Skewed X Chromosome Inactivation And Imprinted/X-linked Gene Expression In Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) can be maintained in culture in a self-renewing state and can be unlimitedly passed from generation to generation. These cells can also be induced to differentiate into all three embryonic germ layers, which hold great promise for restoring damaged tissues and in treating diseases. HESCs are important materials for study of epigenetic mechanisms, genetic and epigenetic stability in hESC is most necessary factor to ensure the safety and quality of those cells for regenerative medicine. X chromosome inactivation (XCI) is one of the first measurable epigenetic phenomenons. In this study, we investigated XCI status of hESC by using methylation specific PCR based on the human androgen receptor (HUMARA) gene and by real-time PCR or immunofluorescence analysis in six hESC lines which were established in our laboratory. Among six distinct hESCs, all lines had active and inactive X chromosomes in very early passage during the undifferentiated stages, irrespective of their karyotype. Four lines have an extremely skewed XCI pattern whereas one line exhibits random XCI status. The skewed XCI pattern in the hESCs can maintained stably upon differentiation. Two active X chromosomes were found in the triploid hESCs, and the XCI pattern of those cells is varied after long-term culture. Proper regulation of imprinted genes and X-linked genes were also found in hESCs. In addition, unstability genetic and epigenetic status was found in a parthenogenetic human ES cells. These results remind us that more careful epigenetic and functional analyses of hESCs should to be conducted to demonstrate whether they are safe and effective for regenerative medicine.Part ICulturing and identification of biological characteristic of human embryonic stem cell lines established from discared embryosObjective: Six well-established female hESC lines were cultured for the follow-up propuse to providing experimental materials. Biological characterizations of these cells were identificated.Materials and Methods:1. Maintained the hESCs at the undifferentiated stage, and the ESC colonies were fixed to the four-well plates for staining of ESC markers such as SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81.2. HESCs were incubated for Giemsa staining every 10 passages. At least 20 cells were examined in each group for the chromosome analysis.3. Genomic DNA and total RNA were extracted from undifferentiated hESCs from different passges and EBs from day0, day3, day7 and day20, respectively.4. Expression of Class I molecular markers in hESC lines were detected by RT-PCR5. In vitro differentiation: After suspension-cultured for 7 days, the formation of embryoid bodies (EBs) was examined and Total RNA of the EB cells was extracted for analysis of the differentiated marker genes.6. In vivo differentiation: The colonies were collected and subcutaneously injected into SCID-beige mice. The resulting teratoma was examined histologically using standard protocol.7. Short tandem repeat analysis: Genomic DNA was extracted from each cell line, and STR analysis was conducted using the Promega PowerPlex 16 System Kit.Results:1. Chromosome analysis showed that FY-hES-7, -8, -10 and -11 had normal 46, XX karyotype, FY-3PN had 69, XXX karyotype, while FY-hES-5 was an unbalanced Robertsonian translocations with 46, XX, +13, der(13;13)(q10;q10).2. All six lines strongly expressed TRA-1-60, TRA-1-81, and SSEA-4, but not SSEA-1.3. OCT-4, NANOG, TDGF1, SOX2, EBAF, THY-1, FGF4 and REX-1, which are specific marker genes for undifferentiated hESC, were expressed in all cell lines.4. All cell lines had the differentiation ability in vitro and in vivo.5. Each cell line showed distinct 16 STR loci indicating that they were derived from different embryos.Conclusion: 1. In order to making the results more reliable, all cells were cultured under the same conditions. It is demonstrated that the culture method and culture syetem in our lab are appropriate.2. The hESC lines established in our lab are healthy, showing normal hESC characteristics and differentiation ability.3. STR loci analysis is vital for the correct identification and verification of hESCs in culture. It gives the district separate cell lines a sound files for the future applications.4. The hESCs with abnormal karyotypes may be derived from discarded embryos and cells can undergo self-correction during subsequent culture. In the present study, self-correction was not observed in the triploid hESC line (FY-3PN) and unbalanced Robertsonian translocations cell line (FY-hES-5).Partâ…¡X inactivation and imprinted / X-linked gene expression in female hESCs Chapter OneXCI pattern in hESCs with normal and abnormal karyotypesObjective:To explore whether the XCI pattern in the hESCs with normal and abnormal karyotypes during propagation and differentiation is stable and to compare the difference of XCI pattern among these cells.Materials and Methods:1. Extracted DNA was amplified to analyze the polymorphism patterns of the HUMARA gene in female hESCs. Only the heterozygous cell lines were further analyzed for random or non-random XCI pattern.2. Genomic DNA was bisulfite treated using the EpiTect Bisulfite Kit (QIGEN). Then a methylation-specific PCR was performed, and sample tubes were loaded into a Genetic Analyzer 3100 for fragment analysis.3. The expression levels of XIST gene of hESCs at different times were investagete by using Real-time PCR amplification. 4. Undifferentiated hESC colonies were fixed to the four-well plates for immuno-fluorescent staining of histone H3 lysine 27 trimethylation (H3K27me3) (Cell Signaling, USA).Results:1. Of the six investigated cell lines, five (FY-hES-5, -8, -10, -11 and FY-3PN) were heterozygous while one (FY-hES-7) was homozygous at the HUMARA locus. Three different HUMARA allele loci were found in FY-3PN.2. At the undifferentiated stage, all of the six female hESC lines had both active and inactive X chromosome alleles.3. For skewing XCI analysis, four cell lines (FY-hES-5, -8, -11 and FY-3PN) had extremely skewed XCI patterns. One line (FY-hES-10) had a random XCI pattern. The XCI pattern in these cells can be maintained stability after differentiation. Two active X chromosomes in FY-3PN varied after long-term culture, and the XCI pattern of FY-3PN varied from random to skewing.4. The XIST expression level in all of the hESCs can be detected at the undifferentiated stage. Variable XIST expression levels in these cells after differentiation were also found.5. Immunofluorescence staining of H3K27me3 methylation of hESC at undifferentiated stage suggested that XCI was initiated before differentiation.Conclusion:1. MS-PCR of the HUMARA gene has become a standard and effective method to analysis of XCI skewing.2. Two types of XCI pattern were found in the female hESCs. Most hESCs fell into one category where the XCI pattern was extremely skewed while the other category includes hESCs with random XCI.3. Unlike XCI in mouse ESC, hESCs had initiated XCI at the undifferentiated stage. The skewed or random XCI patterns in diploid hESCs can be maintained stabe duing the cell propagation and differentiation.4. The variable XIST gene expression levels suggestted that the ability to capture or maintain XCI markers in distinct cell lines may not be equivalent. Most of the cells have initiated XCI prior to differentiation, and completed XCI after differentiation.5. Methylation H3K27me3 indicate that these cells initiated XCI at undifferentiated stage.6. The hESCs with abnormal karyotypes have undergone XCI prior to differentiation; the XCI pattern in these cells was similar to the pattern of hESCs with normal karyotypes.7. Selective pressure may lead to epigenetic fluidity in hESCs, especially in triploid hESCs when cultured in vitro.8. Two active X chromosomes in the FY-3PN may lead to twice X-linked gene expression activity than those in normal hESCs, which may be an important epigenetics factor to causes abortion in the triploid embryos.Chapter TwoImprinted/ X linked genes expression and methylation status of gene promoter region in hESCsObjective:To explore expression levels of X-linked gene PGK1, X-linked tumor suppressor gene and oncogene and autosomal imprinted gene in hESCs with skewed XCI pattern, and to investigate whether there is a relationship between the expression levels of these genes and the shewed XCI patterns. Another aim is to explore whether the methylation status of promoter region of important genes located on X chromosome or autosomal was stable during propagation and differentiation.Materials and Methods:1. Quantitative real-time PCR was used to detect the temporal expression of gene PGK1, X chromosome tumor suppressor gene RBBP7 and oncogene GPC4 at different times.2. Paternally imprinted gene H19, IGF2R and maternally imprinted gene SNRPN were analyzed using real-time PCR at different times.3. The expression levels of OCT4, NANOG were investigated in these cells at different times after spontaneous differentiation.4. Genomic DNA was bisulfite treated using the EpiTect Bisulfite Kit (QIGEN). Then a methylation-specific PCR was performed. The products were analyzed by 7% polyacrylamide gel electrophoresis.Results:1. PGK1 showed up-regulated expression following the time after differentiation in both hESCs with normal and abnormal karyotypes.2. RBBP7 and GPC4 showed up-regulated expression after differentiation in the hESCs with abnormal karyotype whereas down- regulated in normal hESCs.3. Similar imprinted gene expression levels were found in the hESCs with normal and abnormal karyotypes: H19, IGF2R showed up-regulated expression following the time after differentiation whereas SNRPN showed down- regulated.4. In the normal hESCs, the expression levels of OCT4, NANOG were significantly decreased after differentiation. In contrast, the decreased levels were not obviously in the hESCs with abnormal karyotypes.5. Stable maintenance of unmethylated and methylated alleles of the XIST and SNRPN gene promoter region occurred in all of the cell lines irrespective of their different XCI patterns and pluripotent stages.Conclusion:1. The expression levels of PGK1 and imprinted genes in normal and abnormal hESCs are similar, indicate that an appropriate order of these genes expression in hESCs.2. The up-regulated expression levels in oncogene and tumor suppressor gene in abnormal hESCs may be a great hurdle to the application of hESCs, and these cells have owned stronger self-renewal and weaker differentiation ability when compared to the normal hESCs.3. Stable DNA methylation status and normal imprinting pattern were preserved in the hESCs with normal and abnormal cells.Partâ…¢Primary study of genetic and epigenetic stability in one hpESC lineObjective:1. To investigate the genetic sability of a hpESC line cultured in vitro.2. To study the XCI status and imprinted gene expression levels in the hpESCs during propagation and differentiation. Materials and Methods:1. Culture the hpESCs in vitro, genomic DNA from the cells and oocyte donator was performed to analyze STR and HLA typing.2. HpESCs were incubated for Giemsa staining every 10 passage. At least 20 cells were examined in each group for the chromosome analysis.3. Extracted DNA was amplifie to analyze the polymorphism patterns of the HUMARA.4. Genomic DNA was bisulfite treated and a methylation-specific PCR was performed.5. H19, IGF2 and SNRPN were selected for investigation at different times in hpESCs.6. The expression levels of OCT4 gene were investigated at different times after spontaneous differentiation.Results:1. HLA typing indicated that the HLA-A, -B and DRB loci of hpESCs were hemizygous and matched to the oocyte donor. The 16 STR loci also indicated that the hpESC line was originated from the donor's ooctye.2. The genetic status of hpESC was unstable. At passage 20, the karyotype of these cells was 46, XX, and has a 46, XX/45, X mosaicism at p30. At p60, a normal 46, XX karyotype was maintained again.3. Polymorphism analysis showed that hpESC was homozygous at HUMARA locus.4. DNA methylation of both X chromosomes was globally reduced in hpESCs at p20. But XCI in these cells was initatied at p60. The skewed or random XCI pattern cannot be distigulished because of it was homozygous at the HUMARA locus.5. No expression of XIST gene was detected at early passages. After long-term culture, epigenetic status of these cells changed, XIST gene expression could be detected accompanied by the occurrence of XCI.6. No expression of paternally pimprinted gene SNRPN and IGF2 can be detected.7. The expression of OCT4 can be detected in hpESCs even after 20 days differentiation, which may be a reason for it weak ability to differentiation.Conclusion:1. The precious cell line in our laboratory is indeed a hpESC line.2. The homozygous hpESCs have instability genetic characteristics.3. Two active X chromosomes may be the possibility reason for its X chromosome instability. Selection and self-correttion of X chromosome was happened when the cells were cultured in vitro, and finally the X chromosome in hpESCs initiated inactivation process.