrine sweat gland cells were subcutaneously implanted into the inguinal regions of nude mice. Two weeks later, the Matrigel plugs were removed and evaluated for series of detection items. RESULTS Sweat gland cells developed into sweat gland-like structures in the Matrigel plugs based on: (1) de novo formation of tubular-like structures with one or more hollow lumens, (2) expression of epithelial and sweat gland markers (pancytokeratin, CK5/7/14/19, α-SMA and CEA), (3) basement membrane formation, (4) myoepithelial cells presenting in and encompassing the tubular-like structures, (5) cellular polarization, evident by the expression of tight junction proteins (claudin-1 and ZO-2), anchoring junctions (desmoglein-1 and -2 and E-cadherin) and CEA in the luminal membrane, (6) expression of proteins related to sweat secretion and absorption (Na(+)-K(+)-ATPase α/β, Na(+)-K(+)-2Cl-cotranspoter 1, Na(+)/H(+) exchanger 1, aquaporin-5, epithelial sodium channel, cystic fibrosis transmembrane conductance regulator, potassium channel and vacuolar-type H+-ATPase), and (7) about 20% of the tubular-like structures are de novo coils and 80% are de novo ducts. CONCLUSIONS This study provides not only an excellent model to study eccrine sweat gland development, cytodifferentiation and reconstitution, but also an in vivo model for regeneration of eccrine sweat glands.

Localization of Na+–K+-ATPase α/β, Na+–K+–2Cl-cotransporter 1 and aquaporin-5 in human eccrine sweat glands

In order to evaluate the function of the repaired or regenerated eccrine sweat glands, we must first localize the proteins involved in sweat secretion and absorption in normal human eccrine sweat glands. In our studies, the cellular localization of Na(+)-K(+)-ATPase α/β, Na(+)-K(+)-2Cl-cotransporter 1 (NKCC1) and aquaporin-5 (AQP5) in eccrine sweat glands were detected by immunoperoxidase labeling. The results showed that Na(+)-K(+)-ATPase α was immunolocalized in the cell membrane of the basal layer and suprabasal layer cells of the epidermis, the basolateral membrane of the secretory coils, and the cell membrane of the outer cells and the basolateral membrane of the luminal cells of the ducts. The localization of Na(+)-K(+)-ATPase β in the secretory coils was the same as Na(+)-K(+)-ATPase α, but Na(+)-K(+)-ATPase β labeling was absent in the straight ducts and epidermis. NKCC1 labeling was seen only in the basolateral membrane of the secretory coils. AQP5 was strongly localized in the apical membrane and weakly localized in the cytoplasm of secretory epithelial cells. The different distribution of these proteins in eccrine sweat glands was related to their functions in sweat secretion and absorption.

Combination of keratins and alpha-smooth muscle actin distinguishes secretory coils from ducts of eccrine sweat glands.

Eccrine sweat glands are comprised of secretory coils and ducts, which are distinct in morphology and function. To better understand the roles of the two parts in development, homeostasis, wound repair and regeneration of eccrine sweat glands, we must distinguish between them. In this study, the localization of keratins and alpha-SMA in human eccrine sweat glands was examined by immunofluorescence staining. Based on the differential localization of keratins and alpha-SMA in different cell types, four pairs of antibodies (K5/K7, K5/alpha-SMA, K14/K7 and K14/alpha-SMA) were used to differentiate secretory coils from ducts by double-immunofluorescence staining. Immunofluorescence staining showed that myoepithelial cells of secretory coils expressed K5, K14 and alpha-SMA, whereas secretory cells of secretory coils expressed K7, K8, K15, K18 and K19. Ductal cells expressed K5, K8, K14 and K19. Double-staining showed that the secretory coils were K5(+)/K7(+), K5(+)/alpha-SMA(+), K14(+)/K7(+) and K14(+)/alpha-SMA(+), whereas ducts were K5(+)/K7(-), K5(+)/alpha-SMA(-), K14(+)/K7(-) and K14(+)/alpha-SMA(-). In conclusion, by combining use of keratins and alpha-SMA antibodies, secretory coils can be easily differentiated from ducts in morphology.

The cellular localization of Na+/H+ exchanger 1, cystic fibrosis transmembrane conductance regulator, potassium channel, epithelial sodium channel γ and vacuolar-type H+-ATPase in human eccrine sweat glands

The secretory portions of human eccrine sweat glands secrete isotonic fluid into the lumen and then the primary fluid is rendered hypotonic during its passage to the skin surface. During the processes of sweat secretion and absorption, many enzymes and proteins play important roles. In the study, the cellular localizations of Na(+)/H(+) exchanger 1 (NHE1), cystic fibrosis transmembrane conductance regulator (CFTR), potassium channel (KC), epithelial sodium channel γ (γENaC) and vacuolar-type H+-ATPase (V-ATPase) in human eccrine sweat glands and epidermis were detected using immunofluorescence labeling. The results revealed that in the secretory coils, the basolateral membranes showed evidence of CFTR, NHE1 and KC activities, the apical membranes showed the activities of KC and NHE1, and the nucleus showed γEaNC and V-ATPase activities; in the duct, the peripheral and luminal ductal cells showed evidence of CFTR, NHE1 and KC, the apical membranes showed the activities of CFTR and NHE1, and the nucleus showed γEaNC, V-ATPase and KC activities. The cellular localization of these proteins in eccrine sweat glands is helpful to better understand the mechanisms of sweat secretion and absorption.

BrdU-label-retaining cells in rat eccrine sweat glands over time.

Cell proliferation and turnover are fueled by stem cells. In a previous study, we demonstrated that rat eccrine sweat glands contained abundant bromodeoxyuridine (BrdU)-label-retaining cells (LRCs). However, morphological observations showed that eccrine sweat glands usually show little or no signs of homeostatic change. In this study, we account for why the homeostatic change is rare in eccrine sweat glands based on cytokinetic changes in BrdU-LRC turnover, and also determine the BrdU-labeled cell type. Thirty-six newborn SD rats, were injected intraperitoneally with 50mg/kg BrdU twice daily at a 2h interval for 4 consecutive days. After a chase period of 4, 6, 8, 12, 24 and 32 weeks, rats were euthanized, and the hind footpads were removed and processed for BrdU immunostaining, and BrdU/α-SMA and BrdU/K14 double-immunostaining. BrdU-LRCs were observed in the ducts, secretory coils and mesenchymal cells at all survival time points. The percentage of BrdU(+) cells in rat eccrine sweat glands averaged 4.2±1.2% after 4 weeks of chase, increased slightly by the 6th week, averaging 4.4±0.9%, and peaked at 8 weeks, averaging 5.3±1.0%. Subsequently, the average percentage of BrdU(+) cells declined to 3.2±0.8% by the 32nd week. There was no difference in the percentage of BrdU-LRCs among the different survival time points except that a significant difference in the percentage of BrdU-LRCs detected at 24 weeks versus 8 weeks, and 32 weeks versus 8 weeks, was observed. We concluded that the BrdU-LRCs turnover is slow in eccrine sweat glands.

Basic Fibroblast Growth Factor Influences Epidermal Homeostasis of Living Skin Equivalents through Affecting Fibroblast Phenotypes and Functions

Aims: To elucidate the possible mechanisms of how basic fibroblast growth factor (bFGF) influences epidermal homeostasis in a living skin equivalent (LSE) model. Methods: Several wound healing-related growth factors were analyzed at protein and mRNA levels for dermal fibroblasts of induced alpha-smooth muscle actin (α-SMA)-positive or α-SMA-negative phenotypes. During culturing an LSE model by seeding normal human keratinocytes on a fibroblast-populated type I collagen gel, bFGF or neutralizing antibody for keratinocyte growth factor (KGF) was added to investigate its effects on fibroblast phenotypes and, subsequently, epidermal homeostasis by histology and immunohistochemistry. Results: The α-SMA-positive phenotype of fibroblasts induced by transforming growth factor beta-1 (TGF-β1) markedly suppressed the expression of KGF and hepatocyte growth factor (HGF), and slightly upregulated vascular endothelial growth factor (VEGF) and TGF-β1 at mRNA and protein levels, compared with α-SMA-negative fibroblasts treated with bFGF. α-SMA expression of fibroblasts at the epidermal-mesenchymal junction of the LSEs was suppressed by the addition of bFGF, and a better-differentiated epidermis was presented. The abrogation of KGF from fibroblasts by the addition of the KGF neutralizing antibody disenabled the LSE culturing system to develop an epidermis. Conclusions: bFGF, through affecting the phenotypes and functions of fibroblasts, especially KGF expression, influenced epidermal homeostasis in an LSE model.

Efficiency of stem cell based therapy in the treatment of diabetic foot ulcer: a meta-analysis

Diabetic foot ulcer is a chronic, refractory, frequent complication in diabetic patient. Its treatment often requires multidisciplinary joint efforts, diverse strategies have been adopted to address this annoying issue, including stem cell-based therapy/acellular dermal matrix/negative pressure wound therapy etc. However, consensus has not been reached. To assess the current evidence regarding the efficiency and potential advantages of stem cell-based therapy compared with conventional standard treatment and/or placebo in the treatment of diabetic foot ulcer. A comprehensive search in PubMed, EmBase, Cochrane Central and Web of Science databases was conducted during December 2016 and a systematic review and meta-analysis of all relevant studies were performed. A total of 7 studies that involved 224 diabetic foot patients, classified as Wagner grades 1-5, were analyzed. The pooled results confirmed the benefits of using the stem cell treatment. Partial and/or complete healing were significantly higher in the stem cell group compared with the control group (77.4% vs. 31.9%; RR: 2.22; 95% CI, 1.65-2.98). Subgroup analysis on ABI and TCP02 also confirmed the results. The present meta-analysis indicates that stem cell-based therapy can enhance the healing of diabetic foot ulcers and is associated with lesser pain, lower amputation rate and improved prognosis compared with normal treatment. Well-designed randomized controlled trials are required in the future in order to confirm and update these findings.