Cellular Phenotype Transdifferentiation Of Human Adipose Derived Mesenchymal Stem Cells To Epithelial Cells

【Background】Skin defects are common problems in surgery. Wound closure as soon as possible to reduce the body's consumption and disorder within the environment is the key to the treatment of skin defects. Autologous skin graft is the most effective way for large area burns patients of full thickness skin defects in burns surgery clinical treatment. However, autologous thin skin graft show contraction and not rubbing, and the thick skin bring out scar and other problems. With the development of tissue engineering, skin substitutes made substantial progress in research. Seed cells amplification in vitro is the most basic tissue engineering work. In 2001, Zuk et al first isolated and cultured, such as self-replicating, stable and proliferation, and differentiation potential of multipotent mesenchymal stem cells in adipose tissue, called adipose derived stem cells. ADSCs from the mesoderm, under certain conditions can differentiate into fat, bone, cartilage, heart cells, liver cells, nerve cells, etc. ADSCs have the advantages of rich source, easily derived, low immunogenicity, so it has a good prospect in tissue engineering, wound healing and gene therapy. Recent studies show that, ADSCs can be induced to other cells with various phenotypes when cultured in vitro to stimulate the process of adding different factors to simulate the skin's micro-environment.Ramos et al confirmed that implanted the purified ADSCs in nude mice, can significantly promote wound healing, and ADSCs has a capacity of epidermal cell regeneration. Human keratinocytes is important to wound healing, and epidermal growth factor (EGF) is the key factor of wound healing process. EGF can promote cell proliferation, epidermal regeneration, angiogenesis, and also could stimulate epithelial cells synthesis and secretion of collagen, hyaluronic acid and other extracellular matrix, promote the growth of connective tissue cells.Shikiji et al successfully applied EGF to induct human epithelial cells that were cultured on the surface of three-dimension collagen, and then formated tube structure of sweat glands. Therefore, our research use pcDNA3.1 (+) / SP + EGF plasmid liposome transfected HaCat cells induced ADSCs into epithelial cells in vitro phenotype, and find the feasibility of its application in tissue engineering provide a theoretical basis. 【Purposes】1,To explore a method for effective isolation and multiplication of human adipose derived mesenchymal stem cells, observe its biological characteristics and verification by morphology and multi-potential differentiation.2,To explore a method that inverts human adipose derived mesenchymal stem cells into epithelial cells.【Contents】1,ADSCs were isolated and cultured by collagenase digestion, and morphological and biological characteristics of cells were observed by inverse microscope and MTT assay.2,ADSCs were identified by flow cytometry and the induction of different cells types.3,ADSCs and HaCat cells transfected with EGF were co-cultured, then the former could be induced into epithelial cells types.4,Use of exogenous EGF to induce ADSCs into epithelial cells types.【Results】1,The ADSCs in both primary and passage culture were fusiform shapes, and stable in growth with active proliferation. ADSCs were in latency for 2 days, converted into growth period on the 3rd day and entered the stationary phase on the 5th day.2,FCM results indicated that the positive rate of CD29, CD31, CD34, CD45, CD90, CD105 were respectively 73.4%, 3.6%, 4.5%, 2.0%, 97.3%, 80.4%. After purposively induced, ADSCs had the phenotype characterisitics of adipocyte or osteocyte.3,HaCat cells transfected with EGF could induce ADSCs to invert to epithelial cells, which will further make ADSCs as ideal seed cells for tissue engineering.【Conclusions】1,The methods of stirring digestion within collagenase can be effectively used to isolate and purify human ADSCs. The cultured ADSCs are stable in growth with active proliferation and share the general biological characterisitics of ADSCs.2,HaCat cells transfected with EGF could induce ADSCs into the epithelial cells phenotypic, which will further make ADSCs as ideal seed cells for tissue engineering.3,After adding EGF, the mRNA expression level of the epidermal cells surface antigen such as CK19, integrin-βin ADSCs were increased with time up, and the expression level reach to the highest after 4 weeks.4,Reasonable and effective use of ADSCs in wound healing research and has broad prospects for treatment.