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Neda Eslahi,1,2,* Mahmoud Reza Hadjighassem,1,3 Mohammad Taghi Joghataei,1,2 Tooba Mirzapour,4 Mehrdad Bakhtiyari,1,2 Malak Shakeri,5 Vahid Pirhajati,1,2 Peymaneh Shirinbayan,6,* Morteza Koruji1,21Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran; 2Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences, Tehran, Iran; 3Department of Neurosciences, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran; 4Department of Biology, Faculty of Science, University of Mohaghegh Ardabili, Ardabil, Iran; 5Department of Animal Science, Agricultural Campus, University of Tehran, Tehran, Iran; 6Pediatric Neuro-Rehabilitation Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran*These authors contributed equally to this articleIntroduction: A 3D-nanofiber scaffold acts in a similar way to the extracellular matrix (ECM)/basement membrane that enhances the proliferation and self-renewal of stem cells. The goal of the present study was to investigate the effects of a poly L-lactic acid (PLLA) nanofiber scaffold on frozen-thawed neonate mouse spermatogonial stem cells (SSCs) and testis tissues.Methods: The isolated spermatogonial cells were divided into six culture groups: (1) fresh spermatogonial cells, (2) fresh spermatogonial cells seeded onto PLLA, (3) frozen-thawed spermatogonial cells, (4) frozen-thawed spermatogonial cells seeded onto PLLA, (5) spermatogonial cells obtained from frozen-thawed testis tissue, and (6) spermatogonial cells obtained from frozen-thawed testis tissue seeded onto PLLA. Spermatogonial cells and testis fragments were cryopreserved and cultured for 3 weeks. Cluster assay was performed during the culture. The presence of spermatogonial cells in the culture was determined by a reverse transcriptase polymerase chain reaction for spermatogonial markers (Oct4, GFRα-1, PLZF, Mvh(VASA), Itgα6, and Itgβ1), as well as the ultrastructural study of cell clusters and SSCs transplantation to a recipient azoospermic mouse. The significance of the data was analyzed using the repeated measures and analysis of variance.Results: The findings indicated that the spermatogonial cells seeded on PLLA significantly increased in vitro spermatogonial cell cluster formations in comparison with the control groups (culture of SSCs not seeded on PLLA) (P≤0.001). The viability rate for the frozen cells after thawing was 63.00% ± 3.56%. This number decreased significantly (40.00% ± 0.82%) in spermatogonial cells obtained from the frozen-thawed testis tissue. Both groups, however, showed in vitro cluster formation. Although the expression of spermatogonial markers was maintained after 3 weeks of culture, there was a significant downregulation for some spermatogonial genes in the experimental groups compared with those of the control groups. Furthermore, transplantation assay and transmission electron microscopy studies suggested the presence of SSCs among the cultured cells.Conclusion: Although PLLA can increase the in vitro cluster formation of neonate fresh and frozen-thawed spermatogonial cells, it may also cause them to differentiate during cultivation. The study therefore has implications for SSCs proliferation and germ cell differentiation in vitro.Keywords: PLLA nanofibers, tissue cryopreservation, testis

Introduction: Destruction of spermatogonial stem cells (SSCs) along the cancer cells is one of the side effects of cancer treatments that can produce infertile patients. To preserve fertility in these patients, one hypothesis was that testicular biopsies are obtained prior to the treatment, and SSCs are isolated and preserved by cryopreservation and finally transplanted back into the patient's testis in a suitable time. Since small testicular biopsies do not contain sufficient SSCs to fully repopulate the testis after transplantation, in vitro propagation of hSSCs will be necessary to obtain an adequate number of cells for successful transplantation. Materials and Methods: hSSCs were isolated from testis biopsies and permitted to amplify in number by self-renewal in vitro in the presence of LIF and bFGF in DMEM for 5 weeks. Various types of human spermatogonia in culture system was identified and compared with testis tissue using morphological criteria, at the ultrastructural level. Results: Although many differences in various types of spermatogonia were identified but approximately were not observed remarkably difference between spermatogonial cells in culture system and testis tissue. Electron and light microscopic studies of hSSCs colonies and also assessment of gene expression did not show differentiated SSCs in the culture system. Conclusion: The results also showed that two-three weeks after culture is probably suitable time for transplantation of SSCs in recipient testis because in this time apoptosis has not been started in colony cells so that the population of apoptotic cells is low in the culture system. [ABSTRACT FROM AUTHOR]

Introduction: For humans, cytotoxic drug therapy to eradicate cancer cells can damage spermatogenesis and lead to infertility. Theoretically, isolation and differentiation of MSC to germ-like cells could provide a source of endogenous germ cells for possible transplantation for patients undergoing chemotherapy in future. Materials and Methods: Pluripotent mesenchymal stem cell (MSC) lines were established from umbilical cord Wharton's jelly. The cells present the high expansion potential of MSC, a typical fibroblast-like morphology and proliferate up to 15 passages without displaying clear changes in morphology. PCR analysis was performed on MSCs at passages 2-5 to further characterize this cell population. The results showed that these cells strongly expressed markers for mesenchymal progenitors such as CD 105 and CD44 and were negative for cell surface markers associated with hematopoietic cells, CD34. MSC formed tadpolelike germ cells after induction with different dosages of retinoic acid and conditioned medium. Results: The present study showed that, mesenchymasl stem cells are fully functional after proliferation and culturing process do not damages the surface properties of these cells. The ability to isolate and culture of MSCs in vitro and to apply transplantation technique may facilitate development of therapeutic strategies for the treatment of infertility in patients who may suffer destruction of germ cells as a result of irradiation or chemotherapy for cancer. Conclusion: Our results may provide a new route for reproductive therapy and a novel in vitro model to investigate the molecular mechanisms that regulate the development of the mammalian germ lineage. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSCs) have the unique ability to self-renew and differentiate into mature spermatozoa. In vitro culture of SSCs, however, presents several challenges, particularly in promoting efficient differentiation. This study investigates the role of metabolic intermediates, such as alpha-ketoglutarate (AKG), on the differentiation of SSCs isolated from the testes of 3-6 day-old mice. SSCs and sertoli cells were extracted using collagenase ІV and trypsin and co-cultured in DMEM/F12 supplemented with 20 % fetal bovine serum (FBS) and glial cell-derived neurotrophic factor (GDNF) for one week. The survival rate of cells was evaluated under influence of different dosages of AKG (0.04, 0.1, 0.4, 4, 10 mM) after 1 and 7 days of culture using the MTT test. The cell viability was significantly increased at the 0.1 mM dose of AKG rather than other groups. This dosage was selected for adding to culture system. In the control group, the cells cultured for three weeks in DMEM/F12 with 10 % FBS, 10⁻6 M retinoic acid, and 40 ng/mL bone morphogenetic protein-4 (BMP-4). The treatment group received the same medium with the addition of 0.1 mM AKG. The presence of Sertoli cell in the culture system was confirmed by SOX9-positive immunocytochemistry. The Colonies that formed on the Sertoli cells exhibited positive alkaline phosphatase activity and reacted positively for Oct4 and GFRa1 immunocytochemistry. The expression of testicular-specific genes (Acrosin and Sycp3) and anti-apoptosis-related genes (Nrf2 and Bcl2) was evaluated after 7 days (as the zero group) and again after 21 days of culture, in treatment (0.1 mM AKG) and control groups. A high expression of Acrosin and Sycp3 expression was observed in AKG-treated group compared to control and zero groups (p ≤ 0.05). The expression of Nrf2 and Bcl2 genes was also significantly increased in the treatment group (p ≤ 0.05). These findings suggest that AKG activates mechanisms of cellular antioxidant response and subsequently increase the expression of differentiation genes in SSCs., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent cells with self-renewal properties, making them an ideal candidate for regenerative medicine. Recently, numerous studies show that about more than 99% of transplanted cells are destroyed because of the stressful microenvironment. Meanwhile, in the target organs, iron overload can produce oxidative stress introducing it as the most important stress factor. The present study was aimed at increasing BM-MSCs' viability against oxidative stress microenvironment using iron depletion by deferoxamine (DFO). Mesenchymal stem cells are isolated and characterized from rat bone marrow. Then, the sensitivity of BM-MSCs against H 2 O 2 -induced oxidative stress was evaluated through half of the inhibitory concentration (IC 50 ) estimation by using MTT assay. The maximum non-inhibitory concentration of DFO on BM-MSCs was determined. The next step was the comparison between DFO pre-treated BM-MSCs and untreated cells against H 2 O 2 -induced apoptosis. BM-MSCs were identified with morphologic and flow cytometry analysis. IC 50 of H 2 O 2 was determined as 0.55 mM at 4 h. Also, the maximum non-inhibitory concentration of DFO was ascertained as 5 μM at 48 h. Our results demonstrated that pretreatment with DFO significantly potentiates BM-MSCs against H 2 O 2 -induced oxidative stress which was confirmed by MTT assay, AO/EB double staining, DAPI staining, and activated caspase 3 quantification as well as western blot test. Expression of cleaved caspase 3 and pAKT/AKT ratio obviously demonstrated DFO can resist the cells against cytotoxicity. These findings may help to develop better stem cell culture medium for MSC-based cell therapy. Moreover, regulation of cell stress can be used in practical subjects.

Since proliferation and differentiation of spermatogonial stem cells (SSCs) in culture system provide successful transplantation in this study, culture of human SSCs was compared to SACS (soft agar culture system), gelatin and control groups. The cells were isolated from seminiferous tubules of non-azoospermia patients (NOA) and cultured in DMEM for 3 weeks. The presence of SSCs in culture system was confirmed by immunocytochemistry of GFR-α1 and ITGα6 antibodies. The proliferated cells were cultured in three mentioned groups in the presence of retinoic acid and Sertoli cells conditioned medium for another 2 weeks. The number of colonies in the SACS group was significantly higher than two other groups. Before 2 weeks of culture, only Oct4 expression was observed in testicular cells (2.32 ± 0.25). After 2 weeks, the expression of Oct4 in the gelatin group was higher than that of the SACS group on day 7. The maximum expression of Stra8 was observed in SACS and gelatin groups after 7 days, but its expression was significantly decreased after 14 days of culture ( p  < .05). The expression of Scp3 and Acrosin genes were higher after 14 days in the SACS group compared to other groups. SACS has positive effects on proliferation and differentiation of hSSCs.

Bone marrow mesenchymal stem cells (BM-MSCs) were first cultured under induction of retinoic acid (RA), Sertoli cells conditioned medium and RA + con (conditioned medium) as treatment groups. The presence of Sertoli cells was confirmed by immunocytochemistry of follicle-stimulating hormone receptor in Sertoli cells and flow cytometry by anti-Gata4 antibody. Cell viability and morphology of nucleus and cytoplasm of BM-MSCs were evaluated by MTT test and DAPI staining respectively. The expression of Oct4, Plzf, Scp3, Caspases 8, 9 and 3 genes was evaluated by RT-PCR. For increasing the accuracy of experiment, the expression of Vasa and SCP3 genes was investigated quantitatively by real-time PCR after 0, 5, 10, 15 days of culture. The results showed that the number of apoptotic cells increased in RA group. The expression of apoptosis genes (Caspases 3, 8 and 9) was also observed in this group all days of culture. Measurement of Vasa and Scp3 genes by RT-PCR confirmed the positive effects of retinoic acid on increasing of genes expression. So, in this study, a group with maximum expression of differentiation genes and minimum expression of apoptotic genes was RA + conditioned medium group. DNA fragmentation was not observed in all groups., (© 2019 Blackwell Verlag GmbH.)