PDX1 Combined With Cytokines Induce The Human Umblical Cord Mesenchymal Stem Cells To Differentiate Into Insulin-producing Cells In Vitro

Objectives:To explore PDX1 combined with cytokines induce the human umblical cord mesenchymal stem cells (MSCs) to differentiate into insulin-producing cells in vitro.Methods:1. Isolation, culture and identification of human umbilical cord mesenchymal stem cells.MSCs were isolated from human umbilical cord by digestion of collagenase.Cell activity was detected by MTT. Cell cycle and the expressions of cell surface antigens were detected by flow cytometry. RT-PCR detected the expression of OCT-3. Dexamethasone solution and insulin induced MSCs to differentiate into adipogenic.Dexamethasone solution,β-glycerol phosphate and vitamin C induced MSCs to differentiate into osteoblasts. After induction, the cells were observed by oil red O staining, alkaline phosphatase staining.2. PDX1 combined with cytokines induced MSCs to differentiate into isletβ-like cells in vitro.Recombined adenovirus vectors inserted with PDX1 (Adxsi-CMV-Pdxl, constructed, package and saved in our institute) transfected MSCs for 7 days. After infected, cells were induced by cytokines----epidermai growth factor(EGF), B27, glucagons-like peptide-1(GLP-1),betacelluin,hepatocye growth factor (HGF), nicotinamide (NIC) and P-Mercaptoethanol (β-Me). The genes'expression related to isletβcells such as pancreatic and duodenal homeobox factor 1(Pdx1), Neurogenin3 (ngn3), insulin, glucose transporter-2, (Glut2) and NKX6.1 were detected by RT-PCR. PDX-1, insulin and C peptide in the induced cells were examined by immunocytochemistry and immumofluorescence method. The expressions of PDX-1, NKX6.1and insulin were examined by Western blot. The levels of insulin secretion and C peptide secretion were examined by chemiluminescence immunoassay. The levels of insulin secretion and C peptide secretiont were examined with 25mmol/L glucose stimulation after one hour. Insulin(+) cell rate was detected by flow cytometry.Results:1. Isolation, culture and identification of human umbilical cord mesenchymal stem cells.It took about 24-72 hours for the primary MSCs attachmen. After passage 4, cells were highly homogeneous, spindle and growing whirlpool-like. MSCs expressed C44 and CD29, but didn't express CD34, CD45, CD106, CD14, CD31 and HLA-DR.89.3% of cells was in G0/G1 phase. After induction, the cells were positive for oil red O staining, lkaline phosphatase staining. And MSCs could express OCT-3.2. Identification of isletβ-like cells.After infected by recombined adenovirus Adxsi-CMV-Pdxl 7 days and combined with cytokines induced 3 days, MSCs were aggregated and islet-like cell clusters formed. Dithizone staining of these cells was positive. After induction 10-17 days, the genes' expression related to isletβcells,such as Pdxl, ngn3, insulin, Glut2 and NKX6.1 could be detected by RT-PCR. PDX-1, insulin and C peptide in the inducted cells could be detected by immunocytochemistry and immumofluorescence method. The expressions of PDX-1, NKX6.1 and insulin could be detected by Western blot. After induction 17 days,the levels of insulin secretion and C peptide secretion were(473.11±51.52)mU/L, (1.61±0.41) ng/mL respectively. The levels of insulin secretion and C peptide secretion were (964.42±68.19)mU/L, (3.72±1.52) ng/mL respectively with 25mmol/L glucose stimulation after one hour. Insulin(+) cell rate was detected by flow cytometry is (11.61±4.83)%。Conclusions:1. Mesenchymal stem cells can be isolated, cultured and expanded from human human umbilical cord and they have the ability to differentiate into adipocyte and osteoblast in vitro.2 Adxsi-CMV-Pdxl combined with cytokines can induce MSCs from human human umbilical cord to differentiate into isletβ-like cells. They can secret insulin and c-p, and have the sensitivity to the stimulation of glucose.