| Objective: The present study was performed to: (1) Initiate a more comprehensive analysis of the transcriptional characteristic of genes and their biologic significance that occurred in development of human fetal skin and sweat glands and discuss the regulatory mechanism of the development of sweat glands in the molecular level. (2) Investigate the patterns of MSCs phenotypic conversion and gene expression in the course of MSCs co-cultured with thermal injured SGCs, and discuss the feasibility of stem cells plasticity in regenerating or rebuilding the losing sweat glands. Look for specific genes expressed in the process of inducing MSCs differentiate into SGCs in the defined medium, and explore the evidences in MSCs trans-differentiation potential and their therapeutic implications in controlling wound healing and SG regeneration.Methods: (1) Human fetal skins were obtained from different bodies regions of abortuses of 8-36 weeks embryonic gestational age (EGA) within half an hour after abortion. The morphological characteristics of skin at different developmental stages were compared using HE methods, and broad-scope gene information of skin and its appendages in the development of human fetus were analyzed using immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and gene chip methods. (2) Human MSCs and SGCs were isolated, cultured and expanded in vitro respectively. The antigens expression of hMSCs and hSGCs were detected by two-steps immunocytochemistry. The confluence SGCs were heat-shocked at 47 ℃ for 40 min and cooled for 1~2 hours at 37℃, then hMSCs were added later and co-cultured with hSGCs. Immunocytochemistry, RT-PCR and Western blotting methods were performed on hMSCs, hSGCs and fusion cells to detect differential genes or proteins. (3) Based on the above-mentioned studies, we investigated the effects of different culturefactors on the plasticity of hMSCs trans-differentiating into hSGCs. Biomarkers of sweat glands cells were detected by flow cytometry and Western blotting methods.Results: (1) Structure changes of normal human fetal skin and sweat glands at different developing stages: skin samples of human fetuses from EGA 8W to EGA 36W were studied. Human periderm was occurred at the pregerm stage (EGA<10W). Basal layer cells of the primary epidermal ridge exhibited focal aggregation and formed hillocks at EGA 12-13W. The first wave of coat hair development is initiated around EGA 12-14W. Sweat glands begin to develop as a cord of epithelial cells and grow from the epidermal ridge to deep dermis on the palms and soles during EGA 13-15W, and on the rest of the body during about EGA 20-22W. Sweat glands of fetuses at the EGA 32-33W already resemble those of adults. Two types are recognized, namely, eccrine and apocrine sweat glands. The average density of eccrine sweat glands varies according to persons and anatomic sites. Children and adults have the similar numbers of eccrine sweat glands. (2) Molecular biologic technology including immunohistochemistry, RT-PCR and gene chip offered tremendous potential for characterizing programming and distinctive spatiotemporal gene expression in developing fetal skin and SG. (3) The isolation technique of sweat glands from pieces of skin under stereomicroscopes greatly contributed not only to the physiological study but also to cytologic and functional studies of sweat glands. Using living sweat glands, elemental analysis of hSGCs was carried out. (4) There was higher percentage of fusion cells in the coculture of hMSCs with heat shoked hSGCs compared with that of spontaneous cell fusion. HSGCs markers and readjusted hMSCs phonotypes are analyzed to address the possibility of fusion. (5) HMSCs had multiple trans-differentiation potentials and could be induced into hSGCs in vitro under defined microenvironment, which have been identified by the methods of flow cytometry, immunocytochemistry and Western blotting.Conclusions: (1) The development of fetal skin is the result of a complex interplay of proliferation, cell-to-cell communication, inductive events, andcellular movements. Gene expressions show specific time- and tissue-dependent patterns and achieve proper regulation of cell proliferation and differentiation in developing epidermis and SG. Gene chip, otherwise known as cDNA microarray expression profiling, and other technologies offer tremendous potential for characterizing gene expression patterns during normal skin and SG developing processes, as well as for identifing of differentially expressed genes that may play an integral role in these processes. The data initiate a more comprehensive understanding spatiotemporal expressions and functions of genes that occurred in developing fetal skin and SG, which is of great benefit to control epidermal stem cell differentiating into defined cell type. (2) Cellular phenotype and gene expression profile could converse toward hSGCs-like during hMSCs cocultured with injured hSGCs or induced hMSCs differentiate into hSGCs in the defined medium in vitro. In addition, the utility of fusion or induction-mediated reprogramming for future cell-based therapies is discussed. The data suggested that hMSCs would surely become a seed cells making for regenerating or rebuilding the SG and trauma skin if we explore more underlying mechanisms about hMSCs transdifferentiation. |
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