Differentiation Of Menchymal Stem Cells Derived From Human Fetal Liver Into Islet β Like Cells By TAT-PDX1 Protein

ObjectivesTo study the basic biological characteristics of the mesenchymal stem cells derived from human fetal liver(hFLMSCs),the ability of TAT-PDX1 protein to induce hFLMSC_s to differentiate into isletβ-like cells in vitro,and the therapeutic effects of these cells when transplanted into the renal subcapsular space of NOD/SCID diabetic mice.MethodsCells were isolated from the aborted fetuses of 12-20 week's gestation.The hepatic tissue was cut,and then pipetted to obtain the hepatocyte suspension.The non-parenchyma mesenchymal cells were collected through two-step centrifugation, and then the red blood cells were removed by hydroxyethyl starch sedimentation to enrich the nuclear cells.The cells in suspension were inoculated and cultured,and MSC_s purification was done by adhesive screening method(because different kinds of cells have the different adherence to the wall of culture flask).The cell cycle and surface markers of hFLMSC_s were identified using immunochemistry and flow cytometry.Karyotype analysis of hFL-MSC_s was made at passage 8 after recovery from cryopreservation for 6 months.The hFLMSC_s were induced by routine inducing protocols to differentiate toward adipocytes and osteoblastes.The hFLMSC_s were induced to differentiate toward isletβlike cells by TAT-PDX1 protein primarily in virto.Morphological change was observed under microscope.When the figures of the islet-like clusters changed,the expression of islet cells related genes were detected by RT-PCR,and the islet-specific proteins were tested by immumofluorescent staining.Dithizone(DTZ) staining was performed to identify the Zinc in the islet-like clusters.In addition,the quantity of insulin secretion and intracellular insulin were examined by chemiluminescence immunoassay.The test of glucose-stimulated insulin release was made to evaluate the function of the islet-like clusters.Western blot was conducted to identify the secretion of insulin.A diabetic model in NOD/SCID mice was established by intraperitoneal injection of streptozotocin.Finally,the islet-like clusters were transplanted into the left renal subcapsular space of diabetic mice.Blood glucose levels were dynamic monitored after transplantation.The changes of diabetes symptoms were observed.The level of insulin and C-peptide was examineded before and after transplantation.The test of intraperitoneal glucose tolerance test was made to evaluate the function of the cells.The grafts from the left kidney of the diabetic mice were detected the expression of isletβcells related genes with immunohistochemistry and RT-PCR.ResultsThe hFLMSC_s can be isolated and purified from human fetal liver.There is over 90%hFL-MSC_s of passage 3 in G0/G1 phase,hFLMSC_s expressed adhesion molecules of CD90,CD105and CD166,but not antigens of hematopoietic CD14,CD34 CD45,and not antigens such as AFP and HLA-DR.The hFLMSC_s showed normal karyotype at passage 10 after cryopreservation for six months.Exposure of hFLMSC_s to routine inductive agents resulted in morphological changes towards adipocytes,osteoblasts.When hFLMSC_s were induced by TAT-PDX1 protein primarily in virto,they changed quickly into round and oval shape and gathered more and more islet-like clusters.RT-PCR showed the islet-like clusters expressed islet-related genes including Neuro D,Nkx2.2,Nkx6.1,Pax4,Pax6,Isl-1 and PDX1.The islet-like clusters were stained crimson red by dithizone indicating the high zinc content in the cells.The immumofluorescence demonstrated that the islet-like clusters were positive for Insulin and C-peptide by Laser scanning confocal microscope.Chemiluminescence immunoassay demonstrated that the insulin accumulation quantity of secretion was increased and maintained at a higher level with the time.The test of glucose-simulated insulin release showed the islet-like clusters could elevate the insulin secretion upon glucose challenge.The results of Western blot showed that insulin were present in the protein extraction of the islet-like clusters.NOD/SCID mice were easily into models of diabetes mellitus by intraperitoneal injection of streptozotocin.After the islet-like clusters were transpanted into the left renal subcapsular space of diabetic mice,the blood glucose levels decrease gradually, but control animals that did not receive transplants exhibited persistent hyperglycemia.There was a significant difference in the level of insulin and C-peptide before and after transplantation.The test of intraperitoneal glucose tolerance showed these cells had the response to glucose.When the left kidneys that contain the transplanted cells were removed,the hyperglycemia reappeared.Immunohistochemistry staining showed the transplanted cells under the kidny capsule were positive for insulin and C-peptide.RT-PCR showed the transplanted cells under the kidny capsule expressed human islet-related genes including Nkx2.2,Nkx6.1,Pax6,Isl-1 and PDX1.ConcusionsThe MSCs from the aborted fetal liver of 12-20 week's gestation can be cultured and expanded in vitro,hFLMSCs strongly expressed CD90,CD105,CD166,but no CD14,CD45,CD34,HLA-DR and AFP was found.The hFLMSC_s have the ability to differentiate into the derivative cell types,moreover,they have a lower immunogenic activity and A long time to maintain a normal karyotype,so provide the ideal source for tissue engineering and cellular therapeutics.The hFLMSC_s can be induced to differentiate into isletβ-like cells with TAT-PDX1 protein primarily and have the ability of Stable secretion of insulin in vitro.When these cells were transplanted into the left renal subcapsular space of diabetic mice,the blood glucose levels decrease gradually and steadily.The grafts cells removed,the hyperglycemia reappeared.