• 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.

• Development and Reproduction
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• v.13 no.4
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• pp.271-280
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• 2009

Human placenta is abundant source of adult stem cells. Especially, amniotic epithelial cells have stem cell characteristics, expressing surface markers normally present on embryonic stem cells and germ cells. However, culturing and expanding amniotic epithelial cells in vitro without feeder cells are difficult due to endogenous characteristics of epithelial cells. In the present study, amniotic epithelial cells are isolated and proliferated in several passages by applying dithiothreitol and a Rho-associated kinase inhibitor in culture media. The cultured amniotic epithelial cells showed the epithelial and stem cell characteristics. In conclusion, human placenta-derived amniotic epithelial stem cells can be a major source of stem cells for medical treatment of various diseases without any controversial issues.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.243-248
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• 2003

Sperrnatogenesis, the process by which the male germ-line stem cells(GSCs; type A spermatogonia) divide and differentiate to produce the mature spermatozoa, occurs in the seminiferous tubules of the testis. The GSCs proliferate actively to produce two types of cells: other GSCs and differentiating spermatogonia. GSCs have unipotentcy, devoted solely to the generation of sperm. The function of GSCs has broad implications for development, disease, and evolution. Spermatogenesis is fundamental for propagation of species and the defects of this system can result in infertility or disease. The ability to identify, isolate, culture, and alter GSCs will allow powerful new approaches in animal transgenesis and human gene therapy relating to infertility. Until recently, research on stem cells in the testis has been limited because of technical difficulties in isolating and identifying these cell populations. Here, we were trying to find out optimal conditions for in vitro culture of GSCs for identifying and isolating GSCs. We collected mouse GSCs from 3-days old mouse by two-step enzyme digestion method. GSCs were plated and grown on mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium (DMEM) containing 15％ fatal bovine serum, 10 mM 2-mercaptoethanol, 1％ non-essential amino acids, 1 ng/$m\ell$ bFGF, 10 $\mu$M forskolin, 1500 U/$m\ell$ human recombinant leukemia inhibitory factor (LIF). Over a period 3∼5 days, GSCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells. After 5th passages, cells within the colonies continued to be alkaline phosphatase and Oct-4 positive and tested positive against a panel of two immunological markers(Integrin $\alpha$ 6 and Integrin $\beta$ 1) that have been recognized generally to characterize GSCs. SSEA-1, SSEA-3, and SSEA-4 also showed positive signals. Based on our data, these GSCs-derived cultures meet the criteria for GSCs itself and even other pluripotent stem cells. We reported here the establishment of in vitro cultures from mouse male GSCs.

• Journal of Periodontal and Implant Science
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• v.32 no.1
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• pp.1-12
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• 2002

The periodontal ligament(PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. PDL-specific protein;PDLs22 had been previously identified as a novel protein isolated from cultured human PDL fibroblasts using subtraction hybridization between human gingival fibroblasts and PDL fibroblasts. The aim of this study was to examine the expression pattern and tissue localization of PDLs22 protein in embryonic and various postnatal stages of developing mouse using immunohistochemical staining. Embryos (E18) and postnatal (P1, P4, P5, P15, P18) were decapitated and the heads were fixed overnight in a freshly prepared solution of 4% paraformaldehyde. Some specimens were decalcified for $2{\sim}4$ weeks in a solution containing 10% of the disodium salt of ethylenediamine-tetraacetic acid (EDTA). Next, tissues were dehydrated, embedded in paraffin and sectioned serially at $6{\mu}m$ in thickness. Polyclonal antiserum raised against PDLs22 peptides, ISNKYLVKRQSRD, were made. The localization of PDLs22 in tissues was detected by polyclonal antibody against PDLs22 by means of immunohistochemical staining. The results were as follows; 1. Expression of PDLs22 protein was not detected in the tooth germ of bud and cap stage. 2. At the late bell stage and root formation stage, strong expression of PDLs22 protein was observed in developing tooth follicle, osteoblast-like cells, and subodontoblastic cells in the tooth pulp, but not in gingival fibroblasts, ameloblasts and odontoblasts of tooth germ 3. In erupted tooth, PDLs22 protein was intensely expressed in PDL and osteoblast-like cells of alveolar bone, but not in gingival fibroblasts, mature osteocytes and adjacent salivary glands. 4. In the developing alveolar bone and mid-palatal suture, expression of PDLs22 protein was seen in undifferentiated mesenchymal cells and osteoblast-like cells of developing mid-palatal suture, but not in mature osteocytes and chondrocytes. These results suggest that PDLs22 protein may play an important role in the differentiation of undifferentiated mesenchymal cells in the bone marrow and PDL cells, which can differentiate into multiple cell types including osteoblasts, cementoblasts, and PDL fibroblasts. However, more researches should be performed to gain a better understanding of the exact function of PDLs22 protein which related to the PDL cell differentiation.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.1-7
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• 2005

Telomeres consisting of (TTAGGG)n tandem repeat DNA sequences and associated proteins are essential for chromosome stability and related with cell senescence, apoptosis and cancer. The telomerase is a ribonucleoprotein which act as a template for the synthesis of telomeric DNA. This study was carried out to identify the distribution of telomeres on mouse chromosomes and also to analyze the amount of telomeres and telomerase activity of mouse embryos at early embryonic stages. Germ cells and early embryos from 1 cell to blastocyst stage were analyzed. The amount of telomeres was analyzed by quantitative fluorescence in situ hybridization technique(Q-FISH) using a human telomeric DNA probe, and telomerase activity was measured by telomeric repeat amplification protocol assay(TRAP). In results, the telomeres on mouse chromosomes were distributed at the ends of all autosomes and sex chromosomes. Although the quantity of telomeres varied among chromosomes, most of chromosomes had higher amount in q-arm telomeres than in p-arm telomeres. The results of Q-FISH indicated that the relative amount of telomeres of mouse embryos in each embryonic stage was approximately the same except the higher amount in blastocysts. Using TRAP assay on mouse embryos, telomerase activity was detected in all preimplantation stages from mature oocytes to blastocysts. Especially the telomerase activity was significantly increased at the morula and blastocyst stage. In conclusion, there may be a close association between the amount of telomeres and telomerase activity in early embryonic stages, and analysis of telomere quantity and telomerase activity on early development will be helpful for the investigation of embryogenesis and embryonic cell differentiation in mice.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.1
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• pp.25-33
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• 2006

Objective: The aim of this study was to investigate whether embryonic stem (ES) cells can be established from isolated blastomeres of mouse embryos. Methods: Blastomeres were separated from mouse (C57Bl/6J) 2- or 4-cell embryos. Isolated blastomeres or whole 4-cell embryos were co-cultured with mitosis-arrested STO feeder cells in DMEM supplemented with recombinant murine leukemia inhibitory factor and ES-qualified fetal bovine serum. After the tentative ES cell lines were maintained from isolated blastomeres or whole embryos, some of them were frozen and the others were sub-cultured continually. Characteristics of tentative ES cell lines as were evaluated for specific genes expressions with immunocytochemistry and RT-PCR. Results: One ES cell line (3.0%) was established from isolated blastomere of 2-cell embryo and one cell line (4.0%) from isolated two blastomeres of 4-cell embryo. And five cell lines (16.7%) were established from whole 4-cell embryos. Both cell lines from isolated blastomere and whole embryo expressed mouse ES cell specific markers such as SSEA-1, Oct-4 and alkaline phosphatase. Marker genes of three germ layers were expressed from embryoid bodies of both cell lines. Conclusion: This study suggests that mouse ES cells could be established from isolated blastomeres, although the efficiency is lower than whole embryos. This animal model could be applied to establishment of autologous human ES cells from biopsied blastomeres of preimplantation embryos in human IVF-ET program.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.60-60
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• 2003

Embryonic stem (ES) cells have property of self-renewal and can differentiate into the cells of all three primary germ layers. Recently, many growth factors, alteration of culture condition and gene modifications have been used to differentiate mouse and human ES cells into specific cell types. This study was performed to evaluate the differentiation protocol for human ES cells to the endodermal lineage cells. Human ES cells (Miz-hESl ) were cultured on STO feeder layer mitotically inactivated with mitemycin C, and embryoid bodies (EBs) were formed by suspension culture. Differentiation protocol of EBs consisted of three steps: stage I, culture of EBs for 6 days with ITSFn medium; stage II, culture of stage I cells for 8 days with N2 medium ; stage III, culture of stage II cells for 22 days with N2 medium. mRNA levels of the endodermal lineage differentiation genes were analyzed by semi- quantitative RT-PCR. The Oct-4 expression, a marker of the pluripotent state, was detected in undifferentiated human ES cells but progressively decreased after EBs formation. Differentiating human ES cells expressed marker genes of endodermal differentiation and pancreatic islet cells. GATA4, a-fetoprotein, Glut-2, and Ngn3 were expressed in all stages. However, albumin and insulin were expressed in only stage III cells. The human ES cells can be differentiated into endodermal lineage cells by multiple step culture system using various supplements. We are developing the more effective protocols for guided differentiation of human ES cells.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.243-247
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• 1994

최근 의약적으로 유용한 단백질을 대량 생산키 위한 실현 가능한 방법이 유전자변환 가축의 이용과 관련되어 발전되어 왔다. 이러한 유전자 변환동물은 이종의 단백질을 유즙속으로 분비시키는 생체반응기로서 이용되고 있다. 이러한 전략적 목적을 위해 현재 유전자 변환동물의 생산을 위한 이용에 있어 여러 가지 방법들이 보고되고 있다. 그러나 ES 세포의 사용이 이러한 방법들 사이에서 가장 실질적인 것으로 추정되고 있다. 본 실험에서는 유전자 구축을 위해 사람 황체 호르몬(human luteinizing hormone; hLH)의 전사를 유도하기 위해 각각 2.2 및 0.5 kb의 토끼 $\beta$-casein pronoter 단편을 이용하여 생쥐의 유선에 hLH를 발현시키도록 조절하고 발현이 thynidine kinase(TK) pronoter에 의해 좌우되는 neo 유전자를 selectable marker로서 plasnid속에 삽입하였다. 그 결과 생긴 구축 유전자는 각각 pCas 2.2와 pCas 0.5로 명명하였다. 구축된 유전자로 2$\times$107의 TT-2 ES세포를 170V, 550$\mu$F로 100$\mu$g의 선상 plasmid에 의해 electroporation 시켰다. 감염된 colony들은 250$\mu$g/$m\ell$ G418을 함유하는 ESM 배양액에서 선별 7일 이후에 회수하여 성공적으로 감염된 ES세포는 PCR 및 Southern blot에 의해 확인되었고 그들 중 나머지는 trypsin 처리 후 각각 미세조작과 공배양 기술을 사용하여 ICR 생쥐의 8세포기 수정란 속에 도입하였다. 결국 24시간 동안 37$^{\circ}C$, 5% $CO_2$에서 배양된 배반포를 chimera의 생산을 위해 위임신 유기된 G418 선발처리 이후 400 및 275개의 ES 세포 colony가 생존하였으며, 3개의 ES 세포으 colony 의 genome 속에 임의적으로 plamid가 삽입된 것을 Southern blot에 의해 확인되었다. 총 13 chimera 생쥐가 3 colony로부터 생산되었으나 germ-line chimera는 현재 조사중이다. chimera 생산빈도는 공배양 기술보다 주입방법에서 현저히 높았다.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.281-285
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• 2011

In the present study, we identified differentially methylated region (DMR) upstream of Dnmt1o and Dnmt1s gene in early porcine embryos. Porcine Dnmt1o had at least one DMR which was located between -530 bp to -30 bp upstream from transcription start site of the Dnmt1o gene. DNA methylation analyses of Dnmt1o revealed the DMR to be hypomethylated in oocytes, whereas it was highly methylated in sperm. Moreover, the DMR upstream of Dnmt1o was gradually hypermethylated from oocytes to two cells and dramatically changed in the methylation pattern from four cells to BL stages in an in vivo. In an IVF, the methylation status in the DMR upstream of Dnmt1o was hypermethylated from one cell to eight cells, but demethylated at the Morula and BL stages, indicating that the DNA methylation pattern in the Dnmt1o upstream ultimately changed from stage to stage before the implantation. Next, to elucidate whether DNA methylation status of Dnmt1s upstream is stage-by-stage changed in during porcine early development, we analyzed the dynamics of the DNA methylation status of the Dnmt1s locus in germ cell, or one cell to BL cells. The Dnmt1s upstream was highly methylated in one and eight cells, while less methylated in two, four, morula, and BL cells. Taken together, our data demonstrated that DNA methylation and demethylation events in upstream of Dnmt1o/Dnmt1s during early porcine embryos dramatically occurred, and this change may contribute to the maintenance of genomewide DNA methylation in early embryonic development.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.19-24
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• 2005

The low density lipoprotein receptor-related protein 5 (LRP5) highly expressed in many tissues, including hepatocytes and pancreatic beta cells, can bind to apolipoprotein E. To evaluate in vivo roles of LRP5, we generated LRP5-deficient mice. LRP5 genomic DNA was isolated from TT2 embryonic stem (ES) cells. Targeting vector was constructed to disrupt an exon 18 of the mouse LRP5 gene and transfected into ES cells. Three homologous recombinants at LRP5 locus were identified from 178 G418-resistant clones. Chimeric males generated by morula aggregation technique were mated to C57BL/6 female mice. After achieving germ-line transmission, LRP5+/- females were crossed with LRP5+/- males to obtain LRP5-deficient mice. One line of mice lacking LRP5 gene was confirmed by Southern blotting. Such knock-out mice may serve as an effective animal model to study in vivo function of LRP5 gene.