Culture Epidermal Stem Cells In Vitro And Used In Reconstruction Of Tissue Engineering Skin

Epidermal tissue integrity is maintained by division of cells in the proliferative basal layer to replace differentiated cells in the outmost stratum corneum layer that are continually lost.This hierarchy of cell proliferation and differentiation is maintained by a small subpopulation of stem cells.It has been assumed that epidermal stem cells (ESCs) retain a high capacity for self-renewal throughout adult life and are ultimately responsible for epidermal maintenance and repair. With the development of tissue engineering skin, it become more exigent to find suitably seeding cells. Due to the character of epidermal stem cells, it maybe have valuable for the reseach for seeding cells.Besides, only the stem cells remain and continue to proliferate for the lifetime of the epidermis,which replies that any genetic treatment must be directed towards the stem cell genome.In this study, epidermal stem cells were cultured in vitro and used in reconstruction of tissue engineering skin.1. The difference of epidermal stem cells in adult human and foetus skin: Study special markers of epidermal stem cells: cytokeratin 19 ( Ck19 ) and @1-integrin expression in adult and foetus skin, to compare the keratinocyte stem cells between adult and foetus skin, and to determine the location of ESCs. The results demonstrate that epidermal stem cells exist in the basal layer and hair follicles, and much more in foetus. It suggested the number of ESCs reduce withage and reflect the biological change of skin.2. Experimental study on expression of cytokeratin 19 in cultured human keratinocyte: To explose the existence of epidermal stem cell in cultured human keratinocyte under laboratory conditions, and to observe the growing course of keratinocyte. The results demonstrate the technique for isolation of human keratinocytes can gained nearly all of the cells of basal stratum including epidermal stem cells.3. Selection and identification of human epidermal stem cells in vitro: To select human keratinocyte stem cells in vitro. According to the characteristic of ESCs which adhere to extra-cell medium(ECM) very fast, we selected 4 groups in different time (5 minutes, 20 minutes, 60 minutes and control group) to observe. And all the cells were identificated by immunohistochemistry, used monoclone antibody of β1-integrin and cytokeratin 19, then did image analysis. Furthermore we analysed the cultured cells with transmission electron microscope(TEM). The results demonstrate that the cells of 5 minutes group are shown have a slow cell cycle, characteristics of immaturity, and behaving like clonogenic cells in vitro. These cells have the general properties anticipated for ESCs. So the ESCs can be selected by rapid attachment to extra-cell medium and identified by monoclone antibody of $ i-integrin and Ck19.4. Culture of epidermal stem cells in five different media: The epidermal stem cells were cultured in five different media, FAD, FAD1(+), FAD2(+), FAD3(+), K-SFM and used the same fetous fibroblast as the feeder cells. The proliferation ability was investigated by cell growth curve, MTT detection. Results showed that the epidermal stem cells growed best in FAD(+) supplemented with bFGF and feeder cells.5.Study of biological characteristics of epidermal stem cell: To observe the epidermal stem cells' biological characteristics, which were investigated through phase-contrast microscope, cell growth curve, BrdU detection and FCM analysis.The results showed that the epidermal stem cells retained proliferative capacity and formed larger, more expadnsible clonies in vitro.Besides these, most of the cells show a Go/Gi cell cycle profile.6.Construction of tissue engineering skin of full thickness by use of the epidermal stem cells: To study the epidermal stem cells used to tissue engineering skin. We select 3 groups(the pure ESCs group, kerationocyte group, the two cells mixed group ). Fibroblasts were seeded into bovine type I collagen gel and cultured for 3 days. The 3 sorts of cell...