Establishment And Differentiation Of Induced Pluripotent Stem Cells Of Patients With Premature Ovarian Insufficiency

BACKGROUNDPremature ovarian insufficiency(POI)is a clinical syndrome defined by loss of ovarian activity before the age of 40 years.POI which affects approximately 1% of women by the age of 40 is characterized by menstrual disturbance(amenorrhea or oligomenorrhea)with raised gonadotrophins and low estradiol.A wide spectrum of causes has been considered including genetic,environment,autoimmune and iatrogenic.However,the specific mechanism is still unclear.The current treatments,such as hormone replacement therapy(HRT),psychological and lifestyle interventions,etc.,can only relieve clinical symptoms,prevent cardiovascular disease and osteoporosis,however the problem of decreased fertility still exists.Therefore,finding new methods to clarify the mechanism and improve fertility is an urgent problem to be solved.Induced pluripotent stem cells(iPSCs)using exogenous gene transfection,which from somatic cells is easy to obtain and ethical issues are avoided.The iPSCs derived from POI patients(POI-iPSCs)can be differentiated into primordial germ cell like cells(PGCLCs)and even egg cells,that provide a good cell model for exploring disease mechanism and drug screening,and also provide new ideas for improving the fertility of POI patients.OBJECTIVE1.Establishing iPSCs cell lines derived from PBMCs in patients with POI,thereby providing a method to get standardized and reproducible iPSCs.2.By simulating the in vivo environment,exploring the ability of POI-iPSCs to differentiate into PGCLCs,therefore,provide a wonderful model for exploring the mechanism of POI,high-throughput drug screening,and lay the foundation for PGCLCs transplanting of POI.METHODS1.Peripheral blood samples of 2 POI patients and 2 normal women were collected by venipuncture.Peripheral blood mononuclear cells(PBMCs)were separated by Ficoll-Paque media.Non-integrated plasmids,with four transcription factors of SOX2,KLF4,L-MYC,and LIN28,were used to electroporate T cells,iPSCs clones generated after 17-25 days.2.Immunofluorescence staining,RT-PCR,teratoma formation experiment and embryoid body formation experiment were used to identify the pluripotency of iPSCs.Karyotype analysis researched the influence on chromosome stability during the reprogramming of iPSCs.3.With a two-step method including pre-induction and PGCs induction,iPSCs were differentiated into PGCLCs in vitro.The ratio of Ep CAM/INTEGRINα6double-positive cells was used as the evaluation index,and the induction efficiency of PGCs was detected by Flow Cytometry.RESULT1.We established two POI-iPSCs,named iPSC1001,iPSC1002,and two iPSCs of healthy fertile women,named iPSC1003,iPSC1004.All iPSCs have the typical shape of cell clones with clear boundaries,tightly arranged cells and high nuclear cytoplasm.2.All iPSCs positively express pluripotency transcription factors OCT4,NANOG,SOX2,as well as positively express pluripotency-related genes OCT4,SOX2,C-MYC,LIN28.They have the ability to differentiate into three germ layers and had a normal karyotype.3.Cell morphology changes during the induction process: iPSCs cultured during the pre-induction phase showed a flat epithelial cell morphology with clear cell boundaries.During the induction suspension culture phase,the cells formed embryoids(EBs)with increasing volume.4.There was significantly lower percentage of Ep CAM/INTEGRINα6 double positive cells for POI-iPSCs than that of fertile women hiPSCs differentiation on the process of day 4 and day8(p<0.05).Conclusion1.PBMCs can be constructed into iPSCs cell lines using electroporation by non-integrated plasmid.2.By simulating the signal regulation and molecular environment of early development of human germ cells in vitro,iPSCs can be differentiated into PGCLCs.3.There is no significant difference between POI-iPSCs and iPSCs of healthy women in morphology,function.However,the POI-iPSCs have a lower induction efficiency in the process of inducing into PGCLCs in vitro.