Isolation, Culture And Transfection Of Human Fetal Epidermal Stem Cells And Its Differentiation Into Hair Follicle

[ Background] The main purpose of burns therapy is to heal the burns wound as early as possible and restore the function and appearance of the burnt skin as much as possible. In treating severe burns victims, skin autografting has been useful technique and adopt in most of burns centers. However, this technique confronts with many cruxes, such as lack of donor site, increasing the wound of donor site, contracture of grafted thin split skin and lack of skin appendages, etc. Tissue engineering skin is a new method to repair the burns wound. Up to now, many kinds of tissue engineering skin have been developed, in which the epidermis is substituted with sheets of keratinocytes cultured in vitro or autogenous thin-split graft, while the dermis is substituted with acellular dermis or artificial dermis seeded with fibroblasts. Some of the tissue engineering skins have been commercialized and obtained relative good results clinically. However, all of them have a common disadvantage, that is lack of skin appendages, such as hair follicle, sebaceous gland and sweat gland. As adult stem cells in skin, the epidermal stem cells have high proliferative potential and multipotent, and can differentiate into many kinds of cells in skin and structures such as hair follicle and sweat gland. Dermal papilla cells are necessary in inducing epidermal stem cells to differentiating into hair follicle by supplying optimal condition for the niches.Another crux that confronts us is the immunological rejection of the tissue engineering skin. For example, the acellular dermis (ACM), which is most widely used clinically, were processed by a series procedures to lower its immunity by removing the cellular component and cross-linked the collagens by glutaraldehyde. While the angiogenesis of the grafted ACM needs more time than that of autoskin, this would induce the collagen degenerated and its antigenic determinant exposed, as a result, in some cases ACM caused certain immunological rejection response. As for artificial dermis, the seeded adult homogeneous fibroblast is strong antigen. The fetal skin is low immunogenic, thus cause weak immunological rejection when fetal dermis being used to repair burns wound. Fetal fibroblasts also be seeded in some tissue engineering skin to lessen the immunological rejection.It is reported that epidermal cell is a ideal target cell for gene therapy, but the renewing of epidermis makes it difficult for the target gene to express permanently. As the epidermal stem cells remain for the life, and the genetic information of the stem cells can transfer to their daughter cells, therefore be regarded as optimal target cells for gene therapy.[purpose] the purposes of this study are as follows: To obtain regenerated hair follicle differentiated from human fetal epidermal stem cells induced by dermal papilla cells; To fabricate living tissue engineering skin by using human fetal skin cells as seed cells so as to lessen the immunological rejection; To transfer target gene into epidermal stem cells to investigate the feasibility of epidermal stem cells being used as target cells in gene therapy.[ methods]1.Isolation and culture of human fetal keratinocytes in vitro. Dispase was used to separate the epidermis and dermis. Suspension of epidermal cells was prepared with trypsin digestion and reserved in liquid nitrogen to ensure that all the keratinocytes and epidermal stem cells used in following experiments were from one single individual. Primary culture of keratinocytes was done with DMEM containing serum and the medium was gathered. Subcultures weredone with SFM-KC. Cultured keratinocytes were reserved in liquid nitrogen. Adult keratinocytes were cultured with the same methods mentioned above as control.2. Isolation and culture of human fetal fibroblasts in vitro. Dispase was used to separate the epidermis and dermis. Fibroblasts were cultured with trypsin digestion or tissue culturing and the medium was gathered. Cultured fibroblasts were reserved in liquid nitrogen. Adult fibroblasts...