• Molecules and Cells
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• 제27권6호
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• pp.635-640
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• 2009

Testis-derived germline stem (GS) cells can undergo reprogramming to acquire multipotency when cultured under appropriate culture conditions. These multipotent GS (mGS) cells have been known to differ from GS cells in their DNA methylation pattern. In this study, we examined the DNA methylation status of the H19 imprinting control region (ICR) in multipotent adult germline stem (maGS) cells to elucidate how epigenetic imprints are altered by culture conditions. DNA methylation was analyzed by bisulfite sequencing PCR of established maGS cells cultured in the presence of glial cell line-derived neurotrophic factor (GDNF) alone or both GDNF and leukemia inhibitory factor (LIF). The results showed that the H19 ICR in maGS cells of both groups was hypermethylated and had an androgenetic pattern similar to that of GS cells. In line with these data, the relative abundance of the Igf2 mRNA transcript was two-fold higher and that of H19 was three fold lower than in control embryonic stem cells. The androgenetic DNA methylation pattern of the H19 ICR was maintained even after 54 passages. Furthermore, differentiating maGS cells from retinoic acid-treated embryoid bodies maintained the androgenetic imprinting pattern of the H19 ICR. Taken together these data suggest that our maGS cells are epigenetically stable for the H19 gene during in vitro modifications. Further studies on the epigenetic regulation and chromatin structure of maGS cells are therefore necessary before their full potential can be utilized in regenerative medicine.

• Anatomy and Cell Biology
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• 제56권4호
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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.

• 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.

• 생명과학회지
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• 제17권2호통권82호
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• pp.167-173
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• 2007

생쥐 신경교세포 유래 신경영양인자 유도성 전사인자(mGIF)의 발현조절에 필요한 전사기작을 연구하기 위하여 mGIF cDNA를 탐침자로 이용하여 genomic clone을 분리하였다. 전체 유전자 13-kb 영역 중 전사개시점에서 4-kb 상류영역의 유전자 서열을 파악한 결과, 프로모터 영역에서 TATA box와 CAAT box는 발견할 수 없었으며 G+C content는 높은 것으로 나타났고 여러 개의 Sp1 전사인자 결합영역이 있었다. 또한 mGIF 유전자는 AP2 결합에 필요한 보존적 영역이 있었다. mGIF 유전자의 프로모터 영역의 단편들을 프로모터가 없는 pGL2-Basic 플라스미드의 luciferase 유전자의 상류에 연결하여 서로 다른 5종류의 결손 돌연변이체를 제조하고 NB41A3 세포주를 이용하여 전사활성을 측정하였다. Transient expression assays 결과, 모든 결손 돌연변이체에서 전사활성이 나타났으며 -213과 -129사이에 전사촉진 영역이 존재하며 -806과 -214사이에 전사억제 영역이 있는 것으로 나타났다. 신경세포주인 NB41A3과 신경교세포주인 C6 그리고 간세포주인 HepG2에서 mGIF 유전자 프로모터의 높은 활성이 관찰되었으며, 근육세포주인 C2C12에서는 낮은 활성이 관찰되었다. 따라서 mGIF 유전자는 조직특이적으로 발현하며 도파민 수용체 유전자와 구조적, 기능적 유사성이 있는 것을 알 수 있었다.