Orthotopic transplant fetal rat pancreatic stem cells for treating diabetes mellitus

AIM: To observe the possibility of differentiation of fetal rat pancreatic stem cells into islet-like cell cluster by transplantation of fetal rat pancreatic stem cells into pancreatic parenchyma in diabetic SD rats. METHODS: The pancreatic stem cells (PSCs) were harvested from pancreatic rudiments of SD rat embryos on embryonic day 16. SRY DNA was examined to discriminate gender by fluorescence in situ hybridization (FISH). The pancreatic stem cells were identified by nestin and PDX-1 immunostaining and flow cytometry. Adult SD rats were divided into three groups including 10 pancreatic parenchymal orthotopic transplantation, 10 experimental controls and 10 normal controls. In orthotopic transplantation group, 1×10⁶ male fetal pancreatic stem cells per rat were injected into diabetic rat pancreatic parenchyma while in experimental control group equivalent volume of PBS was injected into diabetic rat pancreatic parenchyma. Glucose and insulin level in serum were monitored periodically. 8 weeks after transplantation pancreata were excised for histological and morphometric analysis. SRY DNA was detected by FISH. Nestin, PDX-1 and insulin mRNA expression in pancreata were detected by RT-PCR, insulin and PDX-1 protein contents were assessed by Western blotting. RESULTS: 5 of 12 fetal rats were male according to FISH. After passaged 3 generations, the PSCs expressed nestin and PDX-1 according to immunostaining while identified by flow cytometry with 74.1% of PSCs expressed nestin. The orthotopic transplantation of PSCs led to stable reduction in hyperglycemia and increase in insulin level in serum (3 weeks after transplantation), culminating (5 weeks post-transplantation) in restoration of normoglycemia which remained steady during the course of experiment without further relapse. Exogenous islet-like cell clusters were found and expressed SRY DNA in the orthotopic transplanted recipients pancreata 8 weeks post-transplantation. The expression levels of insulin mRNA and protein in the orthotopic transplanted recipients pancreata were higher than those in experimental control (P<0.05), and the expressions of PDX-1 mRNA and protein were also higher than those in normal control (P<0.05). CONCLUSION: When orthotopic transplant into pancreatic parenchyma PSCs from fetal rat differentiates into islet-like cell cluster, gains comparable function with normal islets and reverses experimental diabetes.