Abstract:

Objective
To evaluate the effects of the INGAP-related pentadecapeptide (INGAP-PP) combined with chemical molecules and cytokines on the differentiation of human adipose-derived stem cells (hASCs) into islet-like clusters. Methods The hASCs  were isolated,purified and expanded in vitro and differentiated using a four-step protocol that included TSA,INGAP-PP/Scrambled peptide (Scrambled-P),nicotinamide and exendin-4. By using real-time PCR and immunofluorescence technology,the expression levels of genes related with pancreas development were detected. The insulin and C-peptide released by differentiated cells in response to high/low glucose level were detected by ELISA.Results Undifferentiated hASCs expressed oct3/4,key markers of embryo stem cells and at the end of induction,the Oct3/4 expression level decreased to 1/20 of original. The expression levels of genes involved in early pancreas development(Ck19,nestin,pdx-1,and nkx2.2) were increased in both INGAP-PP/Scrambled peptide cultures significantly. The islet-like clusters were obtained in both INGAP-PP/Scrambled peptide groups. The diameters of clusters in the INGAP-pp group were much closer to native islets,which were about 150-200 μm,whereas those in Scramble-pp group were about 50-100 μm. Immunocy- tochemical analysis showed C-peptide/insulin positive cells in INGAP-pp induced group but not in undifferentiated hMSCs. Conclusion By INGAP-pp protocol, the hASCs could be differentiated into islet-like clusters secreting the insulin. It suggests that the hASCs could be substitution of embryo or bone-marrow derived stem cells for future cell therapy applications.

Key words: islet neogenesis associated protein;adipose-derived stem cells;differentiation;islet