In Vitro Human Adipose Derived Stem Cells Differentiate Into Insulin-producing Cells

Objective:To explore a method for effective isolation and cultivation of hADSCs, observe morphological feature, growth kinetics, surface marker expressions as well as differentiation potential. To investigate expression of Insulin,Nestin, CK19in the course of hADSCs differentiate into insulin-producing cells induced by multiple chemical regents in vitro.Methods:hADSCs were isolated and cultured from adult subcutaneously adipose tissue by collagenase type I. Growth curve of hADSCs in3rd and8th passage was measured by cells counting, and vimentin was detected by immunocytochemistry. The phenotypic analysis including CD34. CD45,CD44. CD90,CD105was tested by flow cytometry. hADSCs were observed differentiation potential towards the osteogenic and adipogenic lineages. The hADSCs in3rd~5th were induced towards insulin-producing cells through three developmental stages. Firstly, hADSCs were incubated in H-DMEM supplemented with2-mercaptoethanol and FBS for6days. hADSCs were then induced by H-DMEM containing bFGF,EGF,2-mercaptoethanol,B27and BSA for another6days. Finally, hADSCs were cultured in H-DMEM supplemented with nicotinamide,2-mercaptoethanol,B27and BSA for another6days. In the control group, hADSCs were incubated in H-DMEM. The morphological changes of hADSCs in different medium were observed under phase contrast inverted microscope,and the induced cells were detected by dithizone staining. At days6,12and18, the expression of Insulin,Nestin and CK19was tested by immunohistochemistry.Results:hADSCs appeared fibroblast-like, spindle shape and homogeneous arranged parallel under phase contrast inverted microscope. hADSCs were still in latent phase after being subculture for2~3days, followed by logarithmical proliferation from4 days, reached the growth platform at7days. Serial subcultivation of hADSCs, its growth became slow, and gradually appeared senescent. hADSCs could be subcultured until passage9. Immunohistochemical staining showed hADSCs in P3expressed vimentin. FCM results indicated that the positive rate of CD34,CD45, CD44,GD90,CD105was1.5%,0.0%,100.0%,98.6%,99.5%, respectively. After adipocyte like cells committed induction, hADSCs changed to round and with lipid vacuoles accumulated in the cytoplasm displayed positivie staining with oild-red O. Calcium nodules could be seen after osteogenic induction and alizarin red S staining was positive. At the first stage of being induced towards insulin-producing cells, hADSCs had a good refractive capacity, whereas the number of cells was decreased. Both dithizone staining and immunohistochemistry testing of Nestin,Insulin,CK19were negative at6days. At12days, the induced cells gradually become round, Nestin positive was detected by immunohistochemistry, but Insulin and CK19were negative. Dithizone staining was no signal. After18days of induction, induced cells could be self-assemble to form clusters. Dithizone staining was positive, whereas Nestin, Insulin and CK19was negative. In the control group, dithizone staining and immunohistochemistry detecting were no signal.Conclusion:hADSCs can be effectively isolated from adult subcutaneously adipose tissue with collagenase type I, which possess the multiple differentiation potential. During human adipose derived stem cells differentiate into insulin-producing cells, induced cells appeared Nestin positive tested by immunohistochemistry as well as Dithizone staining was shown to contain insulin-positive cells.