Optimization Of Inducing Human Embryonic Stem Cells Into Insulin-positive Cells

Chapterl The comparison of inducing human embryonic stem cells (hESCs) into Pancreatic endocrine progenitor cellsObjective:Comparing the induction regimens which have been reported in the literature having the higher the efficiency of induciton, to found the best scheme obtained the pancreatic endocrine progenitor cells, under our laboratory conditions.Methods:In vitro, induction of hESCs needs in order to undergo the finite endoderm, the pancreatic endocrine progenitor cells and eventually become insulin positive cells, which is a repeat of the beta cells in vivo development differentiation process. We used the conditions which our laboratory has been set up, inducing hESCs to finite endoderm stage. And then divided into three groups, respectively by induction program which are reported by D ’Amour, Hongkui Deng, Hosoya laboratory, inducing cells into the pancreatic endocrine progenitor cells. Collecting samples in the induction day10and day13, using detection methods of immunofluorescence, flow cytometry, real-time PCR to compare three kinds of induction method, and to find the method which has high efficiency of obtaining the pancreatic endocrine progenitor cells.Results:Only small molecule scheme of Hosoya group during D10to D13the proportion of PDX1+cell apparently increased; D’Amour group showed a significant downward trend; Hongkui Deng group showed basically unbiased. Only small molecule scheme of Hosoya group during D10to D13can continuously detect the large number of NGN3+cells; D’Amour group showed a downward trend, and the proportion of NGN3+cell is very low; Hongkui Deng group barely detectable NGN3+cells.Conclusion:The small molecule scheme of Hosoya group is a better way of gathering the pancreatic endocrine progenitor cells (PDX1+&N GN3+) Chapter2Study on inducing hESCs derived pancreatic endocrine progenitor cells into insulin positive cellsObjective:Comparison of the efficiency of obtains insulin-producing cells, under the condition of basal medium:D/F-12or high glucose-DMEM; the maturation factor solutions or the small molecule scheme of Hosoya.Methods:Using the advantages solution (Small molecule induction regimen) found in the first chapter, the hESCs were induced into the initial phase of the pancreatic endocrine progenitor cells, which was the10th day of induction. The cells were then divided into two groups, one group with the maturation factor of induction, another group continues using the small molecule scheme reported by Hosoya’s laboratory; we were investigated two kinds of induction regimens, under the base medium at different concentrations of glucose. Collecting samples in the induction day18, using detection methods of immunofluorescence, flow cytometry, real-time PCR to compare four kinds of induction method, and to find the method which has high efficiency of obtaining the Ins^cells.Results:High glucose-DMEM added maturation factor group in D18had the highest efficiency of gathering Ins+cells, Its value reaches27.43%±2.97%. D/F-12added maturation factor group, high glucose-DMEM added small molecular group and D/F-12added small molecule group the proportion of Ins+are not reach21%. In D13days we detectded the markers of the pancreatic endocrine progenitor stage, the result showed the relative expression levels of mRNA of PDX1, NGN3, Beta2in the group of maturation factor were several times higher than small molecules groupConclusion:In the maturation factor group, the high glucose can induce β cell proliferation, the low glucose can increas intracellular content of insulin; in the group of small molecules, the concentration of glucose in the medium made no difference of inducing. The reason of high glucose-DMEM added maturation factor group had higher Ins+cells proportion may not be the quantity of pancreatic endocrine progenitor cells(PDX1+&NGN3+), but its expression level of PDX1and NGN3. We can speculate that PDX1、NGN3expression may have a threshold value which is necessary of activate its downstream gene. Chapter3Optimization of inducing hESCs derived pancreatic endocrine progenitor cells into insulin positive cellsObjective:Based on the high glucose-DMEM added maturation factor group, optimization of induction regimens which can efficiently inducing hESCs-derived pancreatic endocrine progenitor cells into insulin-positive cellsMethods:Using the advantages solution (Small molecule induction regimen) found in the first chapter, the hESCs were induced into the initial phase of the pancreatic endocrine progenitor cells, which was the10th day of induction. The cells were then divided into four groups based on the result of the second chapter. The maturation factors in the high glucose-DMEM added maturation factor induction regimen including: Betacellulin, Exendin-4, IGF-1; we made a free combination with these factors, divided into non-factor group, a single factor group, two-factor group, three factors groups. Collecting samples in the induction day18, using detection methods of immunofluorescence, flow cytometry, real-time PCR to compare four kinds of induction method, and to find the method which has high efficiency of obtaining the Ins+cells.Results:In D18, we detected that the three groups have the highest Ins+cells proportion, which were two-factor group Exendin-4&IGF-1, one group of factors of Exendin-4, and IGF-1, their values were:32.63%±8.50%,31.60%±2.16%,30.63%±1.37%. The single factor group Exendin-4in real-time PCR detected the relative mRNA expression level of the islet cell marker genes was significantly higher than the other groups. Immunofluorescence also intuitive reflects its strengths.Conclusion:The optimization based on high glucose-DMEM added maturation factor, finally we Confirmed the high glucose-DMEM added single factor Exendin-4in D18available31.60%±2.16%Ins+cells, which is the highest induction efficiency in our laboratory conditions currently.