Hyaluronic acid/silicon nanoparticle scaffold induces proliferation and differentiation of mouse spermatogonial stem cells transplanted to epididymal adipose tissue.

Spermatogonia stem cells (SSCs) are a unique cell population maintaining male spermatogenesis during life, through their potential for proliferation and differentiation. The application of silicon nanoparticles (SNs) and hyaluronic acid (HA) to induce the differentiation of SSCs seems promising. Herein, we investigate the effect of SN and HA scaffolds on the progression of SSCs spermatogenesis in mice. Initially SSCs were isolated from healthy immature mice and cultured on prepared scaffolds (HA, SN, and HA/SN) in a 3D culture system. Then viability of SSCs cultured on scaffolds was examined using MTT assay and Acridine Orange staining. Then SSCs cultured on scaffolds were transplanted into epididymal adipose tissue (EAT) in mature mice and the result was studied by H&E and IHC staining 8 weeks after transplantation. MTT and Acridine Orange analysis revealed that among three different scaffolds HA/SN based scaffold causes considerable toxicity on SSCs (P < 0.05) while H&E staining showed that culture of SSCs on HA, SN, and HA/SN scaffolds has a positive effect on the progression of SSCs spermatogenesis after transplantation into EAT. IHC staining identified TP1, TEKT1, and PLZF as crucial biomarkers in the spermatogenesis development of SSCs transplanted to EAT. According to the presence of these biomarkers in different experimental groups, we found the most spermatogenesis development in SSCs cultured on HA/SN scaffold (PLZF, P < 0.01) (TEKT1, P < 0.01) (TP1, P < 0.001). Our study showed that, although the cytotoxic effect of the HA/SN scaffold decreases the viability rate of SSCs; however, SSCs that survive on HA/SN scaffold showed more ability to progress in spermatogenesis after transplantation into EAT.

Chimaeras, complementation, and controlling the male germline.

Animal breeding drives genetic progress mainly through the male germline. This process is slow to respond to rapidly mounting environmental pressures that threaten sustainable food security from animal protein production. New approaches promise to accelerate breeding by producing chimaeras, which comprise sterile host and fertile donor genotypes, to exclusively transmit elite male germlines. Following gene editing to generate sterile host cells, the missing germline can be restored by transplanting either: (i) spermatogonial stem cells (SSCs) into the testis; or (ii) embryonic stem cells (ESCs) into early embryos. Here we compare these alternative germline complementation strategies and their impact on agribiotechnology and species conservation. We propose a novel breeding platform that integrates embryo-based complementation with genomic selection, multiplication, and gene modification.

Sperm DNA methylation is predominantly stable in mice offspring born after transplantation of long-term cultured spermatogonial stem cells.

BACKGROUND: Spermatogonial stem cell transplantation (SSCT) is proposed as a fertility therapy for childhood cancer survivors. SSCT starts with cryopreserving a testicular biopsy prior to gonadotoxic treatments such as cancer treatments. When the childhood cancer survivor reaches adulthood and desires biological children, the biopsy is thawed and SSCs are propagated in vitro and subsequently auto-transplanted back into their testis. However, culturing stress during long-term propagation can result in epigenetic changes in the SSCs, such as DNA methylation alterations, and might be inherited by future generations born after SSCT. Therefore, SSCT requires a detailed preclinical epigenetic assessment of the derived offspring before this novel cell therapy is clinically implemented. With this aim, the DNA methylation status of sperm from SSCT-derived offspring, with in vitro propagated SSCs, was investigated in a multi-generational mouse model using reduced-representation bisulfite sequencing. RESULTS: Although there were some methylation differences, they represent less than 0.5% of the total CpGs and methylated regions, in all generations. Unsupervised clustering of all samples showed no distinct grouping based on their pattern of methylation differences. After selecting the few single genes that are significantly altered in multiple generations of SSCT offspring compared to control, we validated the results with quantitative Bisulfite Sanger sequencing and RT-qPCRin various organs. Differential methylation was confirmed only for Tal2, being hypomethylated in sperm of SSCT offspring and presenting higher gene expression in ovaries of SSCT F1 offspring compared to control F1. CONCLUSIONS: We found no major differences in DNA methylation between SSCT-derived offspring and control, both in F1 and F2 sperm. The reassuring outcomes from our study are a prerequisite for promising translation of SSCT to the human situation.

Biopolymer gels as components of protective medium for cryopreservation of spermatogonial stem cells.

Biopolymer gels attract a lot of attention in a field of biothechnology due to their excellent compatibility and degradation. Their application is also promising for cryopreservation of spermatogonial stem cells (SSCs) which is so necessary to preserve the fertility of young patients. The aim of the study was to determine the effectiveness of biopolymer gels as a component of cryopreservation medium for SSCs of immature rats at the stage of exposure to cryoprotectants. It was found that 30-min exposure to cryopreservation media based on collagen or fibrin gel with an addition of 5% Me2SO or 6% glycerol did not lead to significant changes in membrane integrity, cytochrome C content, metabolic, mitochondrial and antioxidant activities in SSCs compared to the control (Leibovitz-based cryomedium). But fibrin gel more than collagen reduced the toxic effects of Me2SO and glycerol on SSCs increasing exposure time up to 45 min without significant changes in cell viability. The same cryoprotectants in Leibovitz-based media showed significant toxicity starting from the 15th minute of exposure. Necrosis was the main cause of cell death at this stage of cryopreservation in all experimental groups. The obtained results can be used to optimize SSC cryopreservation protocols for further cell autotransplantation for spermatogenesis initiation in boys who undergo gonadotoxic therapy in prepubertal age.

Ex-Vivo and In-Vivo Expansion of Spermatogonial Stem Cells Using Cell-Seeded Microfluidic Testis Scaffolds and Animal Model.

AIMS: This study was designed to provide both ex-vivo and in-vivo methods for the extraction and expansion of spermatogonial stem cells (SSCs). METHODS: For in-vivo experiments, azoospermic mouse model was performed with Busulfan. Isolation, culture, and characterization of neonate mouse SSC were also achieved. We performed an in-vivo injection of labeled SSCs to the testes with azoospermia. In ex-vivo experiments, extracted SSCs were seeded on the fabricated scaffold consisting of hyaluronic acid (HA) and decellularized testis tissues (DTT). Immunofluorescence staining with PLZF, TP1, and Tekt 1 was performed for SSCs differentiation and proliferation. RESULTS: Several studies demonstrated efficient spermatogenic arrest in seminiferous tubules and proved the absence of spermatogenesis. Transplanted SSCs moved and settled in the basement covering the seminiferous tubules. Most of the cells were positive for Dil, after 4 weeks. An epithelium containing spermatogonia-like cells with Sertoli-like, and Leydig cells were evident in the seminiferous tubules of biopsies, and the IHC staining was significantly positive, 4 weeks after injection of SSCs. The results of the ex-vivo experiments showed positive staining for all markers, which was significantly enhanced in scaffolds of ex-vivo experiments compared with in-vitro seeded scaffolds. CONCLUSION: Ex-vivo SSC differentiation and proliferation using cell-seeded microfluidic testis scaffolds maybe effective for treatment of the azoospermia.

[Reconstruction of spermatogonial niche for male fertility preservation].

Infertility is one of the late side effects of cancer treatment. Expansion of anti-cancer treatment allow patients to have more life time, however infertility is becoming a matter damaging QOL during the young cancer survivors. The passive strategy such as avoiding the gonad-toxic drug or decreasing the total volume of them and shielding the gonads against cancer therapy has been conducted. To preserve the fertility of young female, ovary tissue cryopreservation is becoming a standard over the world after the success of offspring from cryopreserved ovary tissue autograft was reported. Sperm preservation method is established for the male fertility preservation method, however this is only applicable for sexually matured male patients. For the sake of preserving fertility of sexually immature male patients, many trials using cryopreserved testis tissues or testicular cells have been undergone. Recently, in vitro gametogenesis from stem cell of the human and the mouse to primordial germ cell like cell has been achieved. Here the previous challenges and the latest reports for obtaining functional sperm from immature testis and the reconstruction of spermatogonial niche as a potential approach for preserving fertility procedure are described.

Regeneration of spermatogenesis by mouse germ cell transplantation into allogeneic and xenogeneic testis primordia or organoids.

Gametogenesis requires close interactions between germ cells and somatic cells. Derivation of sperm from spermatogonial stem cells (SSCs) is hampered by the inefficiency of spermatogonial transplantation technique in many animal species because it requires a large number of SSCs and depletion of endogenous spermatogenesis. Here we used mouse testis primordia and organoids to induce spermatogenesis from SSCs. We microinjected mouse SSCs into embryonic gonads or reaggregated neonatal testis organoids, which were transplanted under the tunica albuginea of mature testes. As few as 1 × 104 donor cells colonized both types of transplants and produced sperm. Moreover, rat embryonic gonads supported xenogeneic spermatogenesis from mouse SSCs when transplanted in testes of immunodeficient mice. Offspring with normal genomic imprinting patterns were born after microinsemination. These results demonstrate remarkable flexibility of the germ cell-somatic cell interaction and raise new strategies of SSC manipulation for animal transgenesis and analysis of male infertility.

Hypobaric hypoxia exposure alters transcriptome in mouse testis and impairs spermatogenesis in offspring.

Male fertility relies on continual and robust spermatogenesis. Environmental hypoxia adversely affects reproductive health in humans and animal studies provide compelling evidences that hypoxia impairs spermatogenesis in directly exposed individuals. However, a detail examination of hypoxia induced changes in testicular gene expression is still lacking and spermatogenesis in offspring of hypoxia exposed animals of awaits investigation. In this study, a hypobaric hypoxic chamber was used to simulate hypoxic conditions in mice and effects of hypoxia on spermatogenesis, fertility and testicular gene expression were evaluated. The results showed that hypoxia exposure reduced the number of undifferentiated spermatogonia but did not change the regenerative capacity of spermatogonial stem cells (SSCs) after transplantation. Hypoxia significantly increased the percent of abnormal sperm and these defects were recovered 2 months after returning to the normoxia. Transcriptome analysis of testicular tissues from control and hypoxia treated animals revealed that 766 genes were up-regulated and 965 genes were down-regulated. Surprisingly, expressions of genes that regulate epigenetic modifications were altered, indicating hypoxia-induced damage to spermatogenesis may be intergenerational. Indeed, animals that were sired by hypoxia exposed males exhibited impaired spermatogenesis. Together, these findings suggest that hypoxia exposure alters testicular gene expression and causes long-lasting damage to spermatogenesis.

Surrogate production of genome-edited sperm from a different subfamily by spermatogonial stem cell transplantation.

The surrogate reproduction technique, such as inter-specific spermatogonial stem cells (SSCs) transplantation (SSCT), provides a powerful tool for production of gametes derived from endangered species or those with desirable traits. However, generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily, by SSCT, has not succeeded. Here, using two small cyprinid fishes from different subfamilies, Chinese rare minnow (gobiocypris rarus, for brief: Gr) and zebrafish (danio rerio), we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure. The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis, resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells. Interestingly, the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior. When pou5f3 and chd knockout Gr SSCs were transplanted, Gr-derived genome-edited sperm was successfully produced in zebrafish. This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT, and surrogate production of genome-edited gametes from another species as well. This method is feasible to be applied to future breeding of commercial fish and livestock.

Cdc42 is required for male germline niche development in mice.

Spermatogonial stem cells (SSCs) are maintained in a special microenvironment called a niche. However, much is unknown about components that constitute the niche. Here, we report that Cdc42 is essential for germline niche development. Sertoli cell-specific Cdc42-deficient mice showed normal premeiotic spermatogenesis. However, germ cells gradually disappeared during haploid cell formation and few germ cells remained in the mature testes. Spermatogonial transplantation experiments revealed a significant loss of SSCs in Cdc42-deficient testes. Moreover, Cdc42 deficiency in Sertoli cells downregulated GDNF, a critical factor for SSC maintenance. Cdc42-deficient Sertoli cells also exhibited lower nuclear MAPK1/3 staining. Inhibition of MAP2K1 or depletion of Pea15a scaffold protein downregulated GDNF expression. A screen of transcription factors revealed that Cdc42-deficient Sertoli cells downregulate DMRT1 and SOX9, both of which are critical for Sertoli cell development. These results indicate that Cdc42 is essential for niche function via MAPK1/3-dependent GDNF secretion.

Spermatogonial stem cell transplantation into nonablated mouse recipient testes.

Spermatogonial transplantation has been used as a standard assay for spermatogonial stem cells (SSCs). After transplantation into the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) between Sertoli cells and settle in a niche. Unlike in the repair of other self-renewing systems, SSC transplantation is generally performed after complete destruction of endogenous spermatogenesis. Here, we examined the impacts of recipient conditioning on SSC homing. Germ cell ablation downregulated the expression of glial cell line-derived neurotrophic factor, which has been shown to attract SSCs to niches, implying that nonablated niches would attract SSCs more efficiently. As expected, SSCs colonized nonablated testes when transplanted into recipients with the same genetic background. Moreover, although spermatogenesis was arrested at the spermatocyte stage in Cldn11-deficient mice without a BTB, transplantation not only enhanced donor colonization but also restored normal spermatogenesis. The results show promise for the development of a new transplantation strategy to overcome male infertility.

Male reproductive systems of Macaca mulatta: Gonadal development, spermatogenesis and applications in spermatogonia stem cell transplantation.

Rhesus macaque (Macaca mulatta) is widely applied in animal model construction of infertility, spermatogonia stem cell transplantation and male reproductive diseases. In this review, we describe the seasonal changes of the reproductive system in rhesus macaques, the regular pattern of spermatogenesis and spermatozoa maturation, and the differentiation of spermatogonia and spermatocytes. The duration of the M. mulatta spermatogenesis is approximately 10 days and seminiferous epithelium cycles mainly consist of 12 stages, which provide a suitable model for reproductive studies in non-human primates. Here, we summarize the features of gonadal development and sperm maturation in the rhesus monkeys, which provide important information in the studies of reproductive biology. Rhesus macaque is an excellent animal model in spermatogonia stem cell transplantation. We discuss the applications and progresses of assisted reproductive technologies in sperm liquefaction, semen cryopreservation and spermatogonia stem cell transplantation of rhesus macaques. Besides, we sort out recent proteomic analyses of male reproductive systems and semen samples in rhesus macaques. This review mainly focuses on male reproductive biology and application studies using M. mulatta, which would promote the development of new therapeutic interventions on assisted reproduction and reproductive disease studies in the future.

Production of juvenile masu salmon (Oncorhynchus masou) from spermatogonia-derived sperm and oogonia-derived eggs via intraperitoneal transplantation of immature germ cells.

No effective cryopreservation technique exists for fish eggs and embryos; thus, the cryopreservation of germ cells (spermatogonia or oogonia) and subsequent generation of eggs and sperm would be an alternative solution for the long-term preservation of piscine genetic resources. Nevertheless, in our previous study using rainbow trout, we showed that recipients transplanted with XY spermatogonia or XX oogonia produced unnatural sex-biased F1 offspring. To overcome these obstacles, we transplanted immature germ cells (XX oogonia or XY spermatogonia; frozen for 33 days) into the body cavities of triploid hatchlings, and the transplanted germ cells possessed a high capacity for differentiating into eggs and sperm in the ovaries and testes of recipients. Approximately 30% of triploid recipients receiving frozen germ cells generated normal salmon that displayed the donor-derived black body color phenotype, although all triploid salmon not receiving transplants were functionally sterile. Furthermore, F1 offspring obtained from insemination of the oogonia-derived eggs and spermatogonia-derived sperm show a normal sex ratio of 1:1 (female:male). Thus, this method presented a critical technique for practical conservation projects for other teleost fish species and masu salmon.

Preliminary study on testicular germ cell isolation and transplantation in an endangered endemic species Brycon orbignyanus (Characiformes: Characidae).

We aimed to develop a simplified protocol for transplantation of Brycon orbignyanus spermatogonial stem cells (SSCs) into Astyanax altiparanae testes. Brycon orbignyanus testes were enzymatically digested and SSC purified by a discontinuous density gradient. Endogenous spermatogenesis was suppressed in A. altiparanae using busulfan or by incubation at 35 °C water, and SSCs from B. orbignyanus labeled with PKH26 were injected into their testes via the urogenital papilla. Twenty-two hours post-transplantation, labeled spermatogonia were observed in A. altiparanae tubular lumen. After 7 days, spermatogonia proliferated in the epithelium, and 21 days post-transplantation, sperm was observed in the lumen. Of surviving host fish, nearly 67% of those treated with busulfan and 85% of those held in warm water showed labeled cells in host germinal epithelium. The present study standardized, by a simple and accessible method, germ cell transplantation between sexually mature Characiformes fish species. This is the first report of xenogenic SSC transplantation in this fish order.

Restoration of functional sperm production in irradiated pubertal rhesus monkeys by spermatogonial stem cell transplantation.

BACKGROUND: In male pre-pubertal cancer patients, radiation and chemotherapy impact future fertility by eradication of spermatogonial stem cells (SSCs). In macaques, spermatogenesis could be regenerated by intratesticular transplantation of SSCs, but only a small percentage of spermatozoa produced were of donor origin. Transient hormone suppression with a GnRH antagonist (GnRH-ant) enhanced spermatogenic recovery from transplanted SSCs. OBJECTIVES: To evaluate donor-derived and endogenous spermatogenic recovery after SSC transplantation into irradiated monkeys and to test whether hormone suppression around the time of transplantation facilitates spermatogenic recovery. MATERIALS AND METHODS: Testes of 15 adult rhesus monkeys were irradiated with 7 Gy and 4 months later transplanted, to one of the testes, with cryopreserved testicular cells containing SSCs from unrelated monkeys. Monkeys were either treated with GnRH-ant for 8 weeks before transplantation, GnRH-ant from 4 weeks before to 4 weeks after transplantation, or with no GnRH-ant. Tissues were harvested 10 months after transplantation. RESULTS: Two of the 15 monkeys, a control and a pre-transplantation GnRH-ant-treated, showed substantially higher levels of testicular spermatogenesis and epididymal sperm output in the transplanted side as compared to the untransplanted. Over 84% of epididymal spermatozoa on the transplanted side had the donor genotype and were capable of fertilizing eggs after intracytoplasmic sperm injection forming morulae of the donor paternal origin. Low levels of donor spermatozoa (~1%) were also identified in the epididymis of three additional monkeys. Transplantation also appeared to enhance endogenous spermatogenesis. DISCUSSION AND CONCLUSION: We confirmed that SSC transplantation can be used for restoration of fertility in male cancer survivors exposed to irradiation as a therapeutic agent. The success rate of this procedure, however, is low. The success of filling the tubules with the cell suspension, but not the GnRH-ant treatment, was related to the level of colonization by transplanted cells.

Autologous transplantation of spermatogonial stem cells restores fertility in congenitally infertile mice.

The blood-testis barrier (BTB) is thought to be indispensable for spermatogenesis because it creates a special environment for meiosis and protects haploid cells from the immune system. The BTB divides the seminiferous tubules into the adluminal and basal compartments. Spermatogonial stem cells (SSCs) have a unique ability to transmigrate from the adluminal compartment to the basal compartment through the BTB upon transplantation into the seminiferous tubule. Here, we analyzed the role of Cldn11, a major component of the BTB, in spermatogenesis using spermatogonial transplantation. Cldn11-deficient mice are infertile due to the cessation of spermatogenesis at the spermatocyte stage. Cldn11-deficient SSCs failed to colonize wild-type testes efficiently, and Cldn11-deficient SSCs that underwent double depletion of Cldn3 and Cldn5 showed minimal colonization, suggesting that claudins on SSCs are necessary for transmigration. However, Cldn11-deficient Sertoli cells increased SSC homing efficiency by >3-fold, suggesting that CLDN11 in Sertoli cells inhibits transmigration of SSCs through the BTB. In contrast to endogenous SSCs in intact Cldn11-deficient testes, those from WT or Cldn11-deficient testes regenerated sperm in Cldn11-deficient testes. The success of this autologous transplantation appears to depend on removal of endogenous germ cells for recipient preparation, which reprogrammed claudin expression patterns in Sertoli cells. Consistent with this idea, in vivo depletion of Cldn3/5 regenerated endogenous spermatogenesis in Cldn11-deficient mice. Thus, coordinated claudin expression in both SSCs and Sertoli cells expression is necessary for SSC homing and regeneration of spermatogenesis, and autologous stem cell transplantation can rescue congenital defects of a self-renewing tissue.

Review of injection techniques for spermatogonial stem cell transplantation.

BACKGROUND: Although the prognosis of childhood cancer survivors has increased dramatically during recent years, chemotherapy and radiation treatments for cancer and other conditions may lead to permanent infertility in prepubertal boys. Recent developments have shown that spermatogonial stem cell (SSC) transplantation may be a hope for restoring fertility in adult survivors of childhood cancers. For this reason, several centres around the world are collecting and cryopreserving testicular tissue or cells anticipating that, in the near future, some patients will return for SSC transplantation. This review summarizes the current knowledge and utility of SSC transplantation techniques. OBJECTIVE AND RATIONALE: The aim of this narrative review is to provide an overview of the currently used experimental injection techniques for SSC transplantation in animal and human testes. This is crucial in understanding and determining the role of the different techniques necessary for successful transplantation. SEARCH METHODS: A comprehensive review of peer-reviewed publications on this topic was performed using the PubMed and Google Scholar databases. The search was limited to English language work and studies between 1994 (from the first study on SSC transplantation) and April 2019. Key search terms included mouse, rat, boar, ram, dog, sheep, goat, cattle, monkey, human, cadaver, testes, SSC transplantation, injection and technique. OUTCOMES: This review provides an extensive clinical overview of the current research in the field of human SSC transplantation. Rete testis injection with ultrasonography guidance currently seems the most promising injection technique thus far; however, the ability to draw clear conclusions is limited due to long ischemia time of cadaver testis, the relatively decreased volume of the testis, the diminishing size of seminiferous tubules, a lack of intratesticular pressure and leakage into the interstitium during the injection on human cadaver testis. Current evidence does not support improved outcomes from multiple infusions through the rete testes. Overall, further optimization is required to increase the efficiency and safety of the infusion method. WIDER IMPLICATIONS: Identifying a favourable injection method for SSC transplantation will provide insight into the mechanisms of successful assisted human reproduction. Future research could focus on reducing leakage and establishing the optimal infusion cell concentrations and pressure.

Isolation, characterization, in vitro expansion and transplantation of Caspian trout (Salmo caspius) type a spermatogonia.

Sprmatogonial stem cells (SSCs) are valuable for preservation of endangered fish species, biological experimentation, as well as biotechnological applications. However, the rarity of SSCs in the testes has been a great obstacle in their application. Thus, establishment of an efficient in-vitro culture system to support continuous proliferation of SSCs is essential. The present study aimed to establish an efficient and simple method for in vitro culture of Caspian trout undifferentiated spermatogonial cells. Using a two-step enzymatic digestion, testicular cells were isolated from immature testes composed of mainly undifferentiated spermatogonial cells with gonadosomatic indices of <0.05%. The spermatogonial cells were purified by differential plating through serial passaging. The purified cells indicated high expression of type A spermatogonia-related genes (Ly75, Gfrα1, Nanos2, Plzf and Vasa). Proliferation of purified cells was confirmed by BrdU incorporation. Co-culture of purified cells with testicular somatic cells as a feeder layer, resulted in continuous proliferation of type A spermatogonia. The cultured cells continued to express type A spermatogonia-specific markers after one month culture. The cultured spermatogonia were successfully incorporated into the germline after being intraperitoneally transplanted into sterile triploid rainbow trout hatchlings. These results, for the first time, demonstrated that the somatic microenvironment of the rainbow trout gonad can support the colonization and survival of intraperitoneally transplanted cells derived from a fish species belonging to a different genus. Therefore, the combination of in vitro culture system and xenotransplantation can be considered as a promising strategy for conservation of Caspian trout genetic resources.

Successful transplantation of transfected enriched buffalo (Bubalus bubalis) spermatogonial stem cells to homologous recipients.

Genetic modification of spermatogonial stem cells (SSCs) is an alternative method to pronuclear microinjection and somatic cell nuclear transfer for transgenesis in large animals. In the present study, we optimized the process of homologous SSC transplantation in the water buffalo (Bubalus bubalis) using transfected enriched SSCs generated by a non-viral transfection approach. Firstly, the SSC enrichment efficiencies of extracellular matrix components viz. collagen, gelatin, and Datura stramonium agglutinin (DSA) lectin were determined either individually or in combination with Percoll density gradient centrifugation. The highest enrichment was achieved after differential plating with DSA lectin followed by Percoll density gradient centrifugation. Nucleofection showed greater transfection efficiency (68.55 ±â€¯4.56%, P < 0.05) for enriched SSCs in comparison to fugene HD (6.7 ±â€¯0.25%) and lipofectamine 3000 (15.57 ±â€¯0.74%). The transfected enriched SSCs were transplanted into buffalo males under the ultrasound guidance and testis was removed by castration after 7-8 weeks of transplantation. Persistence and localization of donor cells within recipient seminiferous tubules was confirmed using fluorescent microscopy. Further confirmation was done by flow cytometric evaluation of GFP expressing cells among those isolated from two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated for the first time, generation of buffalo transfected enriched SSCs and their successful homologous transplantation in buffaloes. This study represents the first step towards genetic modifications in buffaloes using SSC transplantation technique.