Studies On The Induction Of Human Adipose-derived Stem Cells Differentiating Into Pancreatic Islet-like Clusters To Cure Type â…  Diabetes

Part I:Objective:To isolate, purify and characterize human adipose tissue-derived stem cells.Methods:With slightly modified protocol demonstrated by zuk et al, human adipose tissue-derived stem cells (hASCs) were isolated and expanded in vitro. FACS analysis was performed to identify the cell surface and cytoplasm antigen.Tolemerase repeated amplification protocol (TRAP) and G-banding Karyotype analysis were performed to evaluate the safety of hASCs cultured in vitro. Results:Since passage 4, hASCs developed a uniform morphology resembling that of fibroblasts, FACS analysis of the fourth passage cells showed purified hASCs expressed high levels of cell surface markers, such as CD73(99.1%),CD90(97.9%), CD105(97.2%). These results are consistent with the definition that mesenchymal stem cells (MSCs) must express CD73, CD90, and CD105. Also, extremely low expression levels of CK-19 and Pdx-1, which supposed to be markers of pancreatic stem cells, were detected. Negative expression of CD45 means no contamination of hemopoiesis stem cells. TRAP test revealed P4, P8, P12 had nomal tolemerase activity and G-banding analysis showed P4, P8, P12 and P16 had normal katyotypes. Conclusions:hASCs could expand in vitro efficiently after being isolated and purified. The hASCs we cultured in the study were consistent to those reported previously. When cultured in vitro, hASCs have stable chromosome structure as devided in vitro and being safe for cytothrapy.Part II:In Vitro differentiation of human adipose tissue-derived stem cells into Insulin/c-peptide positive cells by embryonic pancreas extractObjective:To evaluate the effect of embryo pancreas extract (EPE) from pre-born Sprague-Dawley rats on induction of pancreatic differentiation of human adipose tissue-derived mesenchymal stem cells(hASCs).Methods:By using real-time PCR and immunofluorescence technology, expression levels of genes related with pancreas development of hASCs cultured with EPE for 20 days were detected.Results:undifferentiated hASCs expressed key markers of embryo stem cells. Genes involved in early pancreas development showed increased expression in EPE-treated culture. Immunocytochemical analysis showed C-peptide/insulin positive cells in EPE-treated cells but not in undifferentiated hASCs.Conclusion:hASCs have the potential to differentiate into pancreas cell lineages with EPE induction, so could be substitution of embryo and bone-marrow derived stem cells for future cell therapy applications.Part III:IN Vitro Differentiation of human adipose-derived stem cells intoIslet-like cell clusters by INGAP-pp protocolObjective:To establish a novel method for the differentiation of islet-like clusters from human adipose-derived stem cells with INGAP-related pentadecapeptide (INGAP-PP) combined with chemical molecules and cytokines.Methods:the human adipose-derived stem cells 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, expression levels of genes related with pancreas development. 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, Oct3/4 expression level decreased to 1/20 of original. The expression level of genes involved in early pancreas development increased in both INGAP-PP/Scrambled peptide culture significantly. Islet-like clusters were obtained in both INGAP-PP/Scrambled peptide group, but in INGAP-pp group, the diameters of clusters were much closer to native islets, which were about 150-200μm. Immunocytochemical analysis showed C-peptide/insulin positive cells in INGAP-pp induced group but not in undifferentiated hMSCs.Conclusions:By INGAP-pp protocol, hASCs could differentiate into islet-like clusters more efficiently through Mesenchymal/epithelium transition. It suggests hASCs have the potential to differentiate into pancreas cell lineages by INGAP-pp protocol; so could be substitution of embryo or bone-marrow derived stem cells for future cell therapy applications.Part IV:Objective:To evaluate the effects of transplantation of ILCs induced by INGAP-pp and INGAP-pp injection to cure diabetic rats.Methods:Diabetes was induced in 8-week-old male Sprague-Dawley rats by streptozocin (Sigma,55 mg/kg body weight in citrate buffer, pH 4.4, i.p).48 hours after injection, animals with blood glucose levels≥16.65 mmol/L for two consecutive days were transplanted with ILCs induced by INGAP-pp (200IEQ/l00μ1 High glucose DMEM) into the left subrenal capsule (n=10). As controls, fresh isolated human islets (200IEQ/100μ1 High glucose DMEM) and saline without cells were transplanted respectively. Two transplantation group were administered acrolimus (50ng/kg.d,p.o.) to avoid of immune rejection. To evaluate the effects of INGAP-pp protein on remission of diabetes, we also injected purified INGAP-PP (250μg/0.5mL, i.p.) to diabetic rats twice a day for twenty days. Blood glucose levels of all groups were measured every three days. Glucose tolerance tests were performed after injection of glucose (2g/kg bodywight, i.p.). Diabetic rats transplanted with normal islets and normal rats served as positive controls. Diabetic rats were used as negative control. Results:Blood glucose levels of rats transplanted with INGAP-induced ILCs fell to 7.8±0.8 mmol/L and kept this level for the whole experimental process while diabetic rats transplanted with normal islets, as a control, showed blood glucose levels similar to normal rats after 1-2 weeks. In addition, non-transplanted diabetic rats remained hyperglycemic and died consecutively. Interestingly, the group treated with INGAP-pp protein injection also showed a decrease of blood glucose level after 5-day injections, but never fell to normal level and re-raised 2 weeks later since the day INGAP-PP injection stopped. Diabetic rats transplanted with INGAP-induced ILCs or normal islets survived the whole 69-day process while diabetic rats without treatment suffered from hyperglycemic and died off within 51 days. Diabetic rats undergone INGAP-pp injections also all survived until the end of experiment. Intraperitoneal glucose tolerance tests showed SD rats transplanted with ILCs induced by INGAP-pp had the similar kinetics of the glucose clearance to the group transplanted with normal islets, although the blood glucose levels were higher at all time points. In contrast, untreated diabetic rats revealed no reaction to the high glucose concentrationConclusions:The results of transplantation of ILCs and normal islets to diabetic rats showed ILCs did decrease the blood glucose levels and extended the life-span of diabetic rats. ILCs transplantation reversed diabetes partially in animal models.