Isolation And Identification Of Porcine Fetal Pancreatic Islet Mesenchymal Stem Cells And Their Differentiation Into Insulin Producing Cells

Diabetes mellitus is a devastating disease that has been heavily threatening the health of human beings. Traditional treatment with medicine or insulin injection often does not provide sufficient control of blood glucose and prevent complications of this disease. Islet transplantation offer potential therapeutic options for diabetic patients because this therapy can restore not only the insulin-secreting unit, but also the precise fine tuning of insulin release in response to multiple signals within and outside the islets. However, this therapy has been hampered by the shortage of donor islets. The usage of porcine islet cells is currently viewed as the most promising alternative, as there is a plentiful supply of porcine islet cells; moreover, porcine and human insulins are highly conserved, and porcine normal physiological glucose levels are similar to those in humans. Many studies showed that there are multipotential stem cells exist in pancreas. Under specific conditions, these cells can easily differentiate into functional insulin-producing cells. This study aims to isolate islet mesenchymal stem cells (IMSCs) from porcine fetus older than the age of 2 months, in vitro induce these cells to differentiate into insulin-producing cells, and transplant them into STZ-induced nude mice model for treatment of diabetes to explore the prospects of IMSCs as heterogeneous donor cells.1 Study on isolation and biological properties of fetal porcine pancreatic islet mesenchymal stem cellsDerived from islets, especially fetal islets, IMSCs has greater potential to differentiate into insulin-producing cells. This study was to isolate IMSCs from porcine fetal pancreas older than the age of 2 months by a suspend-to-adhere culture method. Isolated cells were identified by cell growth curve, cell surface antigen analysis, immunohistochemical staining, RT-PCR, karyotype and tumorigenicity analysis, and of in vitro differentiation potential analysis. Results show that, porcine fetal IMSCs can effectively isolate by suspend-to-adhere culture method。Results by detection the genes expression of different passages porcine fetal IMSCs showed that the expression of markers of islet endocrine cells(such as insulin and glucogan)and epithelial cells(such as CK7) of porcine fetal IMSCs was declined gradually, but the expression of mesenchymal cell marker vimentin was enhanced. These results preliminary demonstrated that the porcine fetal IMSCs may derived fromβcells that undergone dedifferentiation process. The population doubling time of porcine fetal IMSCs of passage 14 and 37 were 27.05±1.05 h and 28.35±1.02 h respectively, which showed that the proliferative capacity of porcine fetal IMSCs was not changed after passages. Cell antigen analysis showed that porcine fetal IMSCs express not only the markers of pancreatic stem cells, but also the multipotent markers of embryonic stem cells, and the expression of cell surface antigens of porcine fetal IMSCs was similar to that of bone marrow mesenchymal stem cells. In vitro differentiation potential analysis showed that porcine fetal IMSCs possessed the ability of differentiation into neurocytes and cardiomyocytes. The passage 46 porcine fetal IMSCs showed normal karyotype and formed no tumor in the nude mice. These results indicated that porcine fetal IMSCs has strong proliferation and differentiation capacity in vitro, the characteristics of stem cells, and can provide ample cell resources for tissue engineering and regenerative medicine.2. Differentiation of porcine fetal IMSCs into insulin producing cells in vitro and treatment of diabetes(1) Differentiation of porcine fetal IMSCs into insulin producing cells in vitro: Compared with L-DMEM medium, RPMI 1640 and H-DMEM medium can significantly enhance the secretion of insulin and C-peptide of porcine fetal IMSCs(P<0.01). However, the cells after induction with H-DMEM medium can't regulate the secretion of insulin and C-peptide according to the changes of glucose level, so the RPMI1640 medium was finally chosen as basic inductive medium. Compared with the control group, adding Activin-A could significantly enhance the secretion of insulin and C-peptide of porcine fetal IMSCs (P<0.05 or P<0.01). A two-step induction protocol was devised for the differentiation of porcine fetal IMSCs into insulin-producing cells, in which the serum-free RPMI 1640 medium was used as the basic inductive medium. In the first step, the porcine fetal IMSCs were treated with Activin-A and nicotinamide for 1 week on stick petri dishes. Then the cells were collected by trypsin digestion and seeded onto non-stick petri dishes for another 1 week induction. In this step, exendin-4 and betacellulin were added. In the second step, porcine fetal IMSCs quickly gathered and formed islet-like cell clusters (ICCs), and these ICCs were stained into crimson with DTZ. After 2 weeks induction, ICCs expressed the specific markers of isletβcells (Insulin and Glut2), and the expression of Ngn3, a marker of pancreatic endocrine cells, decreased detected by RT-PCR and immunofluorescent staining. Under stimulation of 25 mmol/L glucose for 2 h, the insulin and C-peptide secretion of porcine fetal IMSCs after 1 week induction were 34.92±6.54μIU/mL/106 cells and 0.23±0.06 ng/mL/106 cells respectively, and that of porcine fetal IMSCs after 2 weeks induction were 287.65±42.24 μIU/mL/106 cells and 0.37±0.11 ng/mL/106 cells respectively, which significantly higher than the uninduced porcine fetal IMSCs(secretion of insulin and C-peptide of this group were 2.16±0.37μIU/mL/106 cells and 0.11±0.03 ng/mL/106 cells respectively) (P<0.05 or P<0.01). These results indicated that porcine fetal IMSCs have the ability to differentiate into insulin producing cells.(2) Transplantation of induced cells for treatment of diabetes:The diabetic nude mice model was successfully established by intraperitoneal STZ injection, and the rate of molding was 100%(17/17). The transplanted cells were labeled with CM-DiL, a fluorescent dye, and the labeling rate of the cells exceeded 90%. After labeling with CM-DiL, the induced porcine fetal IMSCs were transplanted into the left testes of diabetic mice. The body weight and blood glucose level of mice were detected regularly. The left testes of diabetic mice transplanted with induced cells were removed after one week and one month respectively to observe the survival situation of transplanted cells by slice fluorescent staining. The normal mice and diabetic mice injected with PBS or uninduced cells were used as control. The body weight of diabetic mice, including the mice transplanted with induced cells, decreased. However, that of normal mice increased gradually. These results indicated that the body weight of diabetic nude mice was not increased after cell transplantation. After transplantation, the blood glucose levels of diabetic mice injected with PBS or uninduced cells maintained at a high level (>16.7 mmol/L), but that of diabetic mice transplanted with induced cells quickly declined, and it approach to normal level on the sixth day, then it increased gradually and maintained at a high level(>16.7 mmol/L). The glucose level of normal mice maintained at a low level (<8mmol/L). These results indicated that porcine fetal IMSCs has the ability to ameliorate the hyperglycemia of diabetic nude mice。Analysis of slice fluorescent staining showed that there are large amounts of cells, which coexpressed Insulin and CM-DiL, existed in testes after one week transplantation of induced cells. However, the number of these cells declined after one month. These indicated that the transplanted cells could not survive a long time in vivo.3. Construction of a porcineβcell specific promoter system and their detection of insulin producing cells derived from porcine fetal islet-derived mesenchymal stem cellsA porcineβcell specific detection system was established by using the porcine insulin promoter (IP) and pEGFP-1 vector. After transfection, primary fetal porcine islet cells expressed the green fluorescent protein (GFP), while porcine fetal IMSCs did not; this indicated that the expression of GFP has specificity. After induction, the porcine fetal IMSCs transfected with IP-pEGFP vector expressed GFP, and it coexpressed with insulin. RT-PCR and Western blotting analysis showed that after induction, the expression of insulin and GFP increased simultaneously, which indicated that this system can be used for identification of insulin-producing cells derived from porcine stem cells, and has important application value on selection of induction protocols and sorting the insulin-producing cells from induced cells.