Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells Into Insulin-producing Cells

Objective: The purpose of this study is to combine the small molecule compounds such as activin A,nicotinamide,trichostatin A,transferrin selenium,exenatide-4 to gradually induce human umbilical cord mesenchymal stem cells(hUC-MSCs)differentiated into functional insulin-producing cells(IPCs),establishing an efficient and stable chemical induction differentiation scheme,and laying an experimental foundation for stem cell treatment of diabetes.Methods: The hUC-MSCs were isolated,cultured and passaged by adherent culture method,and identified by cell morphology,induction culture of adipogenesis,osteogenesis,chondrogenesis and cell flow cytometry analysis.Using chemical induction method,the fourth to sixth generations are phased in with small molecule compounds such as activin A,basic fibroblast growth factor,epidermal growth factor,trichostatin A,nicotinamide,exenatide-4 hUC-MSCs induce differentiation into IPCs,identify islet cells by dithizone staining,and detect the expression of genes and proteins related to insulin development at various stages of differentiation by agarose gel electrophoresis,q-PCR,and cell immunofluorescence,and by human insulin ELISA analysis detects the function of insulin-producing cells.Results: 1.hUC-MSCs successfully been induced into bone cells,chondrocytes and adipocytes by in vitro differentiation.2.The positive rates of CD90,CD44 and CD73 markers on the surface of hUC-MSCs detected by flow cytometry were all over 90%,and the positive rates of CD34,CD45 and CD14 were below 5%.3.After induction of hUC-MSCs by small molecule compounds for 22 days,staining with dithizone was positive.4.After 22 days of induction by hUC-MSCs,the expressions of β-cell related genes insulin and Glut-2 were up-regulated.5.After induction of hUC-MSCs for 22 days,β-cell expression of protein,peptide C,and Pdx1 was detected by cellular immunofluorescence.6.After 22 days of induction of hUC-MSCs,the in vitro insulin release test was positive by ELISA.Conclusions: hUC-MSCs can be successfully differentiated into functional IPCs in vitro through the chemical induction protocol.