Epigenetic Regulation Of Primordial Germ Cells Induced From Human Pluripotent Stem Cells In Vitro

Part 1 Construction of H1 ESCs Fluorescent Reporter Cell Line and Optimization of PGCLCs Induction Efficiency[Purpose] Due to the inaccessible of human early germ cells,induction of germ cells by h PSCs in vitro provides a model for the study of human early germ cells.However,due to the lack of specific surface markers in early human primordial germ cells,it is difficult to distinguish them from surrounding somatic cells.Therefore,in this experiment,a fluorescent protein gene m Kate2 was inserted into downstream of the PGCs specific gene BLIMP1 termination codon using CRISPR-Cas9 gene editing technology,for detecting the expression of BLIMP1 gene.During the in vitro differentiation of PGCs,the expression of fluorescent protein can be observed by flow cytometry or fluorescence microscopy to determine the differentiation efficiency of PGCs.This reporter cell line is used to detect the induction efficiency of different induction schemes to optimize the induction culture system.[Methods] Three pairs of sg RNAs were designed near the stop codon of BLIMP1 gene and cloned into p X330 vector.After transfecting 293 FT cells,the genome was sequenced to determine the sg RNA sequence that could accurately cause DNA double strand breaks.Then construct a recombinant plasmid,connect the T2A-m Kate2-Lox P-EF1a-neo-Lox P sequence in front of the stop codon of the 5 'homology arm,and mutate the sg RNA homology DNA region to avoid cutting homologous recombination plasmid.Fugene6 transfection reagent was used to simultaneously transfect sg RNA plasmid and homologous recombination plasmid into H1 ESCs.The next day,cells were selected with 50?g/ml G418 for 10 days,and then 800 cells were seeded to 10 cm culture dishes to obtain monoclonal cells.Monoclonal colonies were digested into 48-well plates with Collagenase IV,and passaged into 24-well plates to identify correctly inserted clones by PCR.The G418 resistance gene was removed by transfection of the CRE enzyme plasmid,and the correct clone was identified by PCR and point mutation was confirmed by Sanger sequencing.Flow cytometry was used to identify the pluripotency of knock-in cell lines,and the currently reported PGCLCs induction system was used to optimize the induction medium and cytokines to explore the highest induction efficiency of the PGCLCs induction culture system.RT-q PCR was used to detect whether PGCLCs in EB(Embryoid Bodies)induced the expression of PGCs marker genes on the fourth day.[Results] After the first step of knock-in was verified by PCR,three G418-resistant normal insert clones were obtained,and the gene editing rate was 12.5%.After CRE enzyme removed G418 resistance,there were 13 positive clones,no point mutations were found in Sanger sequencing,and the rate of gene editing was 46.4%.Flow cytometry identified positive cell lines still expressing pluripotency markers such as POU5F1,SOX2,NANOG.After 42 hours of pre-induction with GK15 medium,PGCLCs induction with a RB27 medium with 500ng/ml BMP4 and 10ng/ml LIF can stabilize the induction efficiency of PGCLCs at 35%-60%.And on the fourth day,EB highly expressed PGCs marker genes such as BLIMP1,SOX17,TFAP2 C,NANOS3.[Conclusions] In this section,CRISPR-Cas9 gene editing technology was used to successfully knock in a fluorescent gene before the stop codon of the PGCs marker gene BLIMP1,providing an experimental platform for inducing human pluripotent stem cells to differentiate into PGCs in vitro.Using this cell line to further optimize the existing PGCLCs induction culture system,a highly efficient PGCLCs induction system suitable for our laboratory and cells was explored.Part 2 Establishment of TET Knockout Cell Lines and the Difference in Induction Efficiency to PGCLCs[Purpose] Pluripotent stem cells have the ability to differentiate into all cells,including germ cells,and mouse pluripotent stem cells have been able to differentiate into egg and sperm-like cells in vitro,and they can fertilize in vitro to obtain health offspring.Human primordial germ cells are the progenitor cells of eggs and spermatozoa.During development and differentiation,they undergo epigenetic reprogramming of whole genome demethylation and remethylation,and this process is mainly responsible for TET demethylase.And this process is mainly completed 7 weeks before the development of PGCs.However,due to ethical and material constraints,PGCs cells can hardly be obtained before 7 weeks,so in vitro simulation of PGCs development process provides a good material for understanding epigenetic reprogramming mechanisms.And ESCs induced PGCs have brought us a new way to study the epigenetic regulation mechanism of early germ cells.In this part,CRISPR-Cas9 gene editing technology was used to knock out the TET gene family to study the effect of TET gene family on PGCs differentiation.[Methods] The CRISPR-Cas9 technology was used to knock out the TET gene family in the gene knock-in ESCs cell line,and the BLIMP1-m Kate2 fluorescence reporter system established in the first part was used to detect the induction efficiency of ESCs differentiation into PGCLCs in vitro,and to study the TET gene family epigenetic regulatory mechanisms during PGCs differentiation.Dot-blot was used to detect the content of 5hm C and 5m C in each group of cells,and the expression of PGCs marker was detected by fluorescent quantitative PCR and immunofluorescence staining.Bisulfite sequencing was used to detect the methylation levels in the promoter regions of SOX17 and BLIMP1,the key genes in PGCs specialization.[Results] After knocking out the TET gene family,we found that TET2,TET3 alone and TET2/3 knockout cells had no significant effect on the induction efficiency of PGCLCs.When the combination with TET1 knockout included TET1,TET1/2,and TET1/3,the induction efficiency of PGCLCs decreased,and the TKO(triple-knockout)cell line with simultaneous knockout of TET1/2/3 had no PGCLCs positive cells on the fourth day of induction.Dot-blot experiments revealed that 5hm C modification in h ESCs DNA was significantly down-regulated after TET1 gene knockout,but did not affect the level of 5m C;Compared with WT group the key genes SOX17,BLIMP1,TFAP2 C,NANOS3 expression levels were all low during PGCs differentiation,and the expression of the above genes was significantly down-regulated in TKO cell lines,but there was no significant difference in TET2,TET3 and TET2/3 simultaneous knockout cell lines.The results of TET2/3 DKO TET1 gene heterozygous mutant cell line and WT group were consistent.The results of immunofluorescence staining were consistent with the results of RT-q PCR.Bisulfite sequencing revealed that the methylation level of the promoter region of SOX17 and BLIMP1 genes in TKO cell lines was higher than that in WT group.[Conclusions] In this part,epigenetic regulation mechanism during early PGCs development was studied through induction of PGCs in vitro.It was found that the deletion of TET demethylase resulted in hypermethylation of SOX17 and BLIMP1 promoters,leading to down-regulation of SOX17 and BLIMP1 expression,and the inability of ESCs to differentiate into PGCs.Therefore,the TET gene family plays an important role in the regulation of PGCs differentiation specific genes SOX17 and BLIMP1 during PGCs development,and TET1 gene plays the most important role in this process.Part 3 Overexpression of Exogenous and Endogenous SOX17 and BLIMP1 Can Reconstitute the Induction of PGCLCs in TKO cell lines[Purpose] It has been reported that simultaneous overexpression of SOX17 and BLIMP1 in pluripotent stem cells can promote the induction of PGCLCs.The lentiviral method using small molecules to induce overexpresses SOX17 and BLIMP1 genes in TKO cell lines or uses d Cas9-TET1 CD technology changes the promoter methylation levels of SOX17 and BLIMP1 genes to initiate the expression of endogenous SOX17 and BLIMP1 to reconstitute the induction of PGCLCs in TKO cell line.[Methods] Construction of doxycycline(DOX)and trimethoprim(TMP)induced lentiviral plasmids expressing SXO17 and BLIMP1.The virus was packaged in 293 FT cells to infect TKO and WT cell lines,and screened with G418 and puromycin to obtain stable cell lines.Flow cytometry was used to detect the induction efficiency of PGCLCs after overexpression of SOX17 and BLIMP1,and the expression of PGCs markers was further verified by fluorescent quantitative PCR and immunofluorescence staining.Bisulfite sequencing was used to detect the methylation levels of SOX17 and BLIMP1 gene promoter regions.Then construct a stable TKO cell line that induces expression of d Cas9-TET1 CD with DOX,and design sg RNA targeting sites in the promoter region of SOX17 and BLIMP1.Determine the sg RNA sequence with the highest expression of SOX17 and BLIMP1 after individual transfection and PCR.The screened sg RNA was constructed as a lentiviral plasmid.Stable cell lines were established after virus infection.The detection method is the same as that of direct overexpression of SOX17 and BLIMP1.[Results] Overexpression of SOX17 and BLIMP1 in WT cell line can efficiently obtain PGCLC positive cells(90%),and overexpression of SOX17 and BLIMP1 in TKO cell line can obtain about 70% of PGCLC positive cells.Quantitative real-time PCR showed that the expression of endogenous SOX17 and BLIMP1 was lower in TKO cell lines,both were exogenous expression of SOX17 and BLIMP1,but other PGCs marker gene expressions such as NANOS3,TFAP2 C,NANOG were up-regulated.The promoter regions of SOX17 and BLIMP1 are still hypermethylated.In the process of PGCLCs induction,d Cas9-TET1 CD technology was used to reduce SOX17 and BLIMP1 promoter methylation modification in TKO cell lines,and PGCLCs positive cells were also induced.Quantitative real-time PCR and immunofluorescence staining showed that SOX17,BLIMP1,TFAP2 C and other PGCs marker gene expression increased,and the methylation level of SOX17 and BLIMP1 promoter regions was lower than that of TKO cell lines without DOX,but still higher than WT group resulted in lower PGCs induction efficiency than the WT group.[Conclusions] The epigenetic modification of the PGCs specific genes SOX17 and BLIMP1 promoters by the TET gene family during PGCs differentiation regulates the differentiation of PGCs,confirms the relationship between epigenetic modification of DNA methylation and regulation of transcription factors,and further determines that SOX17 and BLIMP1 play important roles in the differentiation of human PGCs.Part 4 Identification of SOX17 and BLIMP1 Promoter Methyltransferases and Knockout Can Reconstitute TKO Cell Lines into PGCLCs[Purpose] In the third part,we found that overexpression or activation of endogenous SOX17 and BLIMP1 expression in TKO cell lines can reconstitute the induction of PGCLCs,and because the promoter regions of the SOX17 and BLIMP1 genes in TKO cell lines are hypermethylated to inhibit the PGCLCs differentiation.In human cells,there are three methyltransferases that methylate genomic DNA: DNMT1,DNMT3 A,and DNMT3 B.We use Ch IP-q PCR and CRISPR-Cas9 gene editing technology to explore methyltransferase on the SOX17 and BLIMP1 gene promoters to study the effect of epigenetic regulation on PGCs specification.[Methods] Ch IP-q PCR was used to determine the enzymes that methylate the SOX17 and BLIMP1 promoters regions in WT and TKO cell lines using DNMT1,DNMT3 A,and DNMT3 B antibodies.This enzyme was then knocked out using CRISPR-Cas9 technology to establish a QKO(quadruple-knockout)cell line.The QKO cell line was induced to PGCLCs,the induction efficiency was detected by flow cytometry,the expression of PGCs markers was detected by fluorescent quantitative PCR and immunofluorescence staining,the expression of 5hm C and 5m C was detected by Dot-blot,bisulfite sequencing was used to detect the methylation degree of PGCs specific genes.[Results] The results of Ch IP-q PCR showed that DNMT3 B was methylated the promoter region of SOX17 and BLIMP1.After knockout of DNMT3 B gene in TKO cell line,it could partially restore the induction of PGCLCs,and the specific gene expression of PGCs was up-regulated.Immunofluorescence staining also showed that PGCs specific markers were expressed in QKO cell line induced PGCLCs.Dot-blot results showed that 5hm C in QKO and TKO cell line were still significantly lower than the WT group,and 5m C was not significantly different between the three cell lines of WT,TKO,and QKO.Bisulfite sequencing results showed that on the fourth day of EB induction by PGCs,the methylation levels in the promoter regions of SOX17 and BLIMP1 were lower in QKO cells than in TKO cells,but still higher than in the WT group.[Conclusions] During the specification of PGCs,TET demethylases demethylate the promoter regions of SOX17 and BLIMP1 genes to guide the cells in the direction of PGCs.DNMT3 B methylase is responsible for the methylation of the promoter regions of SOX17 and BLIMP1 genes to inhibit the development of cells in the direction of PGCs.Moreover,transcription factors in PGCs differentiation are also linked to epigenetic regulation,so that we can have a more comprehensive understanding of the regulatory mechanism of PGCs.