Differentiation of human spermatogonial stem cells using a human decellularized testicular scaffold supplemented by platelet-rich plasma.

BACKGROUND: Effective culture systems for attachment, migration, proliferation, and differentiation of spermatogonial stem cells (SSCs) can be a promising therapeutic modality for preserving male fertility. Decellularized extracellular matrix (ECM) from native testis tissue creates a local microenvironment for testicular cell culture. Furthermore, platelet-rich plasma (PRP) contains various growth factors for the proliferation and differentiation of SSCs. METHODS: In this study, human testicular cells were isolated and cultured for 4 weeks, and SSCs were characterized using immunocytochemistry (ICC) and flow cytometry. Human testicular tissue was decellularized (0.3% SDS, 1% Triton), and the efficiency of the decellularization process was confirmed by histological staining and DNA content analysis. SSCs were cultured on the human decellularized testicular matrix (DTM) for 4 weeks. The viability and the expression of differentiation genes were evaluated by MTT and real-time polymerase chain reaction (PCR), respectively. RESULTS: Histological evaluation and DNA content analysis showed that the components of ECM were preserved during decellularization. Our results showed that after 4 weeks of culture, the expression levels of BAX, BCL-2, PLZF, and SCP3 were unchanged, while the expression of PRM2 significantly increased in the cells cultured on DTM supplemented with PRP (ECM-PRP). In addition, the expression of GFRA1 was significantly decreased in the ECM group compared to the control and PRP groups. Furthermore, the MTT test indicated that viability was significantly enhanced in cells plated on DTM supplemented with PRP. CONCLUSION: Our study demonstrated that DTM supplemented with PRP can provide an effective culture system for the differentiation and viability of SSCs.

Recovering Spermatogenesis By Protected Cryopreservation Using Metformin and Transplanting Spermatogonial Stem Cells Into Testis in an Azoospermia Mouse Model.

Cryopreservation and transplantation of spermatogonial stem cells (SSCs) may serve as a new method to restore male fertility in patients undergoing chemotherapy or radiotherapy. However, SSCs may be damaged during cryopreservation due to the production of reactive oxygen species (ROS). Therefore, different antioxidants have been used as protective agents. Studies have shown that metformin (MET) has antioxidant activity. The aim of this study was to assess the antioxidant and antiapoptotic effects of MET in frozen-thawed SSCs. In addition, the effect of MET on the proliferation and differentiation of SSCs was evaluated. To this end, SSCs were isolated from mouse pups aged 3-6 days old, cultured, identified by flow cytometry (ID4, INTEGRIN ß1+), and finally evaluated for survival and ROS rate. SSCs were transplanted after busulfan and cadmium treatment. Cryopreserved SSCs with and without MET were transplanted after 1 month of cryopreservation. Eight weeks after transplantation, the recipient testes were evaluated for the expression of apoptosis (BAX, BCL2), proliferation (PLZF), and differentiation (SCP3, TP1, TP2, PRM1) markers using immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction. The findings revealed that the survival rate of SSCs was higher in the 500 µm/mL MET group compared to the other groups (50 and 5000 µm/mL). MET significantly decreased the intracellular ROS production. Transplantation of SSCs increased the expression level of proliferation (PLZF) and differentiation (SCP3, TP1, TP2, PRM1) markers compared to azoospermia group, and their levels were significantly higher in the MET group compared to the cryopreservation group containing basic freezing medium (p < 0.05). MET increased the survival rate of SSCs, proliferation, and differentiation and decreased the ROS production and the apoptosis rate. Cryopreservation by MET seems to be effective in treating infertility.

Effect of a Freezing Medium Containing Melatonin on Markers of Pre-meiotic and Post-meiotic Spermatogonial Stem Cells (SSCs) After Transplantation in an Azoospermia Mouse Model Due to Testicular Torsion.

Spermatogonial stem cells (SSCs) are essential to the initiation of spermatogenesis. Cryopreservation, long-term maintenance, and auto-transplantation of SSCs could be a new treatment for infertility. The aim of this study was to add melatonin to the basic freezing medium and to evaluate its effect on the efficiency of the thawed SSCs after transplantation into the testicles of azoospermic mice. SSCs were isolated from newborn NMRI mice, and the cells were enriched to assess morphological features. The thawed SSCs were evaluated for survival, apoptosis, and ROS level before transplantation, and the proliferation (MVH and ID4) and differentiation (c-Kit, SCP3, TP1, TP2, and Prm1) markers of SSCs were examined using immunofluorescence, western blot, and quantitative real-time polymerase chain reaction (PCR) after transplantation. It was found that the survival rate of SSCs after thawing was significantly higher in the melatonin group compared with the cryopreservation group containing basic freezing medium, and the rate of apoptosis and level of ROS production also decreased significantly in the cryopreservation group with melatonin (p < 0.05). The expression of proliferation and differentiation markers after transplantation was significantly higher in the cryopreservation group with melatonin compared to the cryopreservation group (p < 0.05). The results suggest that adding melatonin to the basic freezing medium can effectively protect the SSCs by increasing the viability and reducing the ROS production and apoptosis and improve the transplantation efficiency of SSCs after cryopreservation, which will provide a significant suggestion for fertility protection in the clinic.