Reprogramming Of Human Umbilical Cord Mesenchymal Stem Cells In Vitro To Promote Ex-parcreatic Cell Differentiation

Objective:To explore the potentiality of inducing human umbilical cord mesenchymal stemcells(huMSCs)to differentiation into pancreatic precusor cells in vitro by ectopic expression ofpdx1, ngn3,mafa.Methods:1 .Isolate and primary culture huMSCs, Characteristic of huMSCs and the markers ofimmunogenicity were tested by flow cytometry2 .Adenoviral expansion and preparation:Using AD293 cells to expand adenovirus and dilutedgradient,then test the concentration,stored in -70 centi-degree.3. Ectopic expression of key transcription factors (pdx1,ngn3,mafa) of the development ofβ-cell using adenvirus as gene delivering vector. Immunocytochemical methods were used todetect adenovirus transfected into huMSCs.The proliferation of huMSCs were indentified byCCK-8. 4. 4.To certify ectopic expression of key transcription factors (pdx1,ngn3,mafa) inhuMSCs differentiated into pancreatic precursor cells:The morphology of huMSCs weremonitored by under inversion phase contrast microscope after infection withadenovirus.Reverse Transcription-Polymerase Chain Reaction and Quantititative ReverseTranscription-Polymerase Chain Reaction was used to detect human gene glucagon,neuroD,pdx1,nkx2.2.Result1. Five to seven days later,fibroblast-like cells migrated out from the adherent fragment . Arandomly selected cell population at 3-5 passage was characterized by a number of markersOct4(45.2%),CD29(89.64%),CD59(91.19%),CD80(0.11%),CD86(0.08%),CD40(0.03%),CD40L(0.07%).2. The morphology of huMSCs do not changed after transfected adenovirus for three or sevendays;Fluorescent microscope showed adenovirus were transfected into huMSCs.The resultof CCK-8 showed that the tendency of proliferation were no significant differencescompared with control group at multiplicity of infection (MOI) equal to 50. 3. Reverse Transcription-Polymerase Chain Reaction showed glucagon and nueroD1 gene,andgene sequencing match about 93.21%,93.91%.Quantititative ReverseTranscription-Polymerase Chain Reaction showed that human gene glucagon(GCG) ,pdx1and nkx2.2 expression.3.1 The combination of three gene (pdx1,mafa,ngn3) transfected huMSCs for 3 or 7daysincreased the GCG gene 21-folds,the secondary was the combination with mafa andngn3(14-folds,15-folds).GCG gene expression for inducing 3 and 7days had nodifference.Mafa was the key transcription factors in inducing GCG gene.3.2 Pdx1 gene increased the most about 15-folds when activated by three gene together,andcultured until 7 days the level of gene did not elevate.Ectopic gene mafa transfectedhuMSCs individually increased pdx1 gene for 6-folds,but combination with pdx1 orngn3 had no promotion. Pdx1 increased endogenous pdx1 gene for 15 folds three day toseven day,and enhanced the transcription level by the time.3.3 Nkx2.2 increased 8-folds when transfected with ad-mafa,the secondary was thecombination ngn3(4.5-folds), induced for 7days each group had nodifference.Comparesion with inducing for 3days and 7 days, nkx2.2 gene levelincreased except ngn3 individually and mafa and ngn3 together transfected huMSCs,px1 induced individually can increase for 11-fold. Pdx1 and enhanced the transcriptionlevel by the time.ConclusionEctopic expression of pdx1,ngn3 and mafa in huMSCs in vitro converts cells into pancreaticprecursor expression endogenous gene GCG,pdx1,nkx2.2,neuroD. Our research show ectopicgene activated endogenous gene exsist variability in time and different gene combination haveantagonism,when endogenous gene expression to some level,do not increased by the time.Ourreseach provide the methods and fundament for the differentiation of HuMSCs toinsulin-producing cell.