The Research Of The Passage Methods And Genetic Stability Of The HESCs

Human embryonic stem cells(hESCs) derives from the inner cell mass(ICM) ofblastocyst during embryo development. They have unique properties includingself-differentiation, self-renewal, and multi-directional differentiation. The research ofhESCs are concerned more and more by the science researchers.The aim to research the hESCs is to applied to clinical therapy, but we need toclear the safety ahead of time. Exploring the genetic stability of the hESCs is vital tothe safety. A lot of factors can effect the genetic stability of the hESCs, such as themethod of passage, the culturing time and so on. Because of the long-term subcultureof the hESCs, the genetic stability of the hESCs will change. And the differentmethods of passage will make it change too, such as enzymatic bulk passage andmechanical method. Genetic stability variations during hESC cultivation have beenfound by G-banding karyotype analysis. Recently, SNP(Single nucleotidepolymorphism) is gradually used in hESCs because of its more accurate detection.Human Omni ZhongHua-8Bead Chip is invented by Illumina Company, which canconver the common and rare genetic variants of the Chinese, and accurately responsethe genetic variation characteristics. Using the SNP in hESCs which is root in theembryos from Chinese can be more conducive to evaluate genomic stability variationof the hESCs.This study aims to explore different methods of passage, analyze the moleculargenetic change of the hESCs by SNP during long term passage, and at last provide the theoretical basis and practical basis for the hESCs. Main innovation point of thisstudy: using Human Omni ZhongHua-8Bead Chip to detect the genetic stabilitychange of the hESCs root in the embryos from Chinese.ObjectiveTo explore different methods of passage adapt to the hESCs form our embryonicstem cell research center, and the changes of genetic stability during subculture.Methods1. Use the hESC established from the embryos from the First Affiliated Hospitalof Zhengzhou University, the pluripotency of which has been identified. Use differentmethods of passage, such as the traditional mechanical method, collagenase IVenzymatic method, trypsin enzymatic method and so on. And then plant the stem cellon the cell seeder which is mixed by mouse embryonic fibroblast and human foreskinfibroblast at the ratio of1:12. Extract the genomic DNA from the P35/P60of Zh2line for high resolutionHuman Omni ZhongHua-8Bead Chip to detect the molecular genetic changes.Results1. The collagenase IV enzymatic method takes a long time, and is difficult tocontrol the time of the enzymic digestion and the inoculum density; Because the0.25%EDTA-trypsin enzymatic method disperses the hESCs to single-cell state, it isnot suitable to grow after planting to the cell seeder; The mechanical method canartificially operate to get rid of the differential clone and cut the clone into differentsize as required, so it was more suitable.2. Using the Omni ZhongHua-8Bead Chip can detect mutations in1130sitesand930mutation genes between the Zh2-P35and the Zh2-P60. Among them, thevariation of chromosome19is the largest. And the genes involves multiple pathways,which are relevant to the growth, differentiation, apoptosis of the cell, the organdevelopment such as nerves, eyes, kidneys, sex, and the pathways such as ECG,Caand so on. Conclusions1.The mechanical method of passage is more adapted to the hESCs than theenzymatic bulk passage.2. After the long-term subculture, the hESCs can not keep the genetic stability,and exist genomic variations. The mutated genes involve multiple pathways by whichwe can ascertain the biological function of them.