PDX1Combined With Cytokines And Controlled Microenvironment Induce Human Placenta MSCs To Differentiate Into β-like Cells

Objective:To establishment the effective methods of isolation, culture, amplification andidentification of human placental MSCs in vitro, and to explore its biologicalcharacteristics and differentiation potential.Method:4-5cm2of heparinized human placental from fetus and purified by density gradientcentrifugation. The human placental MSCs were purified by their adhesiveness. Themorphology of MSCs were observed by phase contrast microscopy, and their expansionfashion at passage3-4was revealed by growth curve. The cell surface markers weredetected by Flow Cytometry and their expressions of stem cell marker SSEA-4and Oct-4were examined by RT-PCR. Their differentiation potential was examined by boneinduction and adipose induction.Result:Primary and passage human placental MSCs showed typically fibroblast-like morphologyand had maintined highly proliferative capacity. The cells showed bone and adiposedifferentiation after21days’ induction. The cells expressed mRNA of SSEA-4and Oct-4,were positive for CD44, CD105.Conclusion:We have isolated and purified human placental MSCs successfully, and successfulidentified them. objective:To construct recombinant adenovirus vector contained human pancreatic and duodenalhomeobox factor1and explore pancreatic and duodenal homeobox factor1(PDX1) Genecombined with cytokines modified mesenchymal stem cells (MSCs) from human placentato differentiate into insulin-producing cells in vitro.Methods:Pdx1with the BglII/XhoI enzyme digestion from the pUC57-PDX1was ligated intopShuttle-GFP-CMV recombinant shuttle vector to obtain the pShuttle-GFP-CMV-PDX1recombinant shuttle plasmid. Then shift pShuttle-GFP-CMV-PDX1to the pAdxsi vector toobtain the pAdxsi-GFP-PDX1virus plasmid. The recombinant plasmid was packaged andamplified in293cells. After24hours, the expression of pdx1was detected in transfectedcell by RT-PCR, immunofluorescence, immunohistochemistry and western blot.Recombined adenovirus vectors inserted with PDX1(Adxsi-CMV-Pdx1) transfectedMSCs for7days. After infected, cells were induced by cytokines----epidermal growthfactor (EGF), B27, glucagon-like peptide-1(GLP-1), beta-cellulin, nicotinamide(NIC),β-Mercaptoethanol. The expression of PDX1, insulin and glucose transporter-2(Glut2)were detected by RT-PCR. PDX-1and insulin were demonstrated by western blot. Insulinand C peptide in the induced cells were examined by immunocytochemistry andimmumofluorescence. The levels of insulin secretion and C peptide secretion wereexamined by chemiluminescence immunoassay. The levels of insulin secretion and Cpeptide secretion were examined with25mmol/L glucose stimulation after one hour.Insulin(+)cell rate was detected by flow cytometry. Results:The Pdx1gene has been inserted into correctly shuttle plasmid and the recombinantadenovirus vector has been successfully constructed according to the results of sequenceand enzyme digestion identification. The adenovirus was transfected into HUCMSCs,RT-PCR verified that Pdx1mRNA was positive in placenta MSCs, the expression of Pdx1protein in the nuclear of placenta MSCs was detected by immunofluorescence assay,immunohistochemistry and western blot. Human placenta mesenchymal stem cells caninfect by Adxsi-CMV-Pdx1efficiently. Specific dithizone staining of these cells waspositive. The protein and genes’ expression related to islet β cells, such as Pdx1, insulinand Glut2could be detected by RT-PCR, immumofluorescence, immunocytochemistry andWestern blot method. After induction10day, the secretion of insulin and C peptide in thetest group were264.32±21.19mU/L、0.21±0.12ng/mL respectively. And in the17day thesecretion of insulin and C peptide in the test group were482.22±56.41mU/L、2.32±0.54ng/mL respectively. The secretion of insulin and C peptide were972.43±65.28mU/L、3.52±0.93ng/mL respectively with25mmol/L glucose stimulation after one hour. Thesecretion of insulin in the control group were (6.81±2.71)mU/L,(11.37±3.25)mU/L and(11.25±1.26) mU/L in the10d,17d and17d+high glucose respectively, the secretion of Cpeptide have not been examined. There are statistically significant differences in the twogroups and in different times of test group (P<0.05). Insulin(+)cell rates were12.31±5.62%and0.00%, in the test group and the control group respectively, which were detected byflow cytometry (P<0.05).Conclusions:The adenovirus vector contained of Pdx1gene is successfully constructed and expressedeffectively in placenta MSCs. Adxsi-CMV-Pdx1combined with cytokines can induceMSCs from human placenta to differentiate into insulin-producing cells in vitro. They cansecret insulin and c-p, and have the sensitivity to the stimulation of glucose. Objective:To investigate the in vivo effect of insulin β-like cells differentiated from human placentaMSCs by micro environmental manipulation on type1diabetes nude mice.Method:The diabetic nude mice were achieved by STZ（60mg/kg）injection. Treated and non-treatedhuman placenta MSCs were transplanted under the renal capsule of diabetic nude micerespectively, Blood glucose, weight and survival time were detected for5and30days aftertransplantation. The graft status was examined by HE staining5days after transplantationand at the end of the observation period. The immunocytochemistry was performed todetect the insulin expression.Result:The transplantation of insulinβ-like cells from human placenta MSCs can decrease thehyperglycemia and prolong the survival time of diabetic nude mice.Conclusion:Differentiated cells transplantation can control diabetes of STZ induced nude mice,indicating human placenta MSCs may be another source of functional β-like cells fordiabetes treatment.