Derivation Of Primodial Germ Cell-like Cells From Induced Pluripotent Stem Cells Of Patients With Idiopathic Azoospermia And Transcriptome Analyses

Part I: Isolation and characterization of human fibroblasts from male patients with idiopathic infertilityObjective We aim to develop a standardized,repeatable method for isolation of fibroblasts from human foreskin tissues,and to identify them according to morphologic,immunohistologic and proliferative criteria.Methods 1.Foreskin specimens were derived from patients under circumcision or microsurgical testicular sperm aspiration at the center of reproductive medicine of Tongji medical college.Written informed consents were obtained from all patients before surgery.The institutional review Board of Tongji medical college approved the study.2.Fibroblasts were isolated from human foreskin with explant adherent culture method.3.Besides cell morphological observation,we used immunofluorescence to detect the expression of VIMENTIN,which was used as a marker for fibroblasts.4.Fluorescence-activated cell sorting was applied to analysis the immunophenotype of the obtained cells.5.We portrayed the growth curve of skin derived fibroblasts in vitro of different passages.6.MTT proliferation assay were adopted to detect the ability of cell proliferation and viability.Results 1.The obtained cells grew as typical morphologic characteristics of fibroblasts,and further identification at molecular level revealed that they strongly expressed intermediate filament protein VIMENTIN.2.Growth kinetics analysis of the cells showed high proliferative properties.Furthermore,cryopreservation had no influence on cell morphology and growth properties.3.Immunophenotypic analysis indicated that the isolated fibroblasts highly expressed mesenchymal surface markers CD73,CD90,CD44 and CD105,and were negative for haematopoietic markers CD45 and CD34.Conclusions We have successfully established a standardized,repeatable method for isolation of fibroblasts from human foreskin tissues and identified their biological characters according to morphologic,immunohistologic and proliferative criteria,which would be meaningful for future clinical trials and regenerative medicine purposes.Part II: Derivation of human induced pluripotent stem cells(iPSCs)from male patients with idiopathic infertilityObjective We aim to generate human iPSCs from male patients with idiopathic infertility and to analyze the pluripotency and differentiation capacity of the derived patient-specific iPSCs.Methods 1.The fibroblasts from part I were reprogrammed with the Yamanaka KOSM(OCT4,SOX2,KLF4 and C-MYC)transcriptional factors using the retroviral vectors to get human iPSCs.2.Immunofluorescence staining for pluripotency markers,NANOG,SOX2,OCT4, TRA-1-60,and SSEA-4,was performed in derived iPSCs.3.Immunohistochemical staining for alkaline phosphatase(AP)was performed in derived iPSCs.4.The qualitative expression of pluripotency genes OCT4,SOX2,KLF4,C-MYC,LIN28,NANOG,REX,TDGF and HTERT was detected by RT-PCR in derived iPSCs.5.The quantitative analysis of pluripotency genes(OCT4,SOX2,and NANOG)by flow cytometry analysis was performed in derived iPSCs.6.The differentiation capacity of derived iPSCs into three germ layers was confirmed by teratoma formation assay in vivo and embryoid body formation in vitro.7.Karyotype analysis.Results 1.We generated two iPSC lines from male patients with idiopathic infertility(1106 and 1122)as well as one normal iPSC line.2.The obtained iPSC lines showed pluripotency verified by the expression of pluripotency markers,OCT4,SOX2,KLF4,C-MYC,LIN28,NANOG,REX,TDGF,HTERT and AP.3.The iPSC lines were demonstrated to have the three germ layers differentiation capacity in vivo by teratoma assay and in vitro by embryoid body formation.4.The iPSC lines also showed normal karyotype.Conclusions We have established hiPSC lines from dermal fibroblasts of two patients(1106 and 1122)with idiopathic non-obstructive azoospermia.These patient-specific iPSC lines can be used to explore the mechanism for idiopathic male infertility.Part III: Differentiation of primodial germ cell-like cells(PGCLCs)from iPSCs of patients with idiopathic azoospermiaObjective We aim to evaluate the differentiation capacity of iPSCs derived from patients with idiopathic non-obstructive azoospermia both in vitro and in vivo.Methods 1.The in vitro differentiation of hiPSCs and h ESCs into PGCLCs was induced with a two-step method,including i Me LC induction and PGC induction.2.PGC-related gene expression dynamics during h PGCLC induction were detected by RT-PCR and immunofluorescence staining.3.Pluripotency gene expression dynamics during h PGCLC induction were detected by RT-PCR and immunofluorescence staining.4.Embryonic lineage gene expression dynamics during h PGCLC induction were detected by RT-PCR and immunofluorescence staining.5.The PGCLC induction efficiency was evaluaged by FACS analysis for Ep CAM /INTEGRIN?6 and c-KIT/INTEGRIN?6 double positive cell percentage.6.Xenotransplantation of patient specific and normal hiPSCs directly into the seminiferous tubules of busulfan-treated immunodeficient mice was performed to evaluate their in vivo differentiation capacity.Results 1.Two days' pre-induction resulted in the induction of hiPSCs into flat epithelial cells with distinct cell borders.then under the floating culture condition,the differentiating cells aggregate to form embryoid bodies.2.Under the stimulation for PGCLC induction,the aggregates derived from all four lines initiated significant upregulation of the early PGC genes(BLIMP1, TFAP2C,NANOS3),but maintained low/no levels for DPPA3 and late PGC genes(DAZL and DDX4).3.The day 4 embryoid bodies derived from all lines upregulated or remained similar levels of key pluripotency genes,OCT4 and NANOG,but became negative for SOX2.compared to undifferentiated cells,the differentiating cells exhibited high levels of genes associated with naive pluripotency such as ZFP42,KLF4 and TFCP2L1,whereas the expression of PRDM14 decreased notably after the pre-induction,except for PGCLCs derived from 1122 iPSCs.4.The day 4 embryoid bodies downregulated endoderm gene GATA6,mesoderm genes RUNX2 and EOMES,and ectoderm gene PAX6,but significantly increased the endoderm gene SOX17.Particularly,the i Me LCs derived from both 1106 and 1122 azoospermic patient specific-hiPSCs showed much lower expression of EOMES than those derived from normal hiPSCs and h ESCs.Additionally,the differentiated cells derived from 1106 iPSCs also exhibited relatively low level of SOX17.5.There was significantly lower percentage of c-KIT/INTEGRIN?6 double positive cells for azoospermic patient specific-iPSCs differentiation than that of normal hiPSCs and h ESCs differentiation on day 4 and day6(p<0.05),however,no differences were detected in Ep CAM/INTEGRIN?6 sorting among different cell lines(p>0.05).6.The azoospermic patient specific hiPSCs are capable of forming early germ cells in vivo,but that,patient derived hiPSCs display limited proliferation potential relative to normal hiPSCs and h ESCs.Conclusions With the stimulation of a series of growth facors and small molecules(ACTA,CHIR,BMP4,LIF,SCF and EGF)to model the developmental environment in vivo,the azoospermic patient specific hiPSCs are capable of forming early germ cells both in vitro and in vivo.But compared with normal hiPSCs and h ESCs,the azoospermic patient specific hiPSCs show differences in embryonic lineage gene expression dynamics,PGCLC induction efficiency and in vivo differentiation capacity,which might be related to the genetic and epigenetic facors for male infertility.Part IV: RNA-Seq analysis of PGCLCs derived from patients with idiopathic azoospermia and comparison with transcriptome of gonadal h PGCs by Guo et al.Objective We aim to examine the global gene expression profile of PGCLCs derived from patients with idiopathic azoospermia and to compare them with transcriptome of gonadal h PGCs by Guo et al.for further research.Methods 1.Ep CAM/INTEGRIN?6 double positive PGCLCs were sorted from day 4 embryoid bodies by FACS.2.Global transcriptome analysis on pre-induction i Me LCs,Ep CAM/INTEGRIN?6 double positive PGCLCs,hiPSCs and h ESCs was performed by RNA sequencing(RNA-seq).3.Bioinformatics analysis of the RNA-seq data.4.We compared the RNA-seq results with the transcriptome data of gonadal h PGCs published by Guo et al.5.Epigenetic-related gene expression dynamics during h PGCLC induction were detected by immunofluorescence staining.Results1.We performed RNA-seq analysis for 24 samples.A total of 127.11 Gb Clean Data was obtained.Each sample had 4.54 Gb of Clean Data and Q30 base percentage is more than 74.59%.The clean reads of each sample were compared with the reference genome and the comparison efficiency ranged from 62.28% to 94.72%.2.At different stages of PGC induction,different cell lines had obviously different gene expression profiles.We performed unsupervised hierarchical clustering(UHC)analysis of the differentially expressed genes.The clustering patterns of patient-specific iPSCs were significantly different from those of normal iPSCs and h ESCs.3.GO enrichment analysis revealed that up-regulated genes in all four cell lines were enriched in BMP signaling pathways,WNT signaling pathways,cell migration regulation,and male gonadal development during the pre-induction of i Me LCs,while the down-regulated genes were enriched in GO terms such as cell adhesion,neural differentiation,cell differentiation,and embryonic development regulation.during the process of PGCLCs induction,the up-regulated genes were enriched in cell migration regulation,cell proliferation,gonadal development and extracellular matrix synthesis,while the down-regulated genes were enriched in neuro development.4.The transcriptome of h PGCLCs differentiated from azoospermic patient specific iPSCs exhibited significant differences from mouse iPSCs.5.During in vitro PGCLC induction for all hiPSC lines and ESCs,a majority of OCT4 positive cells were 5m C negative,whereas almost all of the TFAP2 C positive cells showed robust 5hm C signal.Moreover,the TFAP2 C positive cells from all cell lines presented uniform expression of TET1,and azoospermic patient specific hiPSCs derived OCT4 positive cells exhibited constant expression of DNMT3 A,however,most of the OCT4 positive cells derived from normal hiPSCs and h ESCs were absent for DNMT3 A.Global transcriptome analysis by RNA-seq also suggested analogous initiation of epigenetic reprogram during PGCLC induction from all cell lines.6.The gene expression profiles of PGCLCs derived in vitro were distinct from those of the human gonad PGC.Conclusions According to the transcriptome analysis,iPSCs from patients with idiopathic azoospermia can be differentiated into early PGCLCs,which present relatively low level of some key PGC-related genes.Moreover,PGC induction in vitro still can not completely model the development of PGC in vivo.These differences may be related to the genetic defects of early germ cell development,and further studies are still needed.In addition,h PGCLCs differentiated from azoospermic patient specific iPSCs in vitro initiate DNA methylation and other global epigenetic reprogramming similarly to those from normal hiPSCs and h ESCs.