• Molecules and Cells
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• v.37 no.6
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• pp.473-479
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• 2014

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost-effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.

• Anatomy and Cell Biology
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• v.57 no.2
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• pp.163-171
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• 2024

In the last decade, melatonin has gained recognition as a potent scavenger and an effective antioxidant capable of neutralizing free radicals, including reactive oxygen species. Additionally, it exhibits anti-apoptotic properties. In this review, we will examine a compilation of articles that explore the cellular signaling function of melatonin on spermatogonial stem cells (SSCs) and adjacent cells such as Sertoli and Leydig cells. These cells play a crucial role in the proliferation of SSCs both in vitro and in vivo. In this review, we analyze the function of melatonin in the proliferation of SSCs from other aspects. For this purpose, we examine the articles based on the presence of melatonin on SSCs in four groups: As a supplement in SSCs medium culture, SSCs three-dimensional culture system, SSCs freezing medium, and as a therapeutic factor in vivo. Mechanisms of growth and proliferation of SSCs were considered. The purpose of this study is to investigate the potential effects of melatonin as a powerful antioxidant or growth stimulant for SSCs, both in vivo and in vitro.

• International Journal of Stem Cells
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• v.16 no.1
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• pp.27-35
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• 2023

Background and Objectives: Spermatogonial stem cells (SSCs) are the most primitive cells in spermatogenesis and are the only adult stem cells capable of passing on the genome of a given species to the next generation. SSCs are the only adult stem cells known to exhibit high Oct4 expression and can be induced to self-reprogram into pluripotent cells depending on culture conditions. Epigenetic modulation is well known to be involved in the induction of pluripotency of somatic cells. However, epigenetic modulation in self-reprogramming of SSCs into pluripotent cells has not been studied. Methods and Results: In this study, we examined the involvement of epigenetic modulation by assessing whether selfreprogramming of SSCs is enhanced by treatment with epigenetic modulators. We found that second-generation selective class I HDAC inhibitors increased SSC reprogramming efficiency, whereas non-selective HDAC inhibitors had no effect. Conclusions: We showed that pluripotent stem cells derived from adult SSCs by treatment with small molecules with epigenetic modulator functions exhibit pluripotency in vitro and in vivo. Our results suggest that the mechanism of SSC reprogramming by epigenetic modulator can be used for important applications in epigenetic reprogramming research.

• Reproductive and Developmental Biology
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• v.37 no.3
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• pp.143-148
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• 2013

Spermatogenesis is initiated from spermatogonial stem cells (SSCs) that has an ability of self-renewal and unipotency to generate differentiating germ cells. The objective of this study is to develop the simple method for derivation of SSCs using non-sorting of both spermatogonia and feeder cells. Simply uncapsulated mouse testes were treated with enzymes followed by surgical mincing, and single cells were cultured in stempro-$34^{TM}$ cell culture media at $37^{\circ}C$. After 5 days of culture, aciniform of SSC colony was observed, and showed a strong alkaline phosphatase activity. Molecular characterization of mouse SSCs showed that most of the mouse SSC markers such as integrin ${\alpha}6$ and ${\beta}1$, CD9 and Stra8. In addition, pluripotency embryonic stem cell (ESC) marker Oct4 were expressed, however Sox2 expression was lowered. Interestingly, expression of SSC markers such as Vasa, Dazl and PLZF were stronger than mouse ESC (mESC). This data suggest that generated mouse SSCs (mSSCs) in this study has at least similar biomarkers expression to mESC and mSSCs derived from other study. Immunocytochemistry using whole mSSC colony also confirmed that mSSCs generated from this study expressed SSC specific biomarkers such as c-kit, Thy1, Vasa and Dazl. In conclusion, mSSCs from 5 days old mouse testes were successfully established without sorting of spermatogonia, and this cells expressed both mESC and SSC specific biomarkers. This simple derivation method for mSSCs may facilitate the study of spermatogenesis.

• Anatomy and Cell Biology
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• v.56 no.4
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• pp.508-517
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• 2023

In cancer patients, chemo/radio therapy may cause infertility by damaging the spermatogenesis affecting the self-renewal and differentiation of spermatogonial stem cells (SSCs). In vitro differentiation of stem cells especially mesenchymal stem cells (MSCs) into germ cells has recently been proposed as a new strategy for infertility treatment. The aim of this study was to evaluate the proliferation and differentiation of SSCs using their co-culture with Sertoli cells and conditioned medium (CM) from adipose tissue-derived MSCs (AD-MSCs). Testicular tissues were separated from 2-7 days old neonate Wistar Rats and after mechanical and enzymatic digestion, the SSCs and Sertoli cells were isolated and cultured in Dulbecco's modified eagle medium with 10% fetal bovine serum, 1X antibiotic, basic fibroblast growth factor, and glial cell line-derived neurotrophic factor. The cells were treated with the CM from AD-MSCs for 12 days and then the expression level of differentiation-related genes were measured. Also, the expression level of two major spermatogenic markers of DAZL and DDX4 was calculated. Scp3, Dazl, and Prm1 were significantly increased after treatment compared to the control group, whereas no significant difference was observed in Stra8 expression. The immunocytochemistry images showed that DAZL and DDX4 were positive in experimental group comparing with control. Also, western blotting revealed that both DAZL and DDX4 had higher expression in the treated group than the control group, however, no significant difference was observed. In this study, we concluded that the CM obtained from AD-MSCs can be considered as a suitable biological material to induce the differentiation in SSCs.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.221-228
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• 2014

Despite that porcine spermatogonial stem cells (pSSCs) have been regarded as a practical tool for preserving eternally genetic backgrounds derived from pigs with high performance in the economic traits or phenotypes of specific human diseases, there were no reports about precise definition of niche conditions promoting proliferation and maintenance of pSSCs. Accordingly, we tried to determine niche conditions supporting proliferation and maintenance of undifferentiated pSSCs for short-term. For these, undifferentiated pSSCs were progressively cultured in different composition of culture medium, seeding density of pSSCs, type of feeder cells and concentration of growth factors, and then total number of and alkaline phosphatase (AP) activity of pSSCs were investigated at post-6 day culture. As the results, the culture of $4{\times}10^5$ pSSCs on mitotically in activated $2{\times}10^5$ STO cells in the mouse embryonic stem cell culture medium (mESCCM) supplemented with 30 ng/ml glial cell line-derived neurotrophic factor (GDNF) was identified as the best niche condition supporting effectively the short-term maintenance of undifferentiated pSSCs. Moreover, the optimized short-term culture system will be a basis for developing long-term culture system of pSSCs in the following researches.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.7
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• pp.1015-1022
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• 2007

The spermatogonial stem cell (SSCs) is unique in that it is the only cell in the adult male that can contribute genes to a subsequent generation. Permanent modification of the germ cell line may be realized if stem cells could be cultured, transfected with unique genes, and then transplanted into recipient testes. We developed a culture system that supported long-term viability of SSCs. We used a retrovirus vector (pMSCV including ${\beta}$-galactosidase) to stably transfect spermatogonia following long-term culture using the system developed. Expression of the reporter gene ${\beta}$-galactosidase controlled by the retroviral vector was stable in long-term cultured SSCs. We confirmed the retroviral-mediated ${\beta}$-galactsidase gene could be expressed in germ cells in recipient mice following SSCs transplantation.

• Reproductive and Developmental Biology
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• v.38 no.4
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• pp.137-142
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• 2014

A tremendous increase in the human population has put poultry industry under an increased pressure to meet steep increase in the demand. Poultry is contributing 25% of the total world's meat production and lesser cost of investment per bird makes it more suitable for the further breeding programmes. Major poultry diseases frequently lead to cardiac damage and cause huge economic losses to poultry industry due to mortality. The in vitro embryonic stem cell (ESC) technology has a futuristic approach for homogeneous populace of differentiated cells, for their further transplantations. During in vitro conditions the differentiated cell populace can be used in grafting and transplantation processes to regenerate damaged tissues. Therefore, the current study targeted the use of spermatogonial stem cells (SSCs) in the poultry production system through cardiac regeneration. The current study will also open new boulevard for the similar kind of research in other livestock species for the management of heart diseases.

• Molecules and Cells
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• v.41 no.7
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• pp.631-638
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• 2018

Spermatogonial stem cells (SSCs) derived from mouse testis are unipotent in regard of spermatogenesis. Our previous study demonstrated that SSCs can be fully reprogrammed into pluripotent stem cells, so called germline-derived pluripotent stem cells (gPS cells), on feeder cells (mouse embryonic fibroblasts), which supports SSC proliferation and induction of pluripotency. Because of an uncontrollable microenvironment caused by interactions with feeder cells, feeder-based SSC reprogramming is not suitable for elucidation of the self-reprogramming mechanism by which SSCs are converted into pluripotent stem cells. Recently, we have established a Matrigel-based SSC expansion culture system that allows longterm SSC proliferation without mouse embryonic fibroblast support. In this study, we developed a new feeder-free SSC self-reprogramming protocol based on the Matrigel-based culture system. The gPS cells generated using a feeder-free reprogramming system showed pluripotency at the molecular and cellular levels. The differentiation potential of gPS cells was confirmed in vitro and in vivo. Our study shows for the first time that the induction of SSC pluripotency can be achieved without feeder cells. The newly developed feeder-free self-reprogramming system could be a useful tool to reveal the mechanism by which unipotent cells are self-reprogrammed into pluripotent stem cells.

• Journal of Veterinary Science
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• v.23 no.2
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• pp.35.1-35.13
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• 2022

Background: The testis has been reported to be a naturally O2-deprived organ, dimethyloxaloylglycine (DMOG) can inhibit hypoxia inducible factor-1alpha (HIF-1α) subject to degradation under normal oxygen condition in cells. Objectives: The objective of this study is to detect the effects of DMOG on the proliferation and differentiation of spermatogonial stem cells (SSCs) in Bama minipigs. Methods: Gradient concentrations of DMOG were added into the culture medium, HIF-1α protein in SSCs was detected by western blot analysis, the relative transcription levels of the SSC-specific genes were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Six days post-induction, the genes related to spermatogenesis were detected by qRT-PCR, and the DNA content was determined by flow cytometry. Results: Results revealed that the levels of HIF-1α protein increased in SSCs with the DMOG treatment in a dose-dependent manner. The relative transcription levels of SSC-specific genes were significantly upregulated (p < 0.05) by activating HIF-1α expression. The induction results showed that DMOG significantly increased (p < 0.05) the spermatogenesis capability of SSCs, and the populations of haploid cells significantly increased (p < 0.05) in DMOG-treated SSCs when compared to those in DMOG-untreated SSCs. Conclusion: We demonstrate that DMOG can promote the spermatogenesis activity of SSCs.