Establishment Of Azoospermia Patients Induced Pluripotent Stem Cell Lines And Differentiation Of Them Into Primordial Reproductive Cells In Vitro

PartⅠEstablishment of induced pluripotent stem cells in patients with azoospermia Objective:To establish induced pluripotent stem cells line in patients with azoospermia and to provide a suitable disease model for the further study of the pathogenesis and treatment of the disease.Methods:Using the non-gene integration method of Sendai virus,we established the induction of pluripotent stem cell lines derived from peripheral blood mononuclear cells from patients with azoospermia.(Te SR ? 2)and embryonic stem cell culture medium(Stem Adhere ? Defined Matrix)were used to culture the culture system.Immunofluorescence staining,chromosome karyotype analysis,embryoid body and teratoma were used to investigate the effects of iPSCs Of the dry,pluripotent,in vitro and in vivo differentiation ability to verify.Results:We established iPSCs derived from azoospermia patients with human embryonic stem cell characteristics.Immunofluorescence staining showed that these cells expressed SOX2 and OCT4 as well as TRA-1-60 and SSEA-4 stem cell pluripotency genes;chromosome karyotype analysis showed normal karyotype;embryoid and teratoma experiments showed that,In vitro have the ability to differentiate into three germ layers.Conclusion:to construct the pluripotent stem cell line derived from azoospermia patients by non-gene fusion method,and the cell model was provided for the study and treatment of azoospermia.Part II preliminary study on the differentiation of iPSCs into primordial reproductive cells in vitro Objective:To investigate the induction of pluripotent stem cells(pluripotent stem cells,pa-iPSCs)into primordial germ cell-like cells(PGCLCs)in vitro.Methods:We selected iPSCs into naive iPSCs using the(PD0325901 + CHIR99021 + SB203580 + SP600125,"4i")culture system of,and then pretreated naive iPSCs in vitro with b FGF,TGF-β1 and 1% KSR The cells were cultured in BMP4,LIF,SCF,EGF,and ROCK inhibitor PGCLCs in differentiated cells.The cells were cultured in BMS4,LIF,SCF,EGF,and ROCK inhibitor PGCLCs in 200-4000 cells / well.And specific labeling was used to identify PGCLCs by immunofluorescence staining.Results:We successfully transformed iPSCs into na?ve state iPS cells,and then successfully induced PGCLCs in vitro.Immunofluorescence staining showed that PGCLCs express PGC-specific markers OCT4 and SOX17.Conclusion:The pa-iPSCs were transformed into naive cells by "4i" culture system,and PGCLCs were induced to differentiate into PGCLCs.PGC17 s express PGC-specific markers SOX17 and OCT4,which provided the theory for the treatment of azoospermia in vitro basis.