Study On Induction Of Human Embryonic Stem Cells Differentiation Into Insulin-producing Cells

Islet transplantation orβcells replacement therapy are taken as a promising approach for diabetes treatment,but widespread application of this treatment is restricted by absence of islet source and immunological rejection.Progresses made in human embryonic stem cells(hESCs)field brought hope for settling the shortage of islet source.Many reports have obtained insulin-producing cells from ESCs using different strategies,however,these all have various limitations and there is lack of an optimal protocol producing functionalβcells from ESCs.To explore a high-performance differentiation protocol that can obtaine mature and functional insulin-producing cells from hESCs is the major problem that will be settled in this study.At first,this study explored the condition to culture,amplificate,freeze and reanimate the hESCs line PKUl and the expression of several specific markers of hESCs were detected.Results showed,after high passage,hESCs expressed OCT-4 and Nanog gene,remained positive for OCT-4 and SSEA-4,and the percentage of OCT-4- and SSEA-4-positive cells was respectively 89.38%and 83.44%.Chromatosome karyotype analysis showed the 114 generation hESCs karyotype is normal female(46,XX).Thus,the study successfully established the hESCs culture system that could maintain their stem cell features.In order to realize the morphology and character of islets during embryo development and provide reference for identification of the insulin-producing cells derived from hESCs,the fetal islets gestation age between 14-18 weeks were isolated by enzymatic digestion and cultured in suspension for 3 days.After DTZ staining,fetal islets were changed to tissue culture dish,then some islet markers and precursor cells markers were identified by immunofluorescence.The insulin and C-peptide content in culture supernatant was measured by radioimmunoassay (RIA).Results showed,fetal islets were spherical clusters and were positive for DTZ,insulin and C-peptide were detected in the culture supernatant,and some of the cells in the epithelioid cell colonies were positive for insulin and glucagon, and several precursor cell markers such as PDX-1,CK19 and Nestin.From this, fetal islets of gestation age between 14-17W have some features of islet and some precursor cells.This study designed a proposal of five stages minic embryo pancreas development.Firstly,withdraw the inhibitor of differentiation and hESCs formed embryonic bodies(EBs),after 2 days the EBs were planted to 0.1%gelatin-coated culture dishes and cultured in DF12 medium containing serum replacement(SR) supplemented with bFGF,Activin A and LY294002 for 5 days(stage 2),then the cells were dissociated with trypsin and planted to lminin-coated culture dishes in DF12 medium containing low fetal calf serum(FBS)supplemented with bFGF, KGF,EGF,GLP-1 and nicotinamide(stage 3).After 14 days,bFGF,EGF and KGF were withdrew and replaced by IGF-1 and BTC and cultured for another 14 days(stage 4).The cells were dissociated with collagenaseⅣand transferred into low-attachment plates and cultured in RPMI-1640 medium supplemented with GLP-1 and nicotinamide for 3-5 days(stage 5).Ihe induction effects were identified by RT-PCR,immunofluorescence,DTZ staining and RIA.The indexes markers included definitive endoderm(DE)markers such as SOX17 and FOXA2, pancreatic markers PDX-1 and Ngn3 and islet markers insulin,glucagons and C-peptide,and the insulin and C-peptide content in the last stage culture supernatant and glucose-stimulated insulin and C-peptide release analysis were carried out by RIA.The results showed,the cells induced in the stage 2 for 5d expressed SOX17 and FOXA2 genes,their expression levels were higher than that of the same days EBs,and the FOXA2 level increased with the induction time.The immunofluorescence displayed the cells were positive for SOX17 in nuclei.After stage 3,the cells were positive for PDX-1 and expressed pancreatic precursor marker PDX-1 and islet precursor marker Nng3 gene,even a very low expression of Pax4.After stage 4,the induced cells expressed Insulin gene and expression level raised with induction days,part of them were positive for insulin and C-peptide.At last stage,the hESC- derived cell clusters were spherical just like fetal islets,and were positive for DTZ.RT-PCR showed,the cells expressed Insulin and Glucagon gene.By RIA,insulin and C-peptide content in culture supernatant were similar with that were detected in fetal islets culture supernatant. Intracellular insulin and C-peptide content was 1089.2±217.06μIU/ml and 181.33±30.12ng/ml,and insulin release from the ICCs was regulated by glucose.Thus,results presented in this study provide credence that the five stages protocol differentiated hESCs into mature insulin-producing cells and can offers a promising source of cells for diabetes cell transplantation therapy.