The Role And Mechanism Of KDM2B In The Specification Of Human Primordial Germ Cell-Like Cells

Background:Germ cells are essential to transmit genetic and epigenetic message to the next generation,defects in germline can lead to infertility and many other diseases.Germline specification is a fundamental step for human reproduction,this biological phenomenon possess technical challenges to study in vivo as it occurs immediately after blastocyst implantation(2?3 week embryos).The establishment of in vitro human primordial germ cell-like cells(hPGCLCs)induction system allows sophisticated characterization of human primordial germ cells(hPGCs)development.However,the underlying molecular mechanisms of hPGCLC specification are not fully elucidated.Object:It is important for study of reproductive biology and clinically relevant diseases to understand the developmental and regulatory mechanisms of human germ cells.This study intends to explore specified epigenetic factor correlation with transcription landscape to uncover the mechanisms of hPGCLC specification,and provide a reference for hPGC development and specification mechanisms.Methods:Above all,we acqiured of human primordial germ cell-like cells(hPGCLCs)by hPGCLCs induction system in vitro,and detected the dynamic expression of KDM2B in different time points of hPGCLCs by RNA-seq.Moreover,we establishment of KDM2B Knockout hESC Lines with CRISPR/Cas9 system,and use karyotype assay and microscopic evaluated the chromosome stability of KDM2B KO hESCs.The effect of deletion of KDM2B on self-renewal or pluripotency of hESCs was examined by QPCR,immunofluorescence,subcutaneous tumorigenesis assay,paraffin-embedded sections and hematoxylin-eosin staining.First of all,KDM2B KO hESCs were cultured in 4i medium and inducted to hPGCLCs in vitro.The hPGCLCs-positive cells of different time points were sorted using fluorescent Activated Cell Sorting,and the differentiation efficiency of hPGCLCs was further verified by immunofluorescence.Furthermore,we performed a time dependent RNA-Seq on hESC,day 1 and day 2 with 2 biological replicates during hPGCLC differentiation process to identified the differential expression(DE)gene of KDM2B KO hPGCLCs,and the molecular mechanisms of cell fate determination during hPGCLC development.What's more,we establishment of KDM5B Knockout hESC Lines with CRISPR/Cas9 system,the effect of deletion of KDM5B on pluripotency of hESCs was examined by QPCR.Meanwhile,KDM5B KO hESCs were inducted to hPGCLCs in vitro,the hPGCLCs-positive cells of different time points were sorted using fluorescent Activated Cell Sorting,and the differentiation efficiency of hPGCLCs was further verified by immunofluorescence.To determine whether re-expression of KDM2B could functionally rescue the deficiency of hPGCLC specification,we induced KDM2B expression with KDM2B KO hESCs under the control of the Tet-On system,which would allow Doxycycline(Dox)to activate KDM2B by duration and dosage,then inducted to hPGCLCs.The hPGCLCs-positive cells of different time points were sorted using fluorescent Activated Cell Sorting,and the differentiation efficiency of hPGCLCs was further verified by immunofluorescence.The role of KDM2B in hPGCLC specification was investigated by functional rescue experiment.Results:We found KDM2B was highly expressed in fetal germ cells(FGCs)and hPGCLCs,suggesting an important role for KDM2B in germ cell development.We found the expression of classical pluripotency markers or cell morphology in KDM2B KO hESCs were comparable to WT hESCs,and KDM2B KO hESCs were capable to form teratomas with three somatic lineages when transplanted into immunocompromised mice.These data indicated that KDM2B had no significant impact on the capable to form teratomas of human ESCs.The KDM2B KO hESCs and WT hESCs were cultured in a cocktail of inhibitors with four kinases(4i medium)for 4 days,then cells were incubated with hPGCLCs induction system for 6 days.Meanwhile,The hPGCLCs derived from KDM2B KO hESCs was significantly decreased as compared to WT day 2hPGCLCs,suggesting that loss of KDM2B dramatically impaired hPGCLCs differentiation.These data indicates KDM2B is involved in hPGCLC specification originated from hESCs.Mechanistically,We found that during hPGCLC differentiation,germline genes were more enriched in WT hESCs but heart morphogenesis as well as endodermal genes GATA5 were up-regulated in KDM2B KO hPGCLCs,suggesting the KDM2B KO cells were prone to undergo somatic differentiation after hPGCLC induction(from day 1).Hence,KDM2B might play a role in suppressing somatic gene expression and transformation towards somatic cells during the specification of PGCs.Notably,some epigenetic regulators such as KDM5B,TET1 were significantly up-regulated after hPGCLC induction(from day 2)in KDM2B competent cells,while the activation of these genes' transcription were not observed in KDM2B KO hPGCLCs.These data suggested that KDM2B deletion could cause some epigenetic factors alternation which subsequently affected cell fate determination during hPGCLC development.Our data showed it may be a compensatory effects after KDM2B deletion that the expression of KDM5B,another demethylase of H3K4me3,is upregulated in KDM2B KO hESCs.However,the hPGCLCs derived from KDM5B KO hESCs was no significantly difference compared to WT hPGCLCs,these indicated that KDM5B is not indispensable in hPGCLC specification originated from hESCs.Furthermore,We induced KDM2B expression with KDM2B KO hESCs under the control of the Tet-On system,and activated the expression of KDM2B to the endogenous level.Notably,continuous induction of the KDM2B expression significantly increased the hPGCLC production as compared to non-induced KDM2B expression group.Since the KDM2B KO phenotype can be functionally rescued by KDM2B re-expression,we thus demonstrate the hPGCLC specification defect result from the loss of KDM2B,but not from off-target efects.Conclusions:We demonstrated that KDM2B is not necessary to maintain the pluripotent status of hESCs,however,loss of KDM2B dramatically impaired hPGCLCs differentiation whereas ectopically expressed KDM2B could efficiently rescue such defect,indicating this defect was due to KDM2B's loss in hPGCLC specification.Mechanistically,as revealed by the transcriptional profiling,KDM2B suppressed the expression of somatic genes thus inhibited somatic differentiation during hPGCLC specification.These data collectively indicate that KDM2B is an indispensable epigenetic regulator for hPGCLC specification,shedding lights on how epigenetic regulations orchestrate transcriptional events in hPGC development for future investigation.