The Epigenetic Alteration Of DLk1-dIO3Imprinted Cluster And X Chromosome Inactivation Status During Human Embryonic Stem Cell Early Culture

1:The effect of early culture on genetic stability and gene expression in human embryonic stem cells (hESCs)Abstract:purposes:Genetic and epigenetic alterations are observed in long-term culture (>30passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures.little information is available on whether similar genetic and epigenetic alterations might occur during the early stages of hESC culture (<30passages). Our aim is to investigate possible genetic and gene expression changes during hESCs early culture.Materials and methods:we compared the results of SNP arrays between initial hESCs (-P5) and early hESCs (-P20-P30), then we examined gene expression changes by gene expression profile between initial hESCs and early hESCs using cryopreserved hESCs from initial cultures (<P5).Results:The CNVs analyzed results from SNP chips showed that only few small CNVs changed during the early culture. Only0.10±0.09%of the transcriptome (12-136of the38500genes/isoforms) showed significant changes (>2-fold,t-test, P<0.05) between ihESCs and ehESCs of the same line. Surprisingly, common pattern of gene expression changes between ihESCs and ehESCs was observed across different lines; i.e., MEG3, SNORD114-3and three other expressed sequence tags in the DLK1-DIO3imprinted cluster were significantly down-regulated in most ehESCs. XIST was silenced in most female ehESCs.Conclusions:The early culture caused minor genetic changes and the major gene expression changes during early culture was the silence of XIST in most female hESCs and the significantly down-regulated of DLK1-DIO3imprinted region. 2:Physiological oxygen prevents frequent silencing of the DLK1-DI03cluster during human embryonic stem cells cultureAbstract:purposes:Dlk1-Dio3region are highly conserved across mammals, which located on the14q32in human, the studies in mouse showed that the Dlk1-Dio3were aberrantly silenced in most of the iPSC clones during reprogramme, which compromised the ability to generate entirely iPSC-derived adult mice. Based on the resuts of chapter1that DLK1-DIO3was frequently silenced in early hESCs, we want to answer these questions.1, whether the snoRNAs and miRNAs encoded by DLK1-DIO3cluster would silenced during early culture?2, whether the silence of DLK1-DIO3cluster was common in other hESCs cultured in other lab?3, the regulation mechanism of DLK1-DI03imprinted cluster.4, whether the MEG3was in a normal imprinted pattern?5, The cause and consequence of DLK1-DIO3silencing.Materials and methods:1, DLK1-DIO3imprinted region also encodes a cluster of microRNAs and snoRNAs, including SNORD114-3, some of which are not covered by the gene expression arrays. We performed Solexa deep sequencing for small RNAs in the ihESCs and ehESCs from five of the lines described above to determine whether these additional genes have the same pattern of expression variation as MEG3and SNORD114-3.2, To further test whether aberrant silencing of the DLK1-DIO3cluster is common in other cell lines, we used real-time PCR to examine the expression of MEG’3and SNORD114-3in an additional20hESC lines from our cell bank and obtained64datasets from GEO database. We compared the differentiation capability of ihESCs and ehESCs from three lines with an initial-on-late-off MEG3expression pattern.3, Imprinting of the DLK1-DIO3cluster is known to be regulated by a differentially methylated intergenic region (IG-DMR) and by DMRs in the MEG3promoter region. To further determine whether silencing of the DLK1-DI03cluster in ehESCs correlates with increased DNA methylation of these DMRs, we examined the DNA methylation status in both ihESCs and ehESCs using bisulfite sequencing.4, In order to evaluate the imprinting pattern of the DLK1-DI03cluster, we performed allelic expression analysis in two single nucleotide polymorphism (SNP) sites of MEGS.5, We compared the differentiation capability of ihESCs and ehESCs from three lines with an initial-on-late-off MEG3expression pattern. We evaluated whether the silencing of MEG3was inherited during differentiation6, In searching for the causes for aberrant silencing of the DLK1-DIO3cluster, we noticed that5%oxygen was a critical factor. Further, we cultured newly derived hESC lines under5%oxygen concentrations and detected the MEG3expression and the degree of methylation of regulated region.7, we shifted the hESCs derived under20%oxygen to the20%oxygen and cultured to p20then detected the MEG3expression level.Results:1, The expression pattern of miRNAs and snoRNAs in DLK1-DIO3cluster correlated with that of MEG3and SNOD114-3from ihESCs to ehESCs.2, Most hESC and hiPSC lines used in laboratories worldwide have undergone DLK1-DIO3silencing during early in vitro culture.3, The silencing of the DLK1-DIO3cluster in ehESCs correlates with increased DNA methylation of the differentially methylated regions.4, The SNP sites were expressed in a monoallelic pattern in ihESCs which suggested that the DLK1-DIO3imprinted cluster was normaly imprinted.5, Silencing of the DLK1-DIO3cluster did not seem to compromise the multi-lineage differentiation ability of hESCs. Both the DLK1-DIO3active status in ihESCs and the inactive status in ehESCs were inheritable during differentiation.6, Furthermore, we demonstrated that5%oxygen, instead of the commonly used20%oxygen, is required for preserving the expression of the DLK1-DIO3cluster.7, late exposure to5%oxygen could not recover the expression of MEG3and SNORD114-3in hESCs established under20%oxygen.Conclusions:We identified in this study for the first time a hallmark epigenetic abnormal alteration event-the silence of DLK1-DIO3imprinted cluster during initial culture in most hESC lines, which is irreversible and transmissible into differentiated progeny Further we found that culturing under atmosphere oxygen level (20%) is the cause of this change, and keeping culture under the physiological oxygen level (5%) is required to prevent this alteration. Our study provides new insight into the importance of early culture conditions on maintaining the epigenetic instability of hESCs, and may help to better characterize the genuine molecular profile of hESCs. 3:Tracing the dynamic changes of X chromosome inactivation during the human embryonic stem cells the derivation and early cultureAbstract:purposes:X chromosome inactivation (XCI) is required for dosage compensation of X-linked genes in human female cells. Several researah groups have described the promiscuous XCI status in long-term cultured female human embryonic stem cells (hESCs), meanwhile, the XCI status in human early embryos was controversial. XIST is a critical gene for the initiation and maintain of XCI. Our aim was to evaluate the dynamic changes of XCI during the derivation period and early culture in the female hESCs and to analyse the effect of oxygen concentration.Materials and methods:1, To determine the XCI status of female hESCs during early culture, we assessed the relative expressional levels of XIST and enrichment of H3K27me3staining by Real-time PCR and immunocytochemistry in20hES cell lines and some differentiated progeny cells.2, we evaluated the two X chromosomes activity by two experiments, a) We tested the transcriptional activity of X chromosomes combined DNA FISH using X chromosomes paints probe and immunofluorescence using RNA polymerase Ⅱ antibody, b) We performed late replication assay to judge whether there is an X chromosome replicated in late S phase, as the indicator of the inactivated X chromosome.3, We performed whole genome gene expression profile in12cell lines including10female samples and2male control samples (all including ihESCs and ehESCs).4, we analyze the XCI pattern of ihESCs and ehESCs in five cell lines, chHES26,45,51,137,175using six X-linked single-nucleotide polymorphisms (SNPs) in the transcribed region.5, Further, we cultured newly derived hESC lines under5%oxygen concentration and detected the XCI satus during early culture.6, We analyzed the XCI status during the derivation of female hESCs including the samples day6, day8, day10, day20after fertilization by immunofluorescence for the H3K27me3and Oct4(n=3), which is indicative of inactivated X chromosome and pluripotency cells.Results:1, Here, we found gradual loss of XCI markers XIST expression and H3K27me3dots in female hESCs and their progeny during early culture and almost all the cells lost the XCI markers after P20.2, We further showed that the ihESCs and ehESCs both had an X chromosome without activity from the chromosme level.3, About5%X-linked genes showed more than1.5folds change up-regulation in female ehESCs.4, Furthermore, the results showed that the the ihESCs showed biallelic expression of all six SNPs and the ehESCs showed monoallelic expression in all five cell lines.5, Most female hESCs cultured under5%oxygen concentrations maintained a normal XCI status during early culture.6, Refer to the period of derivation, the day6blastocysts have an inactivated X chromosome (Xi), then after plating to the feeder cells, the blastocysts will reactivated the Xi and then quickly initiated XCI.Conclusions:In conclusion, we traced the XCI dynamic changes during the hESCs derivation period and early culture. The results hinted the XCI states were unstable and prone to change during early culture. Physilogical oxygen culture was an indispensable condition for keeping in a normal XCI state. The blastocysts had a inactivated X, similar to the case in mouse embryo.