The Proliferation And Differentiation Of Adipose-derived Stem Cell Into Cardiomyocyte

Myocardial infarction is one of the diseases with highest morbidity and mortality in the world.The end result of ischemic myocardial damage has long been considered irreversible as cardiomyocytes have been viewed as terminally differentiated and nonproliferating.Clinical studies are under way to investigate the safety and feasibility of cell implantation in patients, an alternative approach to injection or infusion of isolated cells into the heart is using the method of tissue engineering to design artificial cardiac muscle constructs in vitro for later implantation in vivo.Cells,scaffolds and biological factors are three main elements in tissue engineering.Adipose-tissue derived stem cell(ADSC) is a promising cell for tissue engineering because it can be easily and considerably obtained,and it also has the renewable and multi-potential abilities.So we isolated ADSC with improved method,and examined the proliferation and differentiation abilities of ADSC.With improved method,about 5×10~5 stem cells could be obtained from 400～600mg adipose tissue.And ADSCs can be continuously cultured in vitro for up to 1 month without passage and they have several logarithmic growth phases during the culture period.Also the flow cytometry analysis showed that ADSCs expressed high level of stem cell-related antigens(CD13,CD29,CD44,CD105 and CD166),while didn't express hematopoiesis-ralated antigens CD34 and CD45,and human leukocyte antigen HLA-DR was also negative.Meanwhile stem cell related transcription factors,Nanog,Oct-4,Sox-2 and Rex-1,were positively expressed in ADSCs.The expression of alkaline phosphatase was detected in the early osteogenic induction and the calcified nodules were observed by von Kossa staining.Intracellular lipid droplets could be observed by Oil Red staining.And differentiated cardiomyocytes were observed by connecxin43 fluorescent staining.In order to obtain more stem cells,we can subculture ADSCs every 14 days in stead of normal 5 days. ADSCs still keep strong proliferation ability,maintain their phenotypes,and have stronger multi-differentiation potential after 25 passages.Our future approach with ADSC is combinational therapies with genes.Collagen and chitosan all have good biocompatibility and biodegradability,and can be used as scaffold and gene carrier.However,collagen has low mechanical strength,and chitosan is a rigid crystalline structure.If we combine collagen and chitosan to fabricate polymer scaffold, the disadvantages of the two materials should be avoided.We mixed 2%chitosan with 3.55mg/ml rat tail typeⅠcollagen with different volume ratio:9:1,7:3,5:5,3:7 and 1:9 (collagen:chitosan),the mixtures were then freeze-dried and cross-linked with...