Differentiation Of Human Embryonic Stem Cells Into Insulin Producing Cells On Mixed Feeder Layers Cells

Diabetes mellitus is characterized by hyperglycemia, which is the most common metabolic disorder. Although the mechanism of the diabetes mellitus is not fully elucidated, it is mainly related to genetic factors and environmental factors. The oral medication and exogenous insulin replacement therapy is the major method for the diabetes mellitus, but it does not offer a cure and does not prevent the secondary complications associated with diabetes. Recent studies have emphasized the importance of a ideal method of treatment for the diabetes mellitus, Pancreas or islets transplantation have been demonstrated to be an effective and promising method to cure TypeⅠdiabetes and partly TypeⅡdiabetes.Human embryonic stem cells are pluripotent cells and have the ability to differentiate into cells representative of all three body lineages:ectoderm, mesoderm, and endoderm. Take advantage of this ability of pluripotent, human embryonic stem cells differentiate into insulin-producing cells, which offers new directions of cell transplant therapy of the diabetes mellitus.At present, there have been many reports of successful differentiation from hESCs to insulin producing cells. But the efficiencies of differentiation were generally low. This becomes a major obstacle to future researches. So it is imperative to improve the differentiation efficiency. In this study, we differentiated human embryonic stem cells on mixed feeder layers into insulin producing cells through embryoid body formation and adherent monolayer culture without EB formation which have involved supplementation of differentiation medium with a variety of induction and growth factors. To explore a high-performance differentiation protocol that can obtained mature and functional insulin-producing cells for hESCs is the major problem that will be settled in this study.ObjectiveTo get insulin producing cells from human embryonic stem cells by different differentiation protocol and compare the efficiency.Methods1. The experimental material were hESCs lines which were established by the reproductive medical center of the first affiliated hospital of Zhengzhou university, which cultured on mixed feeder layer by the human foreskin fibroblast feeder layer and mouse embryonic fibroblasts feeder layer.2. Grouping for the concentrations of Activin A (25ng/ml,50ng/ml and 100ng/ml) in the differentiation of hESCs into definitive endoderm cells.3. Grouping for the induction protocols was done according to differentiated hESCs through embryoid body formation and adherent monolayer culture without EB formation. The induction protocol is made up four stages.4. Using immunofluorescence positive for Sox-17, diphenyl thiocarbazone staining, RT-PCR to detect the expression of insulin producing cells and the insulin release experiment to detect the secretion of insulin of thess insulin producing cells5. We tried to figure out the better induction protocol for differentiation of hESCs into insulin producing cells on mixed feeder layers cells.Results1. With the extension of differentiation time, the shapes of hESCs have been different. 2. According the results of immunofluorescence positivity for Sox-17, differentia-tion of hESCs in the presence of 50ng/ml Activin A had no significant statistical difference with the presence of 100ng/ml Activin A(P>0.05),25ng/ml Activin A had the least efficient(P<0.05).3. According the results of diphenyl thiocarbazone staining, RT-PCR and the insulin release experiment, we founded differentiation hESCs into insulin producing cells by adherent monolayer culture without EB formation could achieve more insulin producing cells.Conclusion1. Differentiation of hESCs in the presence of 50ng/ml Activin A could achieve definitive endoderm cells as much as in the presence of 100ng/ml Activin A. These cells provide the material for our studies of insulin producing cells differentiation on cellular level.2. The best induction protocol is differentiation hESCs by adherent monolayer culture without EB formation into insulin producing cells through four stages.