Exploring The Role Of PHC1 In The Genomic Integrity Maintenance Of Human Pluripotent Stem Cells

BackgroundRegeneration consists of the very important mechanism that sustain the function of the individual.Dr.Richard.GOSS,one of the leaders in the field of regenerative medicine,once commented on the relationship between Regeneration,Life and Death:"If there is no regeneration,then no life exists in this world.If generation is everywhere,there will be no death." Human pluripotent stem cells(hPSCs)provide a promising future for regenerative medicine.hESCs are capable of proliferating indefinitely in vitro and differentiate into a variety of lineage cells,providing potential seed cells for the treatment of degenerative disease in multiple systems for transplantation.However,hESCs and iPSCs have many clinical bottlenecks,the results showed that DNA replication stress induced by overexpression of reprogramming factors and DNA damage induced by oxidative stress could destroy the genome of iPSCs and generate mutations.On the other hand,hPSCs proliferated rapidly during the process of cellular proliferation,and the cellular metabolism and DNA replication can active reactive oxygen species(ROS)and thus leads to DNA damage and mutagenesis.Transplantation of cells carrying genetic mutations may produce tumors in vivo.[1]Therefore,it is important to study how hPSCs maintain the stability of the genome to obtaining safer hPSCs for cell therapy.In contrast to somatic cell DNA repair studies,there are few reports on how hPSCs maintain genomic stability,and the mechanisms underlying their maintenance of genomic stability have been found to be a lower level of mitochondria in ESCs[2],at the same time,ESCs mainly obtain energy by glycolytic metabolism independent of mitochondrial oxidative phosphorylation,so the ROS level is very low.while ESCs high expression of antioxidant-related genes,so have a stronger resistance to ROS-induced DNA damage;The second is that the ES cells that cannot finish DNA damage repair will be introduced to differentiation and apoptosis[3].Our study demonstrates the role of PHC1 in the maintenance of genomic stability of hESCs.hESCs knocked down by PHC1 have lower expression of OCT4 and NANOG,which indicates its role in hESCs pluripotency maintenance.And they are more sensitive to DNA damage after the treatments of UV radiation and doxorubicin.In addition,previous studies have shown that overexpression of reprogramming factor induced DNA damage response is a major obstacle to somatic cell reprogramming.Our results show that knockdown of PHC1 expression in human somatic cells during reprogramming resulted in increased cell death and reduced reprogramming efficiency,suggesting that PHC1 is involved in the DNA damage response of cells during pluripotency formation.Part I The role of PHC1 in hESCs pluripotency maintenanceObjective:To explore the role of PHC1 in pluripotent maintenance.Methods:Using qPCR and Western blot to check PHC1 expression in hPSCs and differentiated cells.shRNA was constructed to silence PHC1 gene in hESCs.The change of pluripotency marker was examined.Res?lts:PHC1 was highly enriched in hPSCs and we successfully constructed the effective shRNA of PHC1.There were no obvious differences of OCT4?NANOG expression at mRNA level after PHC1 gene silencing.Western Blot and immunofluorescence showed NANOG protein level reduced.Conclusion:PHC1 has a certain effect on the pluripotency maintenance of hESCs.Part II A role of PHC1 on hESCs genome stability maintenanceObjective:To explore the establishment of an in vitro DNA damage model and the reaction of hESCs in DNA damage.Methods:hESCs were treated with ultraviolet(UV)radiation and doxorubicin after PHC1 knockdown.The proliferation of hESCs was measured by cell apoptosis.Res?lts:Compared with the control group,knockdown of PHC1 resulted in decreased stability of hESCs genome and increased sensitivity to DNA damage.Conclusion:PHC1 has a certain effect on the maintenance of hESCs genomic integrity.Part? A role of PHC1 during reprogrammingObjective:To explore the role of PHC1 in overexpression of transcription factor-mediated somatic cell reprogramming.Methods:Human fibroblasts with overexpression of OCT4,SOX2,KLF4 and c-MYC by lentiviral vector was reprogrammed into iPSCs.The effect of silencing PHC1 expression on reprogramming efficiency was tested.Res?lts:Silencing of PHC1 during reprogramming resulted in increased cell cycle,increased apoptosis and reduced reprogramming efficiency.Conclusion:PHC1 is involved in the DNA damage response during reprogramming of human somatic cells,thus affecting reprogramming efficiency.