The Biological Characteristics Of Human Eccrine Sweat Gland Cells And The Study Of Differentiation Of Sweat Gland Cell-derived IPS Cells Into Sweat Gland-like Cells

Treatment of severe full- thickness burn injury is a serious medical and social problem to be solved urgently. There are thousands of millions burn injury patients every year in China. Among those patients, there are about 10% patients get se vere full-thickness burn injury and they were unable to save their lives. Severe burn patients without sweat gland function lost normal function of the skin and faced the bad quality of life, because the scar tissue had replaced the skin. Sweat glands are the important functional appendages in body’s skin tissue, including apocrine sweat glands and eccrine sweat glands. Eccrine sweat gland plays an important role in the regulation of body temperature, secretion of sweat and expelled metabolic waste from the body. Eccrine sweat gland is a type of single tubular gland, containded by the duct and the sedretory portion of the SG. The eccrine sweat gland is a straight channel and the secretory portion of the eccrine sweat gland is coiled tubular structure, narrow center(lumen). Sweat gland development began in the embryonic period of 14-16 weeks, and tobe matured after 24 week, there will be no the newborn sweat gland formation after birth.Skin tissue repair and functional reconstruction are the core and focus o f biomedical research in both China and abroad. In mild burn, the undamaged conduit of sweat gland cells becomes the template to reconstruct eccrine sweat glands and epidermis; in the large area deep burn, due to loss SG tissue, it cannot be regenerated to form SG 3D organization. The present study shows that, the stem cells can differentiate into epidermis and construct epidermal tissue with the protected covering ability; studies have also shown that stem cells can differentiate into sweat gland like cells with sweat gland cell phenotype.However, the functional reconstruction for sweat gland tissue has not been reported and the mechanisms for differentiation of stem cells into sweat gland like cells were not clear.In this study, we established human eccrine sweat gland cell culture methods in vitro, and identified their biological characteristics. Based on this, by constructing human sweat gland cell-derived i PS cells and, taking advantage of SG- i PS cells with the memory of sweat gland cells, we had carr ied out the study of SG- i PS cells differentiating into sweat gland cells. Furthermore, the study explored the key molecular mechanism of SG differentiation, which provides a theoretical foundation for other types of stem cells to be differentiated into sweat gland- like cells. This research makes the functional SG reconstruction and functional skin repair to be possible and may improve the clinical efficacy in treatment of patients with large area burn after study, This study has research innovation and app lication potential.PART ⅠIsolation and culture of human eccrine sweet gland cells in vitro and identification of its biological characteristicsObjective: To establish human eccrine sweat gland cell culture methods in vitro, and analyze their biological characteristics.Methods: Tissues were washed in phosphate buffer solution(PBS) with penicillin and streptomycin for 10 mins at room temperature. After removal of subcutaneous fat, the skin was minced into 1mm3 pieces, and washed again in PBS without penicillin and streptomycin. then the small pieces skins were treated with dispease for 18 h. After dispease treated, epidermis and dermis were separated, use nippers to isolate dermis from tissues, and dermis were treated with collagenase type Ⅳ for 1h at 37℃.After digested, sweat glands were dissociated from surrounding collagen and fat, and taken out one by one with a transferpettor under a ultraviolet-sterilized phase-contrast inverted microscope(×40). Human eccrine sweat gland cells morpholo gy were observed under microscope. Immunohistochemistry analysis to compare the number of sweat gland from different parts of the skin. TEM to analyze the organelle inside the sweat gland cell of skin samples. PCR analysis sweat gland cells gene expression. Immunofluorescence staining detected sweat gland cells specificity marker gene C EA and other keratin K14, K8 and K18 of sweat gland cells cultured in vitro.Result:(1) After 10 days adherent culture, human sweat gland(h SG) cells were grow from tissues, morphology of epithelial- like cells, SG cells were grew closely, similar to the channel-shaped stone pavement. The SG cells had clear cell borders, and their nuclears were clear observed. The SG cells had proliferative capacity cultured in vitro.(2) Human eccrine sweat glands were isolated from several human skin samples, including infant polydactyly skin, adult palm skin, adult neck skin and adult thoracoabdominal skin. After digestion, much more sweat gland tissues could be isolated from infant polydactyly skin compared to other samples, and the result was confirmed by immunohistochemistry.(3) TEM results showed that the sweat glands in the skin consists of two layers of cells in duct section, and there were tonofibrils inside the cells. Sweat glands secrete section were constituted by two types of cells, which are composed of myoepithelial cell and luminal cells.(4) PCR analysis results showed h SG cells not expressed Oct4, Sox2 and Nanog, but expressed K lf4 and c-Myc. For sweat gland related makers, h SG cells expressed keratinocytes maker K14; epithelial cells makers K8, K18 and K19. Specifically, Carcinoembryonic antigen(CEA) was only expressed on h SG cells compared to keratinocytes and h ESCs.(5) The immunofluorescent staining showed that h SG cells have protein expression of K14, K8, K18 and CEA.Conclusions: We isolated a large number of sweat gland tissues from infant polydactyly skin, this can meet the needs of experimental studies. Sweat gland cells have certain proliferative capacity cultured in vitro, cells can subculture 2-3 generations. We identified biological characteristics of sweat gland cells cultured in vitro. Sweat gland cells expressed specific marker CEA, and expressed the epithelial cell marker K14, K8 and K18. Our results lay the fo undation for further study sweat cells.PART ⅡReprogramming of SG cells and differentiation of SG-i PS cells into sweat gland-like cellsObjective: Construction of human eccrine sweat gland cells derived i PS cells, and to identify their biological characteristics. Induce SG-i PS cells differentiated into sweat gland-like cells directly, and identify its biological characteristics and functionality.Methods: 1×10human sweat gland cells were seeded on 60 mm-dish. The cells spread well on culture dishes next day, and were infected with a mixture of four lentiviral in the medium with polybrebe overnight. Three days after infected, the cells were digested by 0.25% trypsin/EDTA and plated 5×104 cells per dish, the dish were pro-coated with MMC treated MEF feeder cells. Single colonies were picked up using pipette under microscope. The colonies were first cultured in 24 well, when colonies grew to large colonies, split to small pieces, and moved to the larger dish. Collected SG cultured condition medium and filtered with 0.22μm filter before using. The SG cells differentiation medium(SGDM) inducing sweat gland cell- medium(SGM) and condition medium(CM) from h SG cells with a ratio of 1:1, supplemented with 50 ng/ml recombinant EGF. 2×105 SG-i PS cells were seeded in 6-well plate and used SGDM to differentiated into sweat gland- like cells(SG- i PS-SG). Cells were collected for realtime-PCR analysis. Flow cytometry analysis was used to detect sweat gland related protein. TEM was used to analyze the organelle inside the SG- i PS-SG cells. SG- i PS-SG cells cultured in 3-dimensional to form sweat gland tubular- like structure.Result:(1) We can observed SG- i PS cells colony exhibited similar morphology to h ESCs. The SG- i PS cells showed up a high nucleus to cytoplasm ratio and prominent nucleoli, and had capacity to form embryoid bodies similar to h ESCs.(2) RT-PCR and immunofluorescence staining results showed that expressed h ESCs makers.(3) SG-i PS cells had proliferative capacity cultured in vitro.(4) SG-i PS-SG cells observed larger than before, nucleus to cytoplasm ratio became low, and appeared obvious boundary between cells. Those characteristics were more like SG cells.(5) RT-PCR analysis, flow cytometry analysis and TEM analysis results show SG- i PS-SG cells biological characteristics like SG cells.(6) SG- i PS-SG cells cultured in gel can form sweat gland tubular- like structures. Immunofluorescence staining results showed the structures expressed sweat gland related proteins.Conclusions: Using reprogramming technology, we induced sweat gland cells turn into SG-i PS cells. Those SG- i PS cells had proliferated like h ESCs, and retained sweat gland memery make SG- i PS cells. This made SG- i PS cells more easily differentiated into SG cells, and can form sweat gland tubular- like structures.PART Ⅲ Investigation on differentiation of sweat gland-like cells and its regulatory mechanismObjective: Based on SG- i PS cells can differentiated into sweat gland-like cells using CM, we analyze molecules which play a key role during the differentiation in the CM. We want to find a suitable culture system for the study of stem cells differentiated into sweat gland-like cells and provide a theoretical basis with possible mechanism, making it possible to rebuild the sweat glands.Methods: 2×104 SG-i PS cells were seeded in 6-well plate, and used different induce medium to compared the effect of differentiation. We collected cells differentiated 7d,14 d and 21 d. We performed RT-PCR analysis and immunofluorescence staining on SG development related makers, including EDA, EDAR, Lef1 et al. We used RT-PCR analysis and immunofluorescence staining to find the source of BMP4 and HGF, compared with SG, SG-Fib and Fib. During the process of differentiated, adding HGF and BMP4 antagonist and additional HGF and BMP4, after 21 days of differentiation, Realtime-PCR analysis was used to detect the expression of sweat gland related genes.Result:(1) In our finding, both EDA and EDAR had a promotion at 7 days differentiation, and at 14 days differentiation had the highest expression, additional, at 21 days differentiation, the expressions were fall back and were close to h SG cells expression, Lef1 expression had the same trend as EDA and EDAR. ERK and β-catenin expression had a correlation in the differentiation process, and at 21 day were close to h SG cells expression, expressions of BMPR1, BMPR2 and c-Met in i PS-SG during early two weeks of differentiation, were greatly increased, BMP4 and HGF may be the important factors in CM for SG differentiation.(2) BMP4 was secreted by both SG and SG-Fib cells. HGF was only secreted by SG-Fib. A interesting phenomenon lead our attention, the SG-Fib cells with long-term cultured lose some ability to secret HGF.(3) The Realtime-PCR data shown the expressions of EDA, EDAR, C EA, and K8, all the makers were reduced by the antagonists, and the combined effect of two antagonists was evident than one. The contributions of BMP4 and HGF were judged by expressions of EDA, EDAR, C EA, and K8. When added one factor the expressions of EDA, EDAR,CEA, and K8 were increased not much. After adding two factors, it had a significant effect similar to SGDM.Conclusions: The data suggested that BMP4 and HGF were the unknown factors working in CM. Base on our experimental results, we drew a diagram to explain the mechanism how BMP4 and HGF working on the differentiation of SG-i PS-SG. BMP4 combine its receptors BMPR1 and BMPR2, then activate ERK pathway, activate Lef1 expression, and active sweat glands development important genes EDA and EDAR. HGF bond its membrane receptor c-Met, increase expression of β-catenin in cytoplasmic, and up-regulate Lef1 expression, affect EDA and EDAR expression.In conclusion, we isolated sweat gland from infant skin samples. Using reprogramming technology, we induced sweat gland cells turn into SG- i PS cells, retained sweat gland memery make SG- i PS cells more easily differentiated into SG cells. We designed a new method induce SG- i PS cells differentiated into SG cells based on the use of SG cell condition medium. O ur i PS-SG cells can formed tubular- like structures just as SG duct by 3D cultured in gel. We determined two key factors(BMP4 and HGF) play an important role in CM. Furthermore, base on our study, it may provide a new method induce stem cells differentiate into sweat gland cells, supplied an opportunity to study mechanism in sweat gland development, and provided a new way to reconstruct functional sweat gland for severe full-thickness burn injury patients.