Isolation and In Vitro Propagation of Human Spermatogonial Stem Cells (SSCs).

Preservation of human spermatogonial stem cells (SSCs) may be suitable for young male patients at risk of male infertility due to various causes, such as gonadotoxic treatment or genetic diseases. With optimal cryopreservation, cell viability can be retained to reestablish spermatogenesis in the future through autologous transplantation or in vitro differentiation of SSCs. This protocol outlines techniques to optimize the SSCs isolation and in vitro culture. With particular emphasis on the microscopic characteristics encountered, this protocol outlines a broader approach to processing tissues with varying morphologies among patients.

Advances of three-dimensional (3D) culture systems for in vitro spermatogenesis.

The loss of germ cells and spermatogenic failure in non-obstructive azoospermia are believed to be the main causes of male infertility. Laboratory studies have used in vitro testicular models and different 3-dimensional (3D) culture systems for preservation, proliferation and differentiation of spermatogonial stem cells (SSCs) in recent decades. The establishment of testis-like structures would facilitate the study of drug and toxicity screening, pathological mechanisms and in vitro differentiation of SSCs which resulted in possible treatment of male infertility. The different culture systems using cellular aggregation with self-assembling capability, the use of different natural and synthetic biomaterials and various methods for scaffold fabrication provided a suitable 3D niche for testicular cells development. Recently, 3D culture models have noticeably used in research for their architectural and functional similarities to native microenvironment. In this review article, we briefly investigated the recent 3D culture systems that provided a suitable platform for male fertility preservation through organ culture of testis fragments, proliferation and differentiation of SSCs.

The developmental dynamics of the human male germline.

Male germ cells undergo a complex sequence of developmental events throughout fetal and postnatal life that culminate in the formation of haploid gametes: the spermatozoa. Errors in these processes result in infertility and congenital abnormalities in offspring. Male germ cell development starts when pluripotent cells undergo specification to sexually uncommitted primordial germ cells, which act as precursors of both oocytes and spermatozoa. Male-specific development subsequently occurs in the fetal testes, resulting in the formation of spermatogonial stem cells: the foundational stem cells responsible for lifelong generation of spermatozoa. Although deciphering such developmental processes is challenging in humans, recent studies using various models and single-cell sequencing approaches have shed new insight into human male germ cell development. Here, we provide an overview of cellular, signaling and epigenetic cascades of events accompanying male gametogenesis, highlighting conserved features and the differences between humans and other model organisms.

Lineage Tracing of Spermatogonial Stem Cells Within the Male Germline.

Spermatogonial stem cells (SSCs) are the fundamental units from which continuous spermatogenesis arises. Although our knowledge regarding the basic properties of SSCs has grown, driven primarily through the advancement of techniques and technologies to study SSCs, the mechanisms controlling their fate remain largely unknown. Among the modern strategies to evaluate SSCs, lineage tracing is among the few established approaches that allow for functional assessment of stem cell capacity. As a result, lineage tracing continues to forge new discoveries underlying the basic attributes of SSCs as well as the molecular factors that govern SSC function. Traditional approaches to lineage tracing with dyes or radioactive labels suffer from progressive loss after successive cell divisions or unintentional label transfer to neighboring cells. To address these limitations, genetic approaches primarily leveraging transgenic technologies have prevailed as the preferred avenue for modern lineage tracing. This chapter will discuss current protocols for effective genetic lineage tracing and address applications of this technology, considerations when designing lineage tracing experiments, and the methods involved in utilizing lineage tracing to study SSCs and other cell populations.

Defining Gene Function in Spermatogonial Stem Cells Through Conditional Knockout Approaches.

Mammalian male fertility is maintained throughout life by a population of self-renewing mitotic germ cells known as spermatogonial stem cells (SSCs). Much of our current understanding regarding the molecular mechanisms underlying SSC activity is derived from studies using conditional knockout mouse models. Here, we provide a guide for the selection and use of mouse strains to develop conditional knockout models for the study of SSCs, as well as their precursors and differentiation-committed progeny. We describe Cre recombinase-expressing strains, breeding strategies to generate experimental groups, and treatment regimens for inducible knockout models and provide advice for verifying and improving conditional knockout efficiency. This resource can be beneficial to those aiming to develop conditional knockout models for the study of SSC development and postnatal function.

Jun-mediated lncRNA-IMS promotes the meiosis of chicken spermatogonial stem cells via gga-miR-31-5p/stra8.

Meiosis, a key step in spermatogenesis, is affected by many factors. Current studies have shown that long noncoding RNAs (lncRNAs) are potential factors regulating meiosis, and their regulatory mechanisms have received much attention. However, little research has been done on its regulatory mechanism in the spermatogenesis of roosters. Here, we found that lncRNA involved in meiosis and spermatogenesis (lncRNA-IMS) was involved in the regulation of Stra8 by gga-miR-31-5p and hindered the inhibition of Stra8 by gga-miR-31-5p. The acquisition and loss of function experiments demonstrated that lncRNA-IMS was involved in meiosis and spermatogenesis. In addition, we predicted and determined the core promoter region of lncRNA-IMS. Prediction of transcription factors, deletion/overexpression of binding sites, knockdown/overexpression of Jun, and dual-luciferase reporter analysis confirmed that Jun positively activated transcription of lncRNA-IMS. Our findings further enrich the TF-lncRNA-miRNA-mRNA regulatory network during male meiosis and provide new ideas for studying the molecular mechanism of meiosis and spermatogenesis in chicken spermatogonial stem cells.

Conserved Transcriptome Features Define Prepubertal Primate Spermatogonial Stem Cells as Adark Spermatogonia and Identify Unique Regulators.

Antineoplastic treatments for cancer and other non-malignant disorders can result in long-term or permanent male infertility by ablating spermatogonial stem cells (SSCs). SSC transplantation using testicular tissue harvested before a sterilizing treatment is a promising approach for restoring male fertility in these cases, but a lack of exclusive biomarkers to unequivocally identify prepubertal SSCs limits their therapeutic potential. To address this, we performed single-cell RNA-seq on testis cells from immature baboons and macaques and compared these cells with published data from prepubertal human testis cells and functionally-defined mouse SSCs. While we found discrete groups of human spermatogonia, baboon and rhesus spermatogonia appeared less heterogenous. A cross-species analysis revealed cell types analogous to human SSCs in baboon and rhesus germ cells, but a comparison with mouse SSCs revealed significant differences with primate SSCs. Primate-specific SSC genes were enriched for components and regulators of the actin cytoskeleton and participate in cell-adhesion, which may explain why the culture conditions for rodent SSCs are not appropriate for primate SSCs. Furthermore, correlating the molecular definitions of human SSC, progenitor and differentiating spermatogonia with the histological definitions of Adark/Apale spermatogonia indicates that both SSCs and progenitor spermatogonia are Adark, while Apale spermatogonia appear biased towards differentiation. These results resolve the molecular identity of prepubertal human SSCs, define novel pathways that could be leveraged for advancing their selection and propagation in vitro, and confirm that the human SSC pool resides entirely within Adark spermatogonia.

Multi-Omics Approaches for Revealing the Epigenetic Regulation of Histone H3.1 during Spermatogonial Stem Cell Differentiation In Vitro.

Epigenetic regulation, particularly post-translational modifications (PTMs) of histones, participates in spermatogonial stem cell (SSCs) differentiation. However, there is a lack of systemic studies of histone PTM regulation during the differentiation of SSCs due to its low number in vivo. Herein, we quantified dynamic changes of 46 different PTMs on histone H3.1 by targeted quantitative proteomics using mass spectrometry during SSCs differentiation in vitro, in combination with our RNA-seq data. We identified seven histone H3.1 modifications to be differentially regulated. In addition, we selected H3K9me2 and H3S10ph for subsequent biotinylated peptide pull-down experiments and identified 38 H3K9me2-binding proteins and 42 H3S10ph-binding proteins, which contain several transcription factors, such as GTF2E2 and SUPT5H, which appear to be crucial for epigenetic regulation of SSC differentiation.

MAST4 controls cell cycle in spermatogonial stem cells.

Spermatogonial stem cell (SSC) self-renewal is regulated by reciprocal interactions between Sertoli cells and SSCs in the testis. In a previous study, microtubule-associated serine/threonine kinase 4 (MAST4) has been studied in Sertoli cells as a regulator of SSC self-renewal. The present study focused on the mechanism by which MAST4 in Sertoli cells transmits the signal and regulates SSCs, especially cell cycle regulation. The expression of PLZF, CDK2 and PLZF target genes was examined in WT and Mast4 KO testes by Immunohistochemistry, RT-qPCR and western blot. In addition, IdU and BrdU were injected into WT and Mast4 KO mice and cell cycle of SSCs was analysed. Finally, the testis tissues were cultured in vitro to examine the regulation of cell cycle by MAST4 pathway. Mast4 KO mice showed infertility with Sertoli cell-only syndrome and reduced sperm count. Furthermore, Mast4 deletion led to decreased PLZF expression and cell cycle progression in the testes. MAST4 also induced cyclin-dependent kinase 2 (CDK2) to phosphorylate PLZF and activated PLZF suppressed the transcriptional levels of genes related to cell cycle arrest, leading SSCs to remain stem cell state. MAST4 is essential for maintaining cell cycle in SSCs via the CDK2-PLZF interaction. These results demonstrate the pivotal role of MAST4 regulating cell cycle of SSCs and the significance of spermatogenesis.

Epigenetic priming as a mechanism of predetermination of spermatogonial stem cell fate.

In the developing mammalian testis, only a small proportion of fetal and neonatal prospermatogonia give rise to the foundational pool of spermatogonial stem cells (SSCs). Multiple lines of evidence have suggested the determination of which prospermatogonia give rise to foundational SSCs is not random, but is rather predetermined, such that foundational SSCs are ensured to develop advantageous characteristics such as enhanced genetic integrity. Here I suggest that differential epigenetic programing contributes to the molecular mechanisms by which an early subset of developing prospermatogonia becomes predetermined to form the foundational pool of SSCs. This would include epigenetic programing that promotes active expression of genes needed to develop advantageous characteristics, as well as differential epigenetic priming, which bookmarks genes that comprise the SSC-specific transcriptome to become activated when foundational SSCs appear in the postnatal testis. I suggest that, together, differential epigenetic programing and epigenetic priming contribute to the molecular mechanisms by which an early subset of developing prospermatogonia becomes predetermined to form the foundational pool of SSCs.

MAP4K4/JNK Signaling Pathway Stimulates Proliferation and Suppresses Apoptosis of Human Spermatogonial Stem Cells and Lower Level of MAP4K4 Is Associated with Male Infertility.

Spermatogonial stem cells (SSCs) serve as a foundation for spermatogenesis and they are essential for male fertility. The fate of SSC is determined by genetic and epigenetic regulatory networks. Many molecules that regulate SSC fate determinations have been identified in mice. However, the molecules and signaling pathways underlying human SSCs remain largely unclear. In this study, we have demonstrated that MAP4K4 was predominantly expressed in human UCHL1-positive spermatogonia by double immunocytochemical staining. MAP4K4 knockdown inhibited proliferation of human SSCs and induced their apoptosis. Moreover, MAP4K4 silencing led to inhibition of JNK phosphorylation and MAP4K4 phosphorylation at Ser801. RNA sequencing indicated that MAP4K4 affected the transcription of SPARC, ADAM19, GPX7, GNG2, and COLA1. Interestingly, the phenotype of inhibiting JNK phosphorylation by SP600125 was similar to MAP4K4 knockdown. Notably, MAP4K4 protein was lower in the testes of patients with non-obstructive azoospermia than those with normal spermatogenesis as shown by Western blots and immunohistochemistry. Considered together, our data implicate that MAP4K4/JNK signaling pathway mediates proliferation and apoptosis of human SSCs, which provides a novel insight into molecular mechanisms governing human spermatogenesis and might offer new targets for gene therapy of male infertility.

A functionalized self-assembling peptide containing E7 and YIGSR sequences enhances neuronal differentiation of spermatogonial stem cells on aligned PCL fibers for spinal cord injury repair.

Background: Spinal cord injury (SCI) induces neuronal death and disrupts the nerve fiber bundles, which leads to partial or complete sensorimotor function loss of the limbs. Transplantation of exogenous neurons derived from stem cells to the lesion site becomes a new neurorestorative strategy for SCI treatment. Spermatogonial stem cells (SSCs) can attain pluripotency features by converting to embryonic stem-like cells in vitro. However, differentiating SSCs into lineage-specific neurons is quite difficult and low efficiency. Methods: Immunofluorescence, immunohistochemistry, Western blotting, whole-cell patch clamp, and behavioral tests were performed to verify that self-assembled hydrogels could improve the directional differentiation efficiency of SSCs and the feasibility of SSC-derived neurons in the treatment of spinal cord injury. Results: We developed a novel self-assembled peptide Nap-FFGEPLQLKMCDPGYIGSR (Nap-E7-YIGSR) coated with aligned electrospun PCL fibers to enhance neuronal differentiation of SSCs. The Nap-E7-YIGSR peptide could evenly self-assemble on the surface of PCL fibers, enhanced the materials's hydrophilicity, and improved the SSC affinity of PCL fibers through the stem cell adhesion peptide sequence EPLQLKM domain. In addition, Nap-E7-YIGSR could effectively induce SSC neuron differentiation by activating the integrin ß1/GSK3ß/ß-catenin signaling pathway. Moreover, implanting the induced neurons derived from SSCs into SCI lesion sites in rats resulted in the formation of new relay circuits, myelination, and synapse formation. Furthermore, SSC-derived neurons could survive and function in the spinal cord injury microenvironment, boosting the recovery of locomotion. Conclusion: The combination of the multifunctional peptide and aligned fibers can potentially trigger SSC differentiation to neurons, facilitating neuronal replacement therapy and promoting functional recovery after SCI.

A homozygous PIWIL2 frameshift variant affects the formation and maintenance of human-induced pluripotent stem cell-derived spermatogonial stem cells and causes Sertoli cell-only syndrome.

BACKGROUND: The most serious condition of male infertility is complete Sertoli cell-only syndrome (SCOS), which refers to the lack of all spermatogenic cells in the testes. The genetic cause of SCOS remains to be explored. We aimed to investigate the genetic cause of SCOS and assess the effects of the identified causative variant on human male germ cells. METHODS: Whole-exome sequencing was performed to identify potentially pathogenic variants in a man with complete SCOS, and Sanger sequencing was performed to verify the causative variant in this man and his father and brother. The pathogenic mechanisms of the causative variant were investigated by in vitro differentiation of human-induced pluripotent stem cells (hiPSCs) into germ cell-like cells. RESULTS: The homozygous loss-of-function (LoF) variant p.His244ArgfsTer31 (c.731_732delAT) in PIWIL2 was identified as the causative variant in the man with complete SCOS, and the same variant in heterozygosis was confirmed in his father and brother. This variant resulted in a truncated PIWIL2 protein lacking all functional domains, and no PIWIL2 expression was detected in the patient's testes. The patient and PIWIL2-/- hiPSCs could be differentiated into primordial germ cell-like cells and spermatogonial stem cell-like cells (SSCLCs) in vitro, but the formation and maintenance of SSCLCs were severely impaired. RNA-seq analyses suggested the inactivation of the Wnt signaling pathway in the process of SSCLC induction in the PIWIL2-/- group, which was validated in the patient group by RT-qPCR. The Wnt signaling pathway inhibitor hindered the formation and maintenance of SSCLCs during the differentiation of normal hiPSCs. CONCLUSIONS: Our study revealed the pivotal role of PIWIL2 in the formation and maintenance of human spermatogonial stem cells. We provided clinical and functional evidence that the LoF variant in PIWIL2 is a genetic cause of SCOS, which supported the potential role of PIWIL2 in genetic diagnosis. Furthermore, our results highlighted the applicability of in vitro differentiation models to function validation experiments.

CHD4 acts as a critical regulator in the survival of spermatogonial stem cells in mice†.

Spermatogenesis is sustained by homeostatic balance between the self-renewal and differentiation of spermatogonial stem cells, which is dependent on the strict regulation of transcription factor and chromatin modulator gene expression. Chromodomain helicase DNA-binding protein 4 is highly expressed in spermatogonial stem cells but roles in mouse spermatogenesis are not fully understood. Here, we report that the germ-cell-specific deletion of chromodomain helicase DNA-binding protein 4 resulted in complete infertility in male mice, with rapid loss of spermatogonial stem cells and excessive germ cell apoptosis. Chromodomain helicase DNA-binding protein 4-knockdown in cultured spermatogonial stem cells also promoted the expression of apoptosis-related genes and thereby activated the tumor necrosis factor signaling pathway. Mechanistically, chromodomain helicase DNA-binding protein 4 occupies the genomic regulatory region of key apoptosis-related genes, including Jun and Nfkb1. Together, our findings reveal the determinant role of chromodomain helicase DNA-binding protein 4 in spermatogonial stem cells survival in vivo, which will offer insight into the pathogenesis of male sterility and potential novel therapeutic targets.

Spliced X-box binding protein 1 (XBP1s) protects spermatogonial stem cells (SSCs) from lipopolysaccharide (LPS)-induced damage by regulating the testicular microenvironment.

XBP1 is a transcription factor that plays a central role in controlling cellular responses to endoplasmic reticulum stress (ERS). Under stress conditions, the transcriptionally active form of XBP1 is generated by splicing of XBP1 mRNA by the ER-resident protein inositol-requiring enzyme-1α (IRE1α). This study aimed to investigate the role of XBP1 in male reproductive disorders. XBP1s-overexpressing goat spermatogonial stem cells (gSSCs) showed higher proliferative ability in vitro and in vivo. These cells also showed higher antioxidant capacity. In comparison, XBP1 knockdown significantly suppressed proliferation. Further analysis showed that XBP1 could stimulate the secretion of IL-6 from macrophages. Overall, the results indicate that XBP1s functions to enhance the proliferation ability and antioxidant capacity of gSSCs, potentially through a mechanism involving the regulation of gSSCs by macrophages.

Regulation of male germline transmission patterns by the Trp53-Cdkn1a pathway.

A small number of offspring are born from the numerous sperm generated from spermatogonial stem cells (SSCs). However, little is known regarding the rules and molecular mechanisms that govern germline transmission patterns. Here we report that the Trp53 tumor suppressor gene limits germline genetic diversity via Cdkn1a. Trp53-deficient SSCs outcompeted wild-type (WT) SSCs and produced significantly more progeny after co-transplantation into infertile mice. Lentivirus-mediated transgenerational lineage analysis showed that offspring bearing the same virus integration were repeatedly born in a non-random pattern from WT SSCs. However, SSCs lacking Trp53 or Cdkn1a sired transgenic offspring in random patterns with increased genetic diversity. Apoptosis of KIT+ differentiating germ cells was reduced in Trp53- or Cdkn1a-deficient mice. Reduced CDKN1A expression in Trp53-deficient spermatogonia suggested that Cdkn1a limits genetic diversity by supporting apoptosis of syncytial spermatogonial clones. Therefore, the TRP53-CDKN1A pathway regulates tumorigenesis and the germline transmission pattern.

miR-106b enhances human mesenchymal stem cell differentiation to spermatogonial stem cells under germ cell profile genes involved in TGF-b signaling pathways.

Mesenchymal stem cells can be differentiated into tissue-specific cells. MicroRNAs (miRNAs) regulate the translation of mRNAs involved in the growth and development of a variety of cells, including primordial germ cells (PGCs). This study evaluated male germ cell differentiation from human MSCs by miR-106b. The MSCs were obtained from human adipose tissue. The differentiation of MSCs into PGCs was accomplished by transfection of a lentiviral vector expressing miR-106b. MSCs were treated with bone morphogenic factor 4 as a control and also as a putative inducer of PGC differentiation. PGC was differentiated into spermatogonial-like cells by retinoic acid. Moreover, Dazl, Plzf, Stra8, Gfra, and Thy1 gene expressions were investigated using real-time PCR. Our results showed that Dazl, Plzf, and Stra8 genes that were treated with BMP4 and miR-106b did not show any significant difference, meaning that miR-106b, like BMP4, is able to differentiate PGC cells from MSCs. In spermatogonial-like cells, Thy1 was significantly unregulated in both the miR-106b and BMP4 groups. Our findings showed that miR-106b regulates the differentiation of MSCs into PGCs. miR-106b influences on the expression of Dazl, Plzf, and Stra8 genes in PGC and Gfra, Stra8, and Thy1 genes.

DNA hypomethylation activation Wnt/TCF7L2/TDRD1 pathway promotes spermatogonial stem cell formation.

Detailed analysis of the regulatory mechanism of spermatogonia stem cell (SSCs) genesis can provide a novel strategy for the application of SSCs in the fields of transgenic animal production and regenerative medicine. Previous studies in this study showed that WNT signaling can positively regulate the formation of SSCs, but the exact regulatory mechanism is not clear. Here, we predicted the target gene of the Wnt/TCF7L2 pathway, namely TDRD1, by bioinformatics analysis. Functional studies revealed that overexpression of TDRD1 during RA-induced SSCs formation in vitro significantly upregulated the expression of reproductive marker genes (Integrinß1 and Integrinα6), and further flow cytometric analysis also confirmed that the formation efficiency of SSCs was significantly increased after overexpression of TDRD1; while interference with TDRD1 showed the exact opposite result. The in vivo experiments were consistent with the results of the in vitro experiments. Interestingly, although Wnt/TCF7L2 can promote the formation of SSCs, its function must be dependent on the expression of TDRD1, which was also repeatedly demonstrated as a target gene of the Wnt/TCF7L2 signaling pathway. Mechanistically, we found a large number of CpG sites in the TDRD1 promoter, and BSP analysis also confirmed that DNA methylation modifications in the TDRD1 promoter were significantly higher in embryonic stem cells than in SSCs, and further dual-luciferase reporter system assays revealed that low DNA methylation modification levels could enhance TDRD1 promoter activity; although previous studies demonstrated that TCF7L2 could enrich in the TDRD1 promoter region, the binding of the two was dependent on low DNA methylation modification. Taken together, we confirmed that low DNA methylation mediates Wnt/TCF7L2 regulation of TDRD1 to promote the formation of SSCs, providing a basis for SSCs in improving animal productivity.

Melatonin in cryopreservation media improves transplantation efficiency of frozen-thawed spermatogonial stem cells into testes of azoospermic mice.

BACKGROUND: Cryostorage of spermatogonial stem cells (SSCs) is an appropriate procedure for long-term storage of SSCs for fertility preservation. However, it causes damage to cellular structures through overproduction of ROS and oxidative stress. In this study, we examined the protective effect of melatonin as a potent antioxidant in the basic freezing medium to establish an optimal cryopreservation method for SSCs. METHODS: SSCs were obtained from the testes of neonatal male mice aged 3-6 days. Then, 100 µM melatonin was added to the basic freezing medium containing DMSO for cryopreservation of SSCs. Viability, apoptosis-related markers (BAX and BCL2), and intracellular ROS generation level were measured in frozen-thawed SSCs before transplantation using the MTT assay, immunocytochemistry, and flow cytometry, respectively. In addition, Western blotting and immunofluorescence were used to evaluate the expression of proliferation (PLZF and GFRα1) and differentiation (Stra8 and SCP3) proteins in frozen-thawed SSCs after transplantation into recipient testes. RESULTS: The data showed that adding melatonin to the cryopreservation medium markedly increased the viability and reduced intracellular ROS generation and apoptosis (by decreasing BAX and increasing BCL2) in the frozen-thawed SSCs (p < 0.05). The expression levels of proliferation (PLZF and GFRα1) and differentiation (Stra8 and SCP3) proteins and resumption of spermatogenesis from frozen-thawed SSCs followed the same pattern after transplantation. CONCLUSIONS: The results of this study revealed that adding melatonin as an antioxidant to the cryopreservation medium containing DMSO could be a promising strategy for cryopreservation of SSCs to maintain fertility in prepubertal male children who suffer from cancer.

Mesenchymal stem cells promote spermatogonial stem/progenitor cell pool and spermatogenesis in neonatal mice in vitro.

Prepubertal cancer treatment leads to irreversible infertility in half of the male patients. Current in vitro spermatogenesis protocols and cryopreservation techniques are inadequate to expand spermatogonial stem/progenitor cells (SSPC) from testicles. Bone marrow derived mesenchymal stem cells (BM-MSC) bearing a close resemblance to Sertoli cells, improved spermatogenesis in animal models. We asked if a co-culture setup supported by syngeneic BM-MSC that contributes to the air-liquid interphase (ALI) could lead to survival, expansion and differentiation of SSPCs in vitro. We generated an ALI platform able to provide a real-time cellular paracrine contribution consisting of syngeneic BM-MSCs to neonatal C57BL/6 mice testes. We aimed to evaluate the efficacy of this culture system on SSPC pool expansion and spermatogenesis throughout a complete spermatogenic cycle by measuring the number of total germ cells (GC), the undifferentiated and differentiating spermatogonia, the spermatocytes and the spermatids. Furthermore, we evaluated the testicular cell cycle phases, the tubular and luminal areas using histochemical, immunohistochemical and flow cytometric techniques. Cultures in present of BM-MSCs displayed survival of ID4(+) spermatogonial stem cells (SSC), expansion of SALL4(+) and OCT4(+) SSPCs, VASA(+) total GCs and Ki67(+) proliferative cells at 42 days and an increased number of SCP3(+) spermatocytes and Acrosin(+) spermatids at 28 days. BM-MSCs increased the percentage of mitotic cells within the G2-M phase of the total testicular cell cycle increased for 7 days, preserved the cell viability for 42 days and induced testicular maturation by enlargement of the tubular and luminal area for 42 days in comparison to the control. The percentage of PLZF(+) SSPCs increased within the first 28 days of culture, after which the pool started to get smaller while the number of spermatocytes and spermatids increased simultaneously. Our findings established the efficacy of syngeneic BM-MSCs on the survival and expansion of the SSPC pool and differentiation of spermatogonia to round spermatids during in vitro culture of prepubertal mice testes for 42 days. This method may be helpful in providing alternative cures for male fertility by supporting in vitro differentiated spermatids that can be used for round spermatid injection (ROSI) to female oocyte in animal models. These findings can be further exploited for personalized cellular therapy strategies to cure male infertility of prepubertal cancer survivors in clinics.