Ruhui Tian

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study.

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.

Derivation of male germ cells from induced pluripotent stem cells in vitro and in reconstituted seminiferous tubules.

Previous studies have demonstrated that mouse‐ and human‐induced pluripotent stem (iPS) cells can differentiate into primordial germ cells in vitro. However, up to now it is not known whether iPS cells would be able to differentiate into male germ cells in vivo. The aim of this study was to explore differentiation potential of iPS cells to male germ cells in vitro and in vivo.

Generation of Haploid Spermatids with Fertilization and Development Capacity from Human Spermatogonial Stem Cells of Cryptorchid Patients

Summary Generation of functional spermatids from azoospermia patients is of unusual significance in the treatment of male infertility. Here, we report an efficient approach to obtain human functional spermatids from cryptorchid patients. Spermatogonia remained whereas meiotic germ cells were rare in cryptorchid patients. Expression of numerous markers for meiotic and postmeiotic male germ cells was enhanced in human spermatogonial stem cells (SSCs) of cryptorchidism patients by retinoic acid (RA) and stem cell factor (SCF) treatment. Meiotic spreads and DNA content assays revealed that RA and SCF induced a remarkable increase of SCP3-, MLH1-, and CREST-positive cells and haploid cells. Single-cell RNA sequencing analysis reflected distinct global gene profiles in embryos derived from round spermatids and nuclei of somatic cells. Significantly, haploid spermatids generated from human SSCs of cryptorchid patients possessed fertilization and development capacity. This study thus provides an invaluable source of autologous male gametes for treating male infertility in azoospermia patients.

Sertoli cells from non-obstructive azoospermia and obstructive azoospermia patients show distinct morphology, Raman spectrum and biochemical phenotype

STUDY QUESTION Are there differences in the morphology, spectrum and biochemical phenotype between Sertoli cells from non-obstructive azoospermia (NOA) patients and those from obstructive azoospermia (OA) patients with normal spermatogenesis? SUMMARY ANSWER Sertoli cells from NOA patients are distinct from those from OA patients in terms of morphological features, Raman spectrum and phenotype including the expression of genes and proteins (e.g. SCF, BMP4 and GDNF). WHAT IS KNOWN ALREADY NOA affects 10% of infertile men and has been diagnosed in 60% of azoospermic men. In contrast with OA patients who have normal spermatogenesis, NOA patients have an impaired spermatogenesis. STUDY DESIGN, SIZE AND DURATION This case-control study included 100 NOA patients (as cases) and 100 OA patients with normal spermatogenesis (as controls). The study was performed between January 2012 and January 2013. PARTICIPANTS/MATERIALS, SETTING AND METHODS Karyotype analysis was performed to check the chromosome content and multiplex PCR was carried out to determine the expression of numerous Y chromosome genes in NOA patients. Human Sertoli cells were then isolated from the testes of NOA and OA patients by two-step enzymatic digestion and differential plating. Transmission electron microscopy was used to determine the ultrastructure of the Sertoli cells and real-time Raman microspectroscopy was used to assess their spectrum. We further compared the two groups of patients for expression of SCF, GDNF and BMP4 in Sertoli cells, using RT-PCR, microarray analysis, immunofluorescence, immunohistochemistry and Western blots. MAIN RESULTS AND THE ROLE OF CHANCE NOA patients had normal chromosome karyotypes and Y chromosome microdeletions were excluded. In morphology, Sertoli cells isolated from NOA patients had a series of abnormal ultrastructural features compared with the control Sertoli cells: (i) existence of small and spindle-shaped nuclei, (ii) smaller diameter, (iii) deficient nucleolus or endoplasmic reticulum and (iv) more vacuoles. Spectral intensities in Sertoli cells of NOA patients were distinct at four typical Raman peaks compared with the control Sertoli cells. In phenotype, SCF, BMP4 and GDNF transcripts and proteins were significantly lower in Sertoli cells of NOA patients than in the control Sertoli cells. LIMITATIONS AND REASONS FOR CAUTION The Sertoli cells of NOA patients were not compared with Sertoli cells of normal fertile men due to the fact that it is hard to obtain adult testes from normal donors. WIDER IMPLICATIONS OF THE FINDINGS This study provides novel insights into understanding the underlying causes for NOA and might offer a basis for developing new therapeutic strategies for patients with NOA.

Differentiation of Induced Pluripotent Stem Cells into Male Germ Cells In Vitro through Embryoid Body Formation and Retinoic Acid or Testosterone Induction

Generation of germ cells from pluripotent stem cells in vitro could have great application for treating infertility and provides an excellent model for uncovering molecular mechanisms controlling gametogenesis. In this study, we explored the differentiation potential of mouse induced pluripotent stem (iPS) cells towards male germ cells. Embryoid body formation and retinoic acid/testosterone induction were applied to promote differentiation of mouse iPS cells into male germ cells in vitro. Quantitative RT-PCR and immunoflourescence were performed to characterize the iPS cell differentiation process, and notably there were different temporal expression profiles of male germ cell-associated genes. The expression of proteins, including MVH, CDH1, and SCP3, was remarkably increased. mRNA expression of Stra8, Odf2, Act, and Prm1 was upregulated in iPS cells by retinoic acid or testosterone induction, whereas Oct-4 transcription was reduced in these cells compared to the controls. Hormones were also measured in the EB medium. DNA content analysis by flow cytometry revealed that iPS cells could differentiate into haploid cells through retinoic acid or testosterone treatment. Collectively, our results suggest that mouse iPS cells possess the potency to differentiate into male germ cells in vitro through embryoid body formation and retinoic acid or testosterone induction.

Dynamics of the Transcriptome during Human Spermatogenesis: Predicting the Potential Key Genes Regulating Male Gametes Generation.

Many infertile men are the victims of spermatogenesis disorder. However, conventional clinical test could not provide efficient information on the causes of spermatogenesis disorder and guide the doctor how to treat it. More effective diagnosis and treating methods could be developed if the key genes that regulate spermatogenesis were determined. Many works have been done on animal models, while there are few works on human beings due to the limited sample resources. In current work, testis tissues were obtained from 27 patients with obstructive azoospermia via surgery. The combination of Fluorescence Activated Cell Sorting and Magnetic Activated Cell Sorting was chosen as the efficient method to sort typical germ cells during spermatogenesis. RNA Sequencing was carried out to screen the change of transcriptomic profile of the germ cells during spermatogenesis. Differential expressed genes were clustered according to their expression patterns. Gene Ontology annotation, pathway analysis, and Gene Set Enrichment Analysis were carried out on genes with specific expression patterns and the potential key genes such as HOXs, JUN, SP1, and TCF3 which were involved in the regulation of spermatogenesis, with the potential value serve as molecular tools for clinical purpose, were predicted.

Mesenchymal stem cells overexpressing Ihh promote bone repair

BackgroundIndian hedgehog (Ihh) signaling pathway is known to play key roles in various aspects of normal endochondral bone development. This study tested the potential roles of high Ihh signaling in the context of injury-induced bone regeneration.MethodsA rabbit tibia defect model was established to test the effects of the implant of Ihh/mesenchymal stem cells (MSCs)/scaffold complex. Computed tomography (CT), gross observation, and standard histological and immunohistological techniques were used to evaluate the effectiveness of the treatment. In vitro studies with MSCs and C3H10T1/2 cells were also employed to further understand the cellular and molecular mechanisms.ResultsWe found that the implanted Ihh/MSCs/scaffold complex promoted bone repair. Consistently, in vitro study found that Ihh induced the upregulation of chondrocytic, osteogenic, and vascular cell markers, both in C3H10T1/2 cells and MSCs.ConclusionsOur study has demonstrated that high Ihh signaling in a complex with MSCs enhanced bone regeneration effectively in a clinically relevant acute injury model. Even though the exact underlying mechanisms are still far from clear, our primary data suggested that enhanced chondrogenesis, osteogenesis, and angiogenesis of MSCs at least partially contribute to the process. This study not only has implications for basic research of MSCs and Ihh signaling pathway but also points to the possibility of direct application of this specific paradigm to clinical bone repair.

VEGF/VEGFR2 Signaling Regulates Germ Cell Proliferation in vitro and Promotes Mouse Testicular Regeneration in vivo

Vascular endothelial growth factor (VEGF) plays fundamental roles in testicular development; however, its function on testicular regeneration remains unknown. The objective of this study was to explore the roles VEGF/VEGFR2 signaling plays in mouse germ cells and in mouse testicular regeneration. VEGF and the VEGFR2 antagonist SU5416 were added to culture medium to evaluate their effects on spermatogonial stem cell line (C18-4 cells) proliferation. Testicular cells obtained from newborn male ICR mice were grafted into the dorsal region of male BALB/c nude mice. VEGF and SU5416 were injected into the graft sites to assess the effects of the VEGF and VEGFR2 signaling pathways on testicular reconstitution. The grafts were analyzed after 8 weeks. We found that VEGF promoted C18-4 proliferation in vitro, indicating its role in germ cell survival. HE staining revealed that seminiferous tubules were reconstituted and male germ cells from spermatogonia to spermatids could be observed in testis-like tissues 8 weeks after grafting. A few advantaged male germ cells, including spermatocytes and spermatids, were found in SU5416-treated grafts. Moreover, VEGF enhanced the expression of genes specific for male germ cells and vascularization in 8-week grafts, whereas SU5416 decreased the expression of these genes. SU5416-treated grafts had a lower expression of MVH and CD31, indicating that blockade of VEGF/VEGFR2 signaling reduces the efficiency of seminiferous tubule reconstitution. Collectively, these data suggest that VEGF/VEGFR2 signaling regulates germ cell proliferation and promotes testicular regeneration via direct action on germ cells and the enhancement of vascularization.

VEGFC/VEGFR3 Signaling Regulates Mouse Spermatogonial Cell Proliferation via the Activation of AKT/MAPK and Cyclin D1 Pathway and Mediates the Apoptosis by affecting Caspase 3/9 and Bcl-2

ABSTRACT We have previously shown that the transcript levels of Vegfc and its receptor Vegfr3 were high in spermatogonia and extremely low in spermatocytes and spermatids. However, it remains unknown about the functions and the mechanisms of VEGFC/VEGFR3 signaling in regulating the fate determinations of spermatogonia. To this end, here we explored the role and signaling pathways of VEGFC/VEGFR3 by using a cell line derived from immortalized mouse spermatogonia retaining markers of mitotic germ cells, namely GC-1 cells. VEGFR3 was expressed in mouse primary spermatogonia and GC-1 cells. VEGFC stimulated the proliferation and DNA synthesis of GC-1 cells and enhanced the phosphorylation of PI3K-AKT and MAPK, whereas LY294002 (an inhibitor for AKT) and CI-1040 (an inhibitor for MAPK) blocked the effect of VEGFC on GC-1 cell proliferation. Furthermore, VEGFC increased the transcripts of c-fos and Egr1 and protein levels of cyclin D1, PCNA and Bcl-2. Conversely, the blocking of VEGFC/VEGFR3 signaling by VEGFR3 knockdown reduced the phosphorylation of AKT/MAPK and decreased the levels of cyclin D1 and PCNA. Additionally, VEGFR3 knockdown not only resulted in more apoptosis of GC-1 cells but also led to a decrease of Bcl-2 and promoted the cleavage of Caspase-3/9 and PARP. Collectively, these data suggested that VEGFC/VEGFR3 signaling promotes the proliferation of GC-1 cells via the AKT /MAPK and cyclin D1 pathway and it inhibits the cell apoptosis through Caspase-3/9, PARP and Bcl-2. Thus, this study sheds a novel insight to the molecular mechanisms underlying the fate decisions of mammalian spermatogonia.

AB199. Human germ cell secreting factor Nodal regulates Sertoli cell functions

Objectives To explore the regulatory effects of germ cells and germ cells secreting factor Nodal on the function of Sertoli cells derived from obstructive azoospermia and non-obstructive azoospermia patients. Design Comparative and controlled study. Materials and methods Human Sertoli cells and germ cells were isolated using two-steps enzymatic digestions from the testes of obstructive azoospermia and non-obstructive azoospermia patients respectively. Expressions of Nodal signaling components in human Sertoli cells and germ cells were identified by PCR and immunochemistry. Human germ cells and Sertoli cells were cocultured in vitro to evaluate their effects on Sertoli cells. Human recombinant nodal and its receptor inhibitor SB431542 were added in the Sertoli cells culture medium to study their effects on Sertoli cell functions. CCK8 measurement was used to evaluate the proliferative activity. Q-PCR and western blot were applied to assess the expression of functional Sertoli cell genes. Results Human germ cells down-regulated blood-testis-barrier associated genes (CLDN11, OCLN) expressions of Sertoli cells in co-culture system. Nodal was expressed in germ cells but not in Sertoli cells, whereas its receptors ALK4, ALK7, and ActR-IIB were detected on Sertoli cells, which indicated Nodal signaling pathway, may play roles in the regulation of germ cells to Sertoli cells. Human recombinant nodal could promote the proliferation of human Sertoli cells, while the proliferative activity was inhibited by SB431542. Nodal could enhance the expressions of functional Sertoli cell genes (GDNF, SCF, BMP4, and ABP), while SB431542 decreased their expressions. In contrast, Nodal decreased the expression of blood-testis-barrier associated genes (CLDN11, OCLN), while SB431542 increase their expressions. Conclusions Human Sertoli cell functions could be regulated by germ cells via paracrine pathway. Human germ cells secrete Nodal which could regulate Sertoli cell functions.