Iron depletion with deferoxamine protects bone marrow-derived mesenchymal stem cells against oxidative stress-induced apoptosis.

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 H2O2-induced oxidative stress was evaluated through half of the inhibitory concentration (IC50) 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 H2O2-induced apoptosis. BM-MSCs were identified with morphologic and flow cytometry analysis. IC50 of H2O2 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 H2O2-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.

Differentiation of spermatogonial stem cells by soft agar three-dimensional culture system.

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.

Germ cell differentiation of bone marrow mesenchymal stem cells.

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.

The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture.

INTRODUCTION: 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 Itgß1), 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.

In vitro colonization of human spermatogonia stem cells: effect of patient's clinical characteristics and testicular histologic findings.

OBJECTIVE: To evaluate the effect of the demographic/clinical characteristics of patients and testicular histologic findings on the in vitro colonization of human spermatogonial stem cells (SSCs). In vitro isolation and proliferation of human SSCs has emerged as a suitable method for the enrichment of spermatogonia germ cells. METHODS: SSCs were isolated from the testicular biopsies of 47 infertile men with nonobstructive azoospermia and co-cultured with a Sertoli cell monolayer. Age, infertility duration, medical/surgical history, testicular size, and testicular histologic findings were recorded. The patients were divided into 2 groups according to the growth/no growth of human SSC colonies in culture. As the main outcome measure, the number and diameter of germ cell-derived colonies were compared between 2 groups in days 8, 13, and 18 after cultivation with respect to the recorded parameters. RESULTS: No difference was found between the 2 groups regarding the demographic/clinical parameters. Maturation arrest at the premeiotic spermatogonia stage was present in a considerably greater proportion in the group with growth of human SSC colonies compared with the group without growth of human SSC colonies (14 [45.1%] of 31 versus 3 [18.7%] of 16; P < .001) on days 8, 13, and 18 after culture. Maturation arrest at premeiotic SSCs was associated with a greater number and larger diameter of germ cell colonies compared with the maturation arrest at primary spermatocyte and secondary spermatocyte/spermatid stages (P < .001). CONCLUSION: Infertile men with testicular histologic findings of maturation arrest at the premeiotic spermatogonia stage were seemingly the most appropriate candidates for testicular biopsy and in vitro propagation of human SSCs, regardless of their demographic/clinical characteristics.