• Reproductive and Developmental Biology
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• 제37권3호
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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.

• Molecules and Cells
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• 제37권6호
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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.

• 한국발생생물학회지:발생과생식
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• 제15권1호
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• pp.41-52
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• 2011

In the present study, embryoid bodies (EBs) obtained from induced pluripotent stem cells (iPSCs) were induced to differentiate into germ lineage cells by treatment with bone morphogenetic protein 4 (BMP4) and retinoic acid (RA). The results were compared to the results for embryonic stem cells (ESCs) and multipotent spermatogonial stem cells (mSSCs) and quantified using immunocytochemical analysis of germ cell-specific markers (integrin-${\alpha}6$, GFR-${\alpha}1$, CD90/Thy1), fluorescence activating cell sorting (FACS), and real time-RT-PCR. We show that the highest levels of germ cell marker-expressing cells were obtained from groups treated with 10 ng/$m{\ell}$ BMP4 or 0.01 ${\mu}M$ RA. In the BMP4-treated group, GFR-${\alpha}1$ and CD90/Thy-1 were highly expressed in the EBs of iPSCs and ESCs compared to EBs of mSSCs. The expression of Nanog was much lower in iPSCs compared to ESCs and mSSCs. In the RA treated group, the level of GFR-${\alpha}1$ and CD90/Thy-1 expression in the EBs of mSSCs Induced pluripotent stem cells, Mouse embryonic stem cells, Multipotent spermatogonial stem cells, Germ cell lineage, Differentiation potential. was much higher than the levels found in the EBs of iPSCs and similar to the levels found in the EBs of ESCs. FACS analysis using integrin-${\alpha}6$, GFR-${\alpha}1$, CD90/Thy1 and immunocytochemistry using GFR-${\alpha}1$ antibody showed similar gene expression results. Therefore our results show that iPSC has the potential to differentiate into germ cells and suggest that a protocol optimizing germ cell induction from iPSC should be developed because of their potential usefulness in clinical applications requiring patient-specific cells.

• 한국수정란이식학회지
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• 제29권3호
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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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• 제29권10호
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• pp.1398-1406
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• 2016

In general, the seminiferous tubule basement membrane (STBM), comprising laminin, collagen IV, perlecan, and entactin, plays an important role in self-renewal and spermatogenesis of spermatogonial stem cells (SSCs) in the testis. However, among the diverse extracellular matrix (ECM) proteins constituting the STBM, the mechanism by which each regulates SSC fate has yet to be revealed. Accordingly, we investigated the effects of various ECM proteins on the maintenance of the undifferentiated state of SSCs in pigs. First, an extracellular signaling-free culture system was optimized, and alkaline phosphatase (AP) activity and transcriptional regulation of SSC-specific genes were analyzed in porcine SSCs (pSSCs) cultured for 1, 3, and 5 days on non-, laminin- and collagen IV-coated Petri dishes in the optimized culture system. The microenvironment consisting of glial cell-derived neurotrophic factor (GDNF)-supplemented mouse embryonic stem cell culture medium (mESCCM) (GDNF-mESCCM) demonstrated the highest efficiency in the maintenance of AP activity. Moreover, under the established extracellular signaling-free microenvironment, effective maintenance of AP activity and SSC-specific gene expression was detected in pSSCs experiencing laminin-derived signaling. From these results, we believe that laminin can serve as an extracellular niche factor required for the in vitro maintenance of undifferentiated pSSCs in the establishment of the pSSC culture system.

• 한국발생생물학회지:발생과생식
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• 제13권4호
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• pp.305-312
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• 2009

단분화성 정원줄기세포의 장기간 체외배양 중에 확립되는 다분화능 정원줄기세포는 배아줄기세포와 유사한 특성을 가져 3배엽성 세포로 체외분화가 가능하며 기형종을 형성할 수 있다. 본 연구에서는 선행 연구를 통해 outbred 생쥐(ICR strain)로부터 확립된 다분화능 정원줄기세포의 형질전환 가능성을 확인하며, 배아 내로 주입하여 유전적 키메라를 형성하는 효율을 배아줄기세포와의 비교를 통하여 검증하고자 하였다. 다분화능 정원줄기세포를 넣은 배아로부터 태어난 산자는 총 47마리(4.8%)가 태어나, 67마리(11.7%)가 태어난 배아줄기세포군에 비해 그 효율이 낮았다(P<0.05). 그러나 산자들 중의 키메라 생쥐의 비율은 다분화능 정원줄기세포 군으로 부터 3마리(6.4%)가 태어나 배아줄기세포 군으로부터 태어난 5마리(7.5%)와 유사하였다(P>0.05). 태어난 유전자변형 생쥐의 장기를 확인한 결과, 췌장, 심장, 뇌, 근육, 위, 피부, 정소에 GFP가 발현되는 것을 확인하였다. 또한 배아의 근육, 위, 뼈 등에서 anti-GFP 항체의 발현을 확인하였다. 이상의 결과를 종합하면 outbred 생쥐로부터 확립된 다분화능 정원줄기세포가 inbred 생쥐로부터 확립된 배아줄기세포와 마찬가지로 키메라 생쥐를 생산할 수 있는 전분화능을 가짐을 확인하였고, 새로운 유전자변형 동물의 생산을 위한 매개체로서의 가능성을 가진 것으로 여겨진다.

• Toxicological Research
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• 제33권2호
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• pp.107-118
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• 2017

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration ($IC_{50}$) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity.