Objectives This study is designed to generate and propagate human spermatogonial stem cells (SSCs) derived from human pluripotent stem cells (hPSCs). Methods hPSCs were differentiated into SSC-like cells (SSCLCs) by a three-step strategy. The biological characteristics of SSCLCs were detected by immunostaining with antibodies against SSC markers. The ability of self-renewal was measured by propagating for a long time and still maintaining SSCs morphological property. The differentiation potential of SSCLCs was determined by the generation of spermatocytes and haploid cells, which were identified by immunostaining and flow cytometry. The transcriptome analysis of SSCLCs was performed by RNA sequencing. The biological function of SSCLCs was assessed by xeno-transplantation into busulfan-treated mouse testes. Results SSCLCs were efficiently generated by a 3-step strategy. The SSCLCs displayed a grape-like morphology and expressed SSC markers. Moreover, SSCLCs could be propagated for approximately 4 months and still maintained their morphological properties. Furthermore, SSCLCs could differentiate into spermatocytes and haploid cells. In addition, SSCLCs displayed a similar gene expression pattern as human GPR125 + spermatogonia derived from human testicular tissues. And more, SSCLCs could survive and home at the base membrane of seminiferous tubules. Conclusion SSCLCs were successfully derived from hPSCs and propagated for a long time. The SSCLCs resembled their counterpart human GPR125 + spermatogonia, as evidenced by the grape-like morphology, transcriptome, homing, and functional characteristics. Therefore, hPSC-derived SSCLCs may provide a reliable cell source for studying human SSCs biological properties, disease modeling, and drug toxicity screening.

Testicular prostheses have been used to deal with anorchia for nearly 80 years. Here, we evaluated a novel testicular prosthesis that can controllably release hormones to maintain physiological levels of testosterone in vivo for a long time. Silastic testicular prostheses with controlled release of testosterone (STPT) with different dosages of testosterone undecanoate (TU) were prepared and implanted into castrated Sprague-Dawley rats. TU oil was applied by oral administration to a separate group of castrated rats. Castrated untreated and sham-operated groups were used as controls. Serum samples from every group were collected to measure the levels of testosterone (T), follicle-stimulating hormone and luteinizing hormone (LH). Maximum intracavernous penile pressure (ICPmax) was recorded. The prostates and seminal vesicles were weighed and subjected to histology, and a terminal dexynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) assay was used to evaluate apoptosis. Our results revealed that the weights of these tissues and the levels of T and LH showed significant statistical differences in the oral administration and TU replacement groups compared with the castrated group (P < 0.05). Compared with the sham-operated group, the ICPmax, histology and TUNEL staining for apoptosis, showed no significant differences in the hormone replacement groups implanted with medium and high doses of STPT. Our results suggested that this new STPT could release TU stably through its double semi-permeable membranes with excellent biocompatibility. The study provides a new approach for testosterone replacement therapy.

Generation of male germ cells from pluripotent cells could provide male gametes for treating male infertility and offer an ideal model for unveiling molecular mechanisms of spermatogenesis. However, the influence and exact molecular mechanisms, especially downstream effectors of BMP4 signaling pathways, in male germ cell differentiation of the induce pluripotent stem (iPS) cells, remain unknown. This study was designed to explore the role and mechanism of BMP4 signaling in the differentiation of mouse iPS cells to male germ cells. Embryoid body (EB) formation and recombinant BMP4 or Noggin were utilized to evaluate the effect of BMP4 on male germ cell generation from mouse iPS cells. Germ cell-specific genes and proteins as well as the downstream effectors of BMP4 signaling pathway were assessed using real-time PCR and Western blots. We found that BMP4 ligand and its multiple receptors, including BMPR1a, BMPR1b and BMPR2, were expressed in mouse iPS cells. Real-time PCR and Western blots revealed that BMP4 could upregulate the levels of genes and proteins for germ cell markers in iPS cells-derived EBs, whereas Noggin decreased their expression in these cells. Moreover, Smad1/5 phosphorylation, Gata4 transcription and the transcripts of Id1 and Id2 were enhanced by BMP4 but decreased when exposed to Noggin. Collectively, these results suggest that BMP4 promotes the generation of male germ cells from iPS cells via Smad1/5 pathway and the activation of Gata4, Id1 and Id2 This study thus offers novel insights into molecular mechanisms underlying male germ cell development.

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.

OBJECTIVE:To isolate, identify and culture human spermatogonial stem cells (SSC) and then obtain purified and enriched human SSCs for research and application.METHODS:We detected the expression of CD90 in the human testis using the immunofluorescence technique and isolated human testicular spermatogenic cells by two-step enzymatic digestion, followed by differential plating and magnetic-activated cell sorting (MACS) with CD90 as an SSC marker. Then we identified the isolated CD90-positive spermatogenic cells by RT-PCR and immunocytochemistry, and meanwhile cocultured them with Sertoli cells in SG medium in vitro.RESULTS:The isolated CD90-positive cells showed a relatively homogeneous characteristic in size and morphology and expressed the genes specific for human SSCs, with high expressions (90.5%) of GFRA1, GPR125, and UCHL1. After coculture with Sertoli cells in the SG medium for 2 weeks, the isolated CD90-positive cells maintained a good activity.CONCLUSION:CD90 can be regarded as a speci- fic marker for human SSCs and used to obtain highly enriched human SSCs by differential plating and MACS. Furthermore, the isolated human SSCs can be cultured in SG medium in vitro.

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.

Spermatogonial stem cells (SSCs) have significant applications in both reproductive and regenerative medicine. However, primary human SSCs are very rare, and a human SSC line has not yet been available. In this study, we have for the first time reported a stable human SSC line by stably expressing human SV40 large T antigen. RT-PCR, immunocytochemistry, and Western blots revealed that this cell line was positive for a number of human spermatogonial and SSC hallmarks, including VASA, DAZL, MAGEA4, GFRA1, RET, UCHL1, GPR125, PLZF and THY1, suggesting that these cells are human SSCs phenotypically. Proliferation analysis showed that the cell line could be expanded with significant increases of cells for 1.5 years, and high levels of PCNA, UCHL1 and SV40 were maintained for long-term culture. Transplantation assay indicated that human SSC line was able to colonize and proliferate in vivo in the recipient mice. Neither Y chromosome microdeletions of numerous genes nor tumor formation was observed in human SSC line although there was abnormal karyotype in this cell line. Collectively, we have established a human SSC line with unlimited proliferation potentials and no tumorgenesis, which could provide an abundant source of human SSCs for their mechanistic studies and translational medicine.

Objective To evaluate the sperm retrieval rate (SRR) in non-obstructive azoospermia (NOA) associated with cryptorchidism and the effect of age at orchidopexy on SRR.Methods A total of 33 NOA after cryptorchidism (inguinal) and 46 NOA without cryptorchidism (as control) matched for testicular volume,follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels.The NOA after cryptorchidism underwent microdissection of testicular sperm extraction (MD-TESE) from September 2012 to March 2014.The SRR and testicular histology between the two groups were compared.The effect of age at orchidopexy (Group A:age 10 and younger; Group B:age older than 10) on testicular volume,hormone levels (FSH and LH) and SRR were analyzed.Results The SRR in NOA after cryptorchidism (70％,23/33) was significantly higher than that in control group (46％,21/46) (P＜0.05).The histology in NOA after cryptorchidism was significantly better than that in control (maturation arrest:64％ versus 39％ ; Sertoli cell-only syndrome:21％ versus 52％) (P＜0.05).No significant differences were found in testicular volume [(8.51±2.73) mlversus (8.80±5.74) ml],FSH [(24.83±7.63) U/Lversus (25.32±5.66) U/L],LH [(9.19±3.49) U/L versus (10.64±5.67) U/L] and SRR [73％ (11/15) versus 67％ (12/18)] between Group A and Group B (P＞0.05).Conclusions The SRR is significantly higher in NOA after cryptorchidism than that in NOA without cryptorchidism,and it might be the result of better histology in NOA associated with cryptorchidism.The age at orchidopexy shows no correlations with testicular volume,hormone levels and SRR in NOA after cryptorchidism.

It is known that spermatogenic disorders are associated with genetic deficiency, although the primary mechanism is still unclear. It is difficult to demonstrate the molecular events occurring in testis, which contains germ cells at different developmental stages. However, transcriptomic methods can help us reveal the molecular drive of male gamete generation. Many transcriptomic studies have been performed on rodents by utilizing the timing of the first wave of spermatogenesis, which is not a suitable strategy for research in fertile men. With the development of separation methods for male germ cells, transcriptome research on the molecular drive of spermatogenesis in fertile men has seen great progress, and the results could be ultimately applied to improve the diagnosis and treatment for male infertility.

This study was designed to explore the regulatory effects of male germ cell secreting factor NODAL on Sertoli cell fate decisions from obstructive azoospermia (OA) and nonobstructive azoospermia (NOA) patients. Human Sertoli cells and male germ cells were isolated using two-step enzymatic digestion and SATPUT from testes of azoospermia patients. Expression of NODAL and its multiple receptors in human Sertoli cells and male germ cells were characterized by reverse transcription-polymerase chain reaction (RT-PCR) and immunochemistry. Human recombinant NODAL and its receptor inhibitor SB431542 were employed to probe their effect on the proliferation of Sertoli cells using the CCK-8 assay. Quantitative PCR and Western blots were utilized to assess the expression of Sertoli cell functional genes and proteins. NODAL was found to be expressed in male germ cells but not in Sertoli cells, whereas its receptors ALK4, ALK7, and ACTR-IIB were detected in Sertoli cells and germ cells, suggesting that NODAL plays a regulatory role in Sertoli cells and germ cells via a paracrine and autocrine pathway, respectively. Human recombinant NODAL could promote the proliferation of human Sertoli cells. The expression of cell cycle regulators, including CYCLIN A, CYCLIN D1 and CYCLIN E, was not remarkably affected by NODAL signaling. NODAL enhanced the expression of essential growth factors, including GDNF, SCF, and BMP4, whereas SB431542 decreased their levels. There was not homogeneity of genes changes by NODAL treatment in Sertoli cells from OA and Sertoli cell-only syndrome (SCO) patients. Collectively, this study demonstrates that NODAL produced by human male germ cells regulates proliferation and numerous gene expression of Sertoli cells.

Parkinson’s disease (PD) is a common neurodegenerative syndrome characterized by loss of midbrain dopaminergic (DA) neurons. Generation of functional dopaminergic (DA) neurons is of unusual significance for treating Parkinson’s disease (PD). However, direct conversion of spermatogonial stem cells (SSCs) to functional DA neurons without being reprogrammed to a pluripotent status has not been achieved. Here, we report an efficient approach to obtain morphological, phenotypic, and functional DA neurons from SSCs using a specific combination of olfactory ensheathing cell-conditioned medium (OECCM) and several defined growth factors (DGF). By following the current protocol, direct conversion of SSCs (both SSC line and primary SSCs) to neural cells and DA neurons was demonstrated by expression of numerous phenotypic genes and proteins for neural cells, as well as cell morphological features. More significantly, SSCs-derived DA neurons acquired neuronal functional properties such as synapse formation, electrophysiology activity, and dopamine secretion. Furthermore, PI3K/Akt pathway and p21/Nolz1 cascades were activated whereas Smurf2 was inactivated, leading to cell cycle exit during the conversion of SSCs into DA neurons. Collectively, this study could provide sufficient neural cells from SSCs for applications in the treatment of PD and offers novel insights into mechanisms underlying neural system development from the line of germ cells.

BACKGROUND:Indian 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.METHODS:A 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.RESULTS:We 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.CONCLUSIONS:Our 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.

Objective The differentiation of male germ cells from iPS cells provides an ideal model for unveiling molecular mechanisms of spermatogenesis. Nodal could promote proliferation of mouse spermatogonial stem/progenitor cells via Smad2/3 and oct-4 activation. The objective of this study was to determine the role of Nodal signaling in the differentiation of iPS cells to male germ cells.

Infertility is a major and largely incurable disease caused by disruption and loss of germ cells. It affects 10-15% of couples, and male factor accounts for half of the cases. To obtain human male germ cells 'especially functional spermatids' is essential for treating male infertility. Currently, much progress has been made on generating male germ cells, including spermatogonia, spermatocytes, and spermatids, from various types of stem cells. These germ cells can also be used in investigation of the pathology of male infertility. In this review, we focused on advances on obtaining male differentiated germ cells from different kinds of stem cells, with an emphasis on the embryonic stem (ES) cells, the induced pluripotent stem (iPS) cells, and spermatogonial stem cells (SSCs). We illustrated the generation of male differentiated germ cells from ES cells, iPS cells and SSCs, and we summarized the phenotype for these stem cells, spermatocytes and spermatids. Moreover, we address the differentiation potentials of ES cells, iPS cells and SSCs. We also highlight the advantages, disadvantages and concerns on derivation of the differentiated male germ cells from several types of stem cells. The ability of generating mature and functional male gametes from stem cells could enable us to understand the precise etiology of male infertility and offer an invaluable source of autologous male gametes for treating male infertility of azoospermia patients.

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.

Objective To investigate the differentiation potential of induced pluripotent stem cells (iPSCs) into male germ cells through retinoic acid (RA) induction in vitro and ectopic transplantation in vivo.Methods Mouse iPSCs were first transferred to bacterial plates to form embryoid body (EB).After 5days,EB was treated by 2 μmol/L RA.After 10 days of cuhure,iPSCs-derived cells were dissociated from EB.The real-time PCR and immunocytochemical assay were used to detect the expression of related gene and protein.Then,they were injected into the male nude mice with testicular cells from neonatal mice.The grafts were obtained for further HE and immunohistochemical staining on 4,6,8,12 weeks after injection.Results After EB formation and RA induction,the expressions of Stra8,Odf2,Act,and Prm1 were upregulated in induction group,which were 4.50± 1.02,1.48±0.04,10.52±0.25,323.28±25.64 times as control group,respectively.On the contrary,the expressions of Oct-4,Dppa3,Piwil2,Tex14,and Scp3 were down-regulated in induction group,which were 0.56±0.02,0.11±0.01,0.50±0.01,0.53±0.14,0.16±0.00times as control group,respectively.Meanwhile,RA-treated EBs also expressed male germ cell markers VASA and SCP3.HE staining showed that the seminiferous tubules could be reconstituted after injecting the iPSCs-derived cells and testicular cells from neonatal mice.Immunofluorescence analysis revealed co-localization of EGFP-positive and VASA-positive cells in the reconstituted tubules.Conclusions Mouse iPSCs could differentiate into male germ cells with EB formation and RA induction.The iPSCs-derived germ cells could settle in the basement membrane of reconstituted tubules,which indicates reconstituted seminiferous tubules may provide a functional niche for exogenous iPSCs-derived male germ cells.

Colorectal cancer is one of the most common and fatal tumors. However, molecular mechanisms underlying carcinogenesis of colorectal cancer remain largely undefined. Here, we explored the expression and function of Nodal in colon cancer stem cells (CCSCs). Nodal and its receptors were present in numerous human colorectal cancer cell lines. NODAL and ALK-4 were coexpressed in human colon cancerous tissues, and NODAL, CD24, and CD44, markers for CCSCs, were expressed at higher levels in human colon cancerous tissues than adjacent noncancerous colon tissues. Human CCSCs were isolated by magnetic activated cell sorting using anti-CD24 and anti-CD44. Nodal transcript and protein were hardly detectable in CD44- or CD24-negative human colorectal cancer cell lines, whereas Nodal and its receptors were present in CCSCs. Notably, Nodal facilitated spheroid formation of human CCSCs, and phosphorylation of Smad2 and Smad3 was activated by Nodal in cells of spheres derived from human CCSCs. Collectively, these results suggest that Nodal promotes the self-renewal of human CCSCs and mediate carcinogenesis of human colorectal cancer via an autocrine manner through Smad2/3 pathway. This study provides a novel insight into molecular mechanisms controlling fate of human CCSCs and offers new targets for gene therapy of human colorectal cancer.

Background Severe shortage of liver donors and hepatocytes highlights urgent requirement of extra-liver and stem cell source of hepatocytes for treating liver-related diseases. Here we hypothesized that spermatogonial stem cells (SSCs) can directly transdifferentiate to hepatic stem-like cells capable of differentiating into mature hepatocyte-like cells in vitro without an intervening pluripotent state. Results SSCs first changed into hepatic stem-like cells since they resembled hepatic oval cells in morphology and expressed Ck8 , Ck18 , Ck7 , Ck19 , OV6, and albumin. Importantly, they co-expressed CK8 and CK19 but not ES cell markers. Hepatic stem-like cells derived from SSCs could differentiate into small hepatocytes based upon their morphological features and expression of numerous hepatic cell markers but lacking of bile epithelial cell hallmarks. Small hepatocytes were further coaxed to differentiate into mature hepatocyte-like cells, as identified by their morphological traits and strong expression of Ck8 , Ck18 , Cyp7a1 , Hnf3b , Alb, Ta t, Ttr , albumin, and CYP1A2 but not Ck7 or CK19. Notably, these differentiated cells acquired functional attributes of hepatocyte-like cells because they secreted albumin, synthesized urea, and uptake and released indocyanine green. Moreover, phosphorylation of ERK1/2 and Smad2/3 rather than Akt was activated in hepatic stem cells and mature hepatocytes. Additionally, cyclin A, cyclin B and cyclin E transcripts and proteins but not cyclin D1 or CDK1 and CDK2 transcripts or proteins were reduced in mature hepatocyte-like cells or hepatic stem-like cells derived from SSCs compared to SSCs. Conclusions SSCs can transdifferentiate to hepatic stem-like cells capable of differentiating into cells with morphological, phenotypic and functional characteristics of mature hepatocytes via the activation of ERK1/2 and Smad2/3 signaling pathways and the inactivation of cyclin A, cyclin B and cyclin E. This study thus provides an invaluable source of mature hepatocytes for treating liver-related diseases and drug toxicity screening and offers novel insights into mechanisms of liver development and cell reprogramming.

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.