| Human embryonic stem cells (hESC) derived from inner cell mass of humanblastocysts can proliferate unlimitly in vitro and have the ability to differentiate intoall three embryonic germ layers. Therefore, hESC hold great promise in regenerativemedicine, studying embryos development and drug discovery. However, there aresome ethically controversy in these hESC lines which derived from fertilized embryos,and one of the main obstacles to their clinical application is that transplantation ofthese cells might lead to immunological rejection. But the derivation ofparthenogenetic embryonic stem cells (hpESC) does not involve the destruction ofviable embryos, and MHC genotypes are mostly homozygous, so it can reduce therisk of immunological rejection and represent one potential source for regenerativemedicine and stem cell therapy. Because lacking of the paternal genes, the epigeneticstability of these hpESC is still unclear. Before used in clinic, hpESC should maintainthe stability of epigenetic during long term cuture in vitro. In this study, weinvestgated the genomic imprinting of two hpESC lines and one nomal hESC linewhich cultured under different oxygen tension by using the whole genome expressionprofile microarray and quantitative real-time PCR, tried to define the influence factorof epigenetic stability is the genetic background of cell lines or the culture system invitro and evaluated the safty of using hESC and hpESC in clinic. In our study, the two hpESC lines can maintain the typical biological characteristics of hESC with normalkaryotype. In addition, hypoxia enhance the expression of pluripotency related genesby increasing the HIF-2a expression level.It was also found that different oxygentension could change the expression levels of imprinted genes in hESC and hpESC,and the varied reaction caused by oxygen tension was depend on the differentimprinted genes. In hypoxia, HIF may be the key factor that regulates the epigeneticstability of human embryonic stem cells. Although under hypoxia, long term culturestill influences the stability of imprinted genes in hESC and hpESC. Therefore, longterm culture is the primary factor that affect the safety of using hESC and hpESC inclinic when compared to oxygen tension.Part â… Culturing and identification of biological characteristic of humanparthenogenetic stem cells under hypoxic conditionObjective:1. Establish a stable hypoxic culture system (5%O2) for further research.2. In5%oxygen tension, Culture and identify the biological characteristic of twohpESC lines (FY-phES-018and chHES-32) and one normal hESC line (FY-hES-7)which derived from fertilized embryos.Material and methods:1. A triple gas incubator which provide a5%O2and5%CO2air mixture, was used forthe hypoxic condition, the traditional normoxic condition(21%O2) as the matchedgroup.2. Culture and passage the FY-phES-018cell line and FY-hES-7cell line whichderived in our lab previously, and the other hpESC line (chHES-32) which obtainedfrom the China National Engineering and Research Center of Human Stem Cell under5%oxygen tension. It was also detected the stem cells specific antigen SSEA-3,SSEA-4, TRA-1-60and TRA-1-81and the capacity of differentiation in vitro.3. HpESC and hESC were maintained in undifferentiated state, then collected the early and late passage cells and extracted the genome RNA and DNA from these cells.4. Expression of molecular marker genes in undifferentiated stem cells were detected byRT-PCR.5. Expression of pluripotency related genes(NANOG and SOX2) as well as the hypoxia-inducible factors (HIF-1a and HIF-2a) in hESC and hpESC under different oxygen weredetected respectively by real-time PCR.6. Chromosome analysis were performed on these three cell lines and Short tandem repeatanalysis were performed on the two hpESC lines.Results:1. The hypoxic culture system was set up successfully. Two hpESC lines and onehESC line can maintain the typical hESC clone morphology and expressed SSEA-3,SSEA-4, TRA-1-60and TRA-1-81. They also have the ability of differentiating intoEB and expressed three embryonic germ layer marker genes.2. NANOG, SOX2, OCT4, THY1and REX-1were all expressed in hESC and hpESC.3. The expression levels of NANOG, SOX2and HIF-2a were significantly higher incells cultured under hypoxia than the cells from the same cell line but cultured undernormoxia (P <0.05). But there was no obviously difference in the expression ofHIF-1a in cells between hypoxia and normoxia (P>0.05).4. Normal karyotype were detected in the two hpESC lines:46,XX. The distinctiveloci between the cells indicated that they were derived from different embryos.Conclusion:1. Under5%oxygen tension, hpESC and hESC can maintain the biologicalcharacteristics of hESC and have the ability of proliferation constantly anddifferentiation.2. Hypoxia enhanced the expression of NANOG and SOX2by increasing theexpression level of HIF-2a. Hypoxia (5%O2) is more close to the physiologicaloxygen concentration where the embryos development, so hypoxic culture systemmaybe better for the growth of human embryonic stem cells, and provided reliablecells for further study. Part â…¡Expression of imprinted genes in human parthenogenetic stem cells underdifferent oxygen tensionObjectiveTo evaluate the clinical application viability of hpESC and offer valuableimformation for depth study on the epigenetics of human embryonic stem cells, weobserved the expression of imprinted genes in undifferentiated hpESC under differentoxygen tension and discussed the effects of hypoxia on the epigenetics of hESC andhpESC, confirmed whether hypoxia is beneficial for maintaining the genomicimprinting stability in hpESC.Material and methods1. Culture two hpESC lines and one hESC line which derived from fertilized embryounder hypoxia and normoxia, respectively.2. The expression profile analysis of hpESC and hESC cultured under differentoxygen tension was performed by using NimbleGen Human Gene ExpressionMicroarrays. Differentially expressed genes were identified between the same passagecells but cultured in different oxygen tension. We also detected the imprinted geneswhich expressed variable after long term culture under hypoxic condition. Comparethe rate of change of imprinted genes expression levels in each stem cell line underdifferent oxygen tension or after long term culture in hypoxia.3. Quantitative real-time PCR was used to validate the expression levels of somedifferentially expressed imprinted genes.4. Imprinted genes in phESCs or hESC under5%O2and21%O2were hierarchicalclustered and GO (Gene ontology) analysis was perform on differentially expressedimprinted genes from the same cell line but cultured under different oxygen tension orafter long term culture.5. Compare the differentially expressed imprinted genes between different hpESClines. Results:1. After long term culture in hypoxic condition, we detected47imprinted genes and30imprinted genes expressed differentially (>2fold),16of which upregulated and14downregulated. GO analysis showed that these imprinted genes were rich fortranscription regulation, organs and systems development and regulate biologicalmetabolic process.2.48imprinted genes were detected in FY-phES-018cells between early-versuslate-passage under5%O2and transcripts of29imprinted genes were differentiallyexpressed,17upregulated and12downregulated;In chHES-32cells, we observedtranscripts of61differentially expressed imprinted genes including34upregulatedand27downregulated in78imprinted genes. GO analysis showed that theseimprinted genes were rich for transcription regulation, organs and systemsdevelopment and morphology, regulate biological metabolic process.3.There were59imprinted genes and37of which were differentially expressed (>2fold) in FY-hES-7late passage cells between5%and21%oxygen tension,23ofwhich upregulated and14downregulated. GO analysis showed that these imprintedgenes were rich for regulating RNA metabolism process, exocellular regional partsand the activity of growth factors.4.53imprinted genes were detected in FY-phES-018cells cultured in21%O2compared with the cells cultured in5%O2, and34imprinted genes differentiallyexpressed.21of the34differentially expressed imprinted genes were downregulatedwhile13were upregulated. There were75imprinted genes detected in chHES-32cellline when comparing the cells cultured in21%O2with that cultured in5%O2and44imprinted genes expressed differentially,21downregulated but23upregulated. GOanalysis showed that these imprinted genes were rich for system formation anddevelopment, organs formation and development, transcription and the activity oftranscription factor, and regulate biological function.5. The rate of change of imprinted genes expression did not differ among the stem celllines under different oxygen tension (P>0.05); the rate of change of imprinted genesdid not differ between hpESC and hESC (P>0.05); there is no significantly difference between hpESC lines (P>0.05). After long term culture under hypoxia, the rate ofchange of imprinted genes expression did not differ among these three cell lines(P>0.05); the rate of change of imprinted genes did not differ between hpESC andhESC(P>0.05); but the rate of change of imprinted genes in chHES-32was higherthan that in FY-phES-018, the difference between the two hpESC lines was significant(P<0.05).6. The results of some imprinted genes from microarray were consistent with thatfrom real time PCR.7. Hierarchical clustering of these imprinted genes illustrated a strong tendency for5%O2cultured FY-phES-018cells to cluster together.It also can be detected inFY-hES-7cell line.But for chHES-32cells, it showed that the same passage cellsunder different O2tension were clustered together.8. Under hypoxic condition, the variation trend of the same imprinted gene but indifferent cell lines was different, these genes including GATA3, CYP1B1, NKX6-2,BRUNOL4, SNURF, MYEOV2, KLF14, LMX1B and PEG3.The variation trends ofsome differentially expressed imprinted genes were not consistent between hpESClines under different oxygen tension and these genes included CSF2, FAM50B,BRUNOL4and FUCA1.Conclusion:1. Different oxygen tension could change the expression levels of imprinted genes inhESC and hpESC, and the varied reaction caused by oxygen tension was depend onthe different imprinted genes.2. HIF may be the key factor that regulates the epigenetic stability in humanembryonic stem cells.3. Although under hypoxia, long term culture still influence the stability of imprintedgenes in hESC and hpESC. Therefore, long term culture is the primary factor thataffect the safty of using hESC and hpESC in clinic when compared to oxygen tension. |
