Dynamic Changes Of Genomic Variation During Establishment,Subculture And Neuron Differentiation Of Human Embryonic Stem Cell Lines

Human embryonic stem cells (hESCs) derives from the inner cell mass (ICM) of blastocyst during embryo development. As a kind of undifferentiated cells with pluripotency, which are considered as one of the most important seed cells in the regenerative medicine. More and more scientists and clinicians have paid attention to the regenerative medicine.Despite the prospective development of hESCs which can promote wound healing and disease treating through transplantation, differentiation and tissue regeneration, it still has some risks and uncertain factors. It is not yet clear that the hESCs genome’s dynamic variation and genetic stability in long-term subculture and induced differentiation process. Therefore, it is necessary to make sure that hESCs are safe and reliable before it is applied in clinical treatment. Further exploration on the genetic stability of hESCs seed cells and related molecular mechanisms, is of great significance for the hESCs scientific research and clinical applications. But at home and abroad it is still a lack of systematic study on genetic variation from line establishment,long-term subculture and induced differentiation process.This study suggests that we should make clear the genetic characteristics of hESCs when the line establishment is built in early period. Whether the genetic variation will increase accompanied subculture and whether the variation will continue to exist in the subsequent induction of differentiation process are the bottlenecks of the hESCs clinical application. This essay makes use of Human discarded embryos from assisted reproductive technology to establish hESCs, and make further molecular cytogenetics detection on hESCs genome DNA using SNP (Single Nucleotide Polymorphism) based on traditional G-banding karyotype to definite the early hESCs genetic variation characteristics. Then hESCs genome is monitored the dynamic changes of genetic variation during long-term subculture process by SNP and methylation status by methylation chip. Each hESCs was detected by SNP,CNV,LOH and methylation so as to give a comprehensive evaluation of the hESCs genome changes in molecular genetics. hESCs was induced differentiation to neural precursor cells by embryonic pathway and extracted genomic DNA to detect the variation characteristics of neural precursor cell gene group which was induced from different pass algebra of clones by SNP. In this way, we study the effect of genome genetic variation caused by differentiation induction process so as to provide the theoretical basis and practical basis for future cell transplantation security.The main innovation of this study:①Using the abnormal zygotes and poor-quality embryos to establish normal karyotype hESCs and using SNP chip to identify hESCs’ molecular cytogenetics.②Study hESCs genome variation on SNP, CNV, LOH and methylation in long-term subculture on mixed feeder layer.③Research neural precursor cells genome SNP, CNV, LOH changes during during the differentiation of hESCs into neural cells. The investigation is new without the same study.PART Ⅰ Establishment of hESCs from Abolished Human Embryo and Identification of Genetic VariationObjectiveEstablishing hESCs lines and detecting them from overall aspects. Establishing cytogenetic genome SNP chips analytical method for hESCs to analyze genetic characteristics. Establishing the genetic information file of hESCs with known diseases associated CNV genome. Methods1. Collecting IVF-ET(in vitro fertilization embryo transfer) the First Affiliated Hospital of Zhengzhou University, or embryos in the sequential co-culture system reaching to the blastocyst stage. All the patients signed informed consent forms. Injecting-embryo transfer (introcyto plasm sperm injection embryo transfer, ICSI-ET) intracytoplasmic sperm into patients’fresh discarded embryos, sequentially culture them to blastocyst stage. All the patients signed informed consent forms.2. Isolating ICM(inner cell mass) of blastocyst by mechanical method, planting ICM on the cell seeder which is mixed by mouse embryonic fibroblast and human foreskin fibroblast at the ratio of1:1, passaging by mechanical method and establishing hESCs lines.3.Indentifying hESCs pluripotency. Detecting the expression of surface antigen SSEA-1,SSEA-4,TRA-1-60and TRA-1-81by immunofluorescence. Detecting the mRNA expression of OCT4(POU5F1), SOX2and NANOG by RT-PCR.4. Detecting hESCs karyotype by traditional G banding technique, and furthermore detecting CNV and LOH on hESCs genome by high resolution Infinium High-Density HumanCytoSNP-12DNA microarray molecular cytogenetic chips. Analyzing the relationship between hESCs and human diseases related molecular genetic background.Results1.772fresh abolished embryos were collected from432patients undergoing IVF/ICSI. Following sequential culture,117blastocysts were formed. Blastulation occupied15.2%, and58of these blastocysts were good (high) quality, occupied7.5%.117ICMs were achieved and6hESC cell lines were established, one of these hESCs cell lines was derived form OPN embryo, one from2PN IV grade embryo, two from1PN embryos and the rest form3PN embryos.2. Surface antigen SSEA-1in6hESCs cell lines showed a negative expression, SSEA-4, TRA-1-60and TRA-1-81showed positive expression, gene mRNA is expressed in OCT4(POU5F1), SOX2and NANOG detected by PCR. 3.6hESCs can differentiate into embryoid bodies in vivo and express three germ layer markers:Nestin, cardiac troponin and alpha fetal protein. hESCs can differentiated teratoma in vitro.4. The karyotype of NO19P25(3PN),NO20P24(3PN) and NO21P17(2PN IV) is46,XY,detecting by G banding technique.5.7deficiency in NO19P25, and NO20P2.2amplification and5deficiency in the NO21P17.2.98MB deficiency in NO20P2411p11.2-11p11.12.6. Xq28in NO20P24has a length of2.2Mb LOH and84related genes.11p11.2p11.12in NO21P17has a length of3.6Mb LOH and16related genes.Conclusions1. Abnormal zygotes can develop into high-quality blastocyst, and establish normal karyotype hESC lines.2. Analyzing molecular cytogenetic features of hESC lines with SNP microarray can provide identification associated with known diseases.PART II The Dynamic Changes of Genomic Variation and Methylation During the Long-term Subculture of Human Embryonic Stem CellsObjectiveThrough long-term subculture of the established hESC lines, the dynamic changes of cultured hESC’s genetic variation such as SNP, CNV, LOH in the process were discussed.Methods1.Zhl-P16and Zh21-P17are unfreezed by vitrification and long-term subcultured on mixed feeder layer MMC by mechanical method every5-7days.2.Genomic DNA is extracted from Zhl line’s P20/P27and Zh21line’s P27/P60/P68respectively for high-resolution genotyping chip testing on SNP1,140,419Illumina SNPs sites of Human omnil-Quad beadchip to analyse dynamic changes of SNP, CNV, LOH and methylation.3.The above DNA sample is used to analyse the dynamic change of methylation by Infinium HumanMethylation27BeadChip.4.CNV, methylation related gene pathway and the function of mutant gene are analyzed through the KEGG metabolic pathway database (http://www.genome.jp/kegg/pathway.html).Results1.Long-term subcultured hESCs (Zhl-P20, Zhl-P27, Zh21-P27, Zh21-P60, Zh21-P68) are all have normal karyotype including G-banding (46, XY) and FISH (diploid, XY).2. Early passages of Zhl P20->27SNP variants was4.01x10-6, early and late stages of passages Zh21P27->60SNP variants was1.46x10-5, the latter was higher than the former.3. The missing ratio of Zh21-P27’s whole-genome CNV is99.38%, the amplification ratio0.62%. ZH1-P20missing ratio was96.77%, the amplification ratio3.23%; Compared with the1000Genomes Project (the abbreviation for the Han people in Beijing of China, CHB),which is91.20%and8.80%, there was no statistically significant difference.4.5CNV>0.5kb was selected from Zh21-P60/27. There are a total of10>1MB of CNVs in the late Zh21-P68/60algebra. CNV gene pathway analysis shows: three genes metabolic pathways, GABRG1, NEGR1and FOLZH1were involved.5. In the process of long-term cultured hESCs reveals that there are25LOH variation more than1Mb in Zh21P27/60and13LOH variation within the same series of P60/68. In Zhl-P20/27, only the3q26.1has a1.48Mb of LOH.6. Methylation analysis in the process of long-term cultured hESCs was taken. Compared to the early algebra Zh21P27,there are more high methylation sites in the late algebra Zh21P68with the number of196and the number of low methylation sites are91.7. Analasis on remarkable difference methylation genes shows that there are10genes in the late passage Zh21P68which has a significant difference with the comparison to the early passage Zh21P27. These different genes are mainy related to the protein kinase signal pathway.Conclusions1.The dynamic changes of SNP, CNV and LOH exist in the long-term subculture process of hESCs. hESCs are able to maintain genomic stability in early passages, while with the increase number of passages, genomic instability is gradually increasing.2. Before clinical application hESCs with karyotype stability should also be monitored genomic chromosome features by high resolution method during appropriate passage interval.3. High methylation sites in advanced passage increased in the long-term hESCs culture process.4. Analysis of genetic variation related gene pathway helps to understand the biological function of the mutated gene.PART IIIStudy on the Genetic Variation of Neural Induction and Differentiation of Human Embryonic Stem Cells in Different PassagesObjectiveFiguring out the effects on genomic variation CNV during neural induction and differentiation process so as to provide experimental data and theoretical basis for the safety of cell transplantation in the future.Methods1. hESCs is differentiated into nerve ball and neural precursor cells in vitro by EBs pathway. Cell morphology is observed by light microscopy differentiation of neural induction.2. Detecting nerve ball marker Nestin and neural precursor cell-specific markers Nestin and MAP2by immunofluorescence.3. Genomic DNA of neurospheres was used to detect the genetic variation of differentiated neural precursor cells through Infinium High Density Human CytoSNP-12DNA BeadChip.Results1.The changes of light microscopic morphology of neural induction and differentiation:undifferentiated hESCs which were feeded in MMC layers had a clonal nest-like growth. When they were cultured in EB medium, the cloning mass transfered into sphere suspended in culture medium. After induced in nerve-induced nutrient medium and NSC medium, the cloning mass transfered into NSC sphere. The adherent differentiated cells gradually turned into neural-like cells in morphology, and they developed bipolar or multipolar and branching like prominent or synapse.2. Immunofluorescence staining analysis on differentiated cells in late differentiation shows a typical Nestin and Map2double positive neural precursor cells.3. Around neural differentiation, early passage Zhl has the loss of heterozygosity9p21.1and single copy increment10p12.31. After inducement, CNV in nerve bulb has increased by0.01and0.02Mb respectively, but associated gene remain the same.4. No.21p41nerve ball is missing a length0.63Mb in13ql3.2-q13.3, three genes NBEA, MAB21L1and MIR548F5related. NO.21P71neurospheres has an amplification length0.75MB in20q11.21, related to the14the gene DEFB115-124REM1, NCRNA00028, HM13, PSIMCT-1, ID1, COX4I2BCL2L1TPX2MYLK2FOXS1TTLL9, PDRG1, and XKR7.5. There are9samples have3.2Mb LOH of Xp11.21p11.1in Zhl clone P37, P27, P32, P33, P34, P38, P42, P44and P49neurospheres. There are4samples which have3.6Mb LOH of11p11.2-p11.12in Zhl P17,P41clone and P41,P71nerve bulbs respectively.Conclusions1.Differentiating Zhl and Zh21hESCs into neurosphere and neural precursor cells via vitro EB pathway successfully.2. CNV can inherit from hESCs in the process of differentiation, while the CNV gene is unchanged, the length will have a morsel of change and early passage will increase slightly, the advanced algebra showed dynamic changes.3.New CNV will be generated during the differentiation, while late passage will grow adaptively.4.Monitoring will not only be implemented on cultured hESCs cells, but also on the corresponding neural differentiation in generation hESCs so as to indentify the cell’s genetic features and ensure genetic safety of transplanted cells.