Experimental Study Of The Biological Characteristics, Cell Surface Molecules, Adipogenic And Osteogenic Potential Of Human Adipose-derived Stem Cells

Objective To investigate the basic biological characteristics of human adipose-derived stromal cells(hADSCs) and their surface protein expression,and to explore their osteogenic and adipogenic potential in vitro.Methods Human adipose tissue was obtained from the abdominal subcutaneous adipose tissue of health patients under the aseptic condition,then the hADSCs were isolated with typeⅠcollagenase digesting method and adherent method and cultured in DMEM containing 10%FBS.The phase-contrast microscope was used to observe hADSCs every day and the viability of 1st,3rd,6th,and 10th passages was assessed by cck-8.The growth cure and doubling time were drawn.Cell cycle and surface proteins include CD 10, CD11b,CD13,CD14,CD18,CD34,CD45,CD55,CD56,CD59,HLA-DR and CD71 on the 1st,3rd,and 6th passage were analyzed by flow cytometry technique,and the data obtained was statistically analyzed by computer with the software SPSS 11.0 for windows,hADSCs from the 3rd passages were induced into osteogenic and adipogenic lineages by different revulsant for 14 days.The viability of hADSCs after induced was also assessed by cck-8.To confirm osteogenesis,cells were examined by RT-PCR for the expression of several genes,including OCN,OPN and OPG and by von Kossa/alkaline phosphatase staining,and the OPG protein expression was further confirmed by Western blotting.Adipogenic differentiation was identified by RT-PCR for the expression of PPARγ,and C/EBPa genes and by Oil Red O staining of intracellular lipid droplets. Results①Morphous of primary and passage hADSCs:hADSCs were fibroblast-like and could rapidly expand.②Growth curve and doubling time of hADSCs:The growth curve of 1st,3rd,6th,and 10th passages were similar,and like "s" shape.The doubling time of hADSCs was about 36 hours.③The Cell cycle of hADSCs:The majority of cells under undifferentiated conditions were in G0/G1 phase(80%).④Surface marks of hADSCs:No significant difference among the 1st,3rd,and 6th passages could be demonstrated concerning the expression of these surface marks.Expressed proteins include CD10,CD13,CD55,CD59 and CD71,while CD11,CD14,CD18,CD34,CD45, CD56 or HLA-DR are not expressed by undifferentiated hADSCs,P＞0.05.⑤After osteogenic induction for 14 days,the OCN,OPN and OPG genes were detected by RT-PCR, and the OPG protein expression was verified by Western-blot assay,too.As we know,no literature regarding OPG gene and protein expression has been published.The results for alkaline phosphatase and Von Kossa staining were also positive.In the adipogenic induction,PPARγ,and C/EBPa genes can also be detected by RT-PCR,and the results for Oil red O staining were positive.Conclusion hADSCs can be isolated from human abdominal subcutaneous adipose tissue and most stayed in G0/G1 phase,hADSCs can express the proteins include CD10, CD13,CD55,CD59 and CD71,and the present method we used to isolate and culture hADSCs has no obviously influence on the expression of the surface proteins.It has the potential to differentiate into osteogenic and adipogenic lineage and may be an ideal seed cell source for tissue engineering.