• 한국발생생물학회지:발생과생식
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• 제14권3호
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• pp.155-162
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• 2010

The trophectoderm is one of the earliest cell types to differentiate in the forming placenta. It is an important for the initial implantation and placentation during pregnancy. Trophoblast stem cells (TBSCs) develop from the blastocyst and are maintained by signals emanating from the inner cell mass. However, several limitations including rarity and difficulty in isolation of trophoblast stem cells derived from blastocyst still exist. To establish a model for trophoblast differentiation, we isolated TBSCs from human term placenta ($\geq$38 weeks) and characterized. Cell cycle was analyzed by measuring DNA content by FACS analysis and phenotype of TBSCs was characterized by RT-PCR and FACS analysis. TBSCs have expressed various markers such as self-renewal markers (Nanog, Sox2), three germ layer markers (hNF68, alpha-cardiac actin, hAFP), trophoblast specific markers (CDX-2, CK7, HLA-G), and TERT gene. In FACS analysis, TBSCs isolated from term placenta showed that the majority of cells expressed CD13, CD44, CD90, CD95, CD105, HLA-ABC, cytokeratin 7, and HLA-G. Testing for CD31, CD34, CD45, CD71, vimentin and HLA-DR were negative. TBSCs were shown to decrease the growth rate when cultured in conditioned medium without FGF4/heparin as well as the morphology was changed to a characteristic giant cell with a large cytoplasm and nucleus. In invasion assay, TBSCs isolated from term placenta showed invasion activities in in vivo using nude mice and in vitro Matrigel system. Taken together, these results support that an isolation potential of TBSCs from term placenta as well as a good source for understanding of the infertility mechanism.

• 식물조직배양학회지
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• 제27권6호
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• pp.423-428
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• 2000

In 1997 when cloned sheep Dolly and soon after Polly were born, it had become head-line news because in the former the nucleus that gave rise to the lamb came from cells of six-year-old adult sheep and in the latter case a foreign gene was inserted into the donor nucleus to make the cloned sheep produce human protein, factor IX, in e milk. In the last few years, once the realm of science fiction, cloned mammals especially in livestock have become almost commonplace. What the press accounts often fail to convey, however, is that behind every success lie hundreds of failures. Many of the nuclear-transferred egg cells fail to undergo normal cell divisions. Even when an embryo does successfully implant in the womb, pregnancy often ends in miscarriage. A significant fraction of the animals that are born die shortly after birth and some of those that survived have serious developmental abnormalities. Efficiency remains at less than one % out of some hundred attempts to clone an animal. These facts show that something is fundamentally wrong and enormous hurdles must be overcome before cloning becomes practical. Cloning researchers now tent to put aside their effort to create live animals in order to probe the fundamental questions on cell biology including stem cells, the questions of whether the hereditary material in the nucleus of each cell remains intact throughout development, and how transferred nucleus is reprogrammed exactly like the zygotic nucleus. Stem cells are defined as those cells which can divide to produce a daughter cell like themselves (self-renewal) as well as a daughter cell that will give rise to specific differentiated cells (cell-differentiation). Multicellular organisms are formed from a single totipotent stem cell commonly called fertilized egg or zygote. As this cell and its progeny undergo cell divisions the potency of the stem cells in each tissue and organ become gradually restricted in the order of totipotent, pluripotent, and multipotent. The differentiation potential of multipotent stem cells in each tissue has been thought to be limited to cell lineages present in the organ from which they were derived. Recent studies, however, revealed that multipotent stem cells derived from adult tissues have much wider differentiation potential than was previously thought. These cells can differentiate into developmentally unrelated cell types, such as nerve stem cell into blood cells or muscle stem cell into brain cells. Neural stem cells isolated from the adult forebrain were recently shown to be capable of repopulating the hematopoietic system and produce blood cells in irradiated condition. In plants although the term$\boxDr$ stem cell$\boxUl$is not used, some cells in the second layer of tunica at the apical meristem of shoot, some nucellar cells surrounding the embryo sac, and initial cells of adventive buds are considered to be equivalent to the totipotent stem cells of mammals. The telomere ends of linear eukaryotic chromosomes cannot be replicated because the RNA primer at the end of a completed lagging strand cannot be replaced with DNA, causing 5' end gap. A chromosome would be shortened by the length of RNA primer with every cycle of DNA replication and cell division. Essential genes located near the ends of chromosomes would inevitably be deleted by end-shortening, thereby killing the descendants of the original cells. Telomeric DNA has an unusual sequence consisting of up to 1,000 or more tandem repeat of a simple sequence. For example, chromosome of mammal including human has the repeating telomeric sequence of TTAGGG and that of higher plant is TTTAGGG. This non-genic tandem repeat prevents the death of cell despite the continued shortening of chromosome length. In contrast with the somatic cells germ line cells have the mechanism to fill-up the 5' end gap of telomere, thus maintaining the original length of chromosome. Cem line cells exhibit active enzyme telomerase which functions to maintain the stable length of telomere. Some of the cloned animals are reported prematurely getting old. It has to be ascertained whether the multipotent stem cells in the tissues of adult mammals have the original telomeres or shortened telomeres.

• Journal of Yeungnam Medical Science
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• 제20권1호
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• pp.92-98
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• 2003

Testicular teratoma is characterized that has more than one germ cell layer in various stages of maturation and differentiation. The incidence of mature teratoma varies from 2 to 9% of all germinal testicular tumor. Only 3 cases of bilateral teratoma have been reported in the literature to date. The teratoma can occurs at all ages but is most common between the age of 15-35 year. It is relatively infrequently seen in childhood and infancy. In these age group, teratoma tends to be a benign. Recently, we experienced one case of bilateral testicular teratoma in 4 month-old infancy. We report a case of bilateral testicular teratoma found infancy with review of related literatures.

• 한국패류학회지
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• 제24권1호
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• pp.1-10
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• 2008

Spermatogenesis and the reproductive cycle in male Spisula sachalinensis were investigated by cytological and histological observations. The morphology of the spermatozoon has a primitive type and is similar to those of other bivalves in that it contains a short midpiece with four mitochondria surrounding the centrioles. But spermatozoon of this species has not axial rod and satellite body in the midpiece. The morphologies of the sperm nucleus type and the acrosome shape of this species have a globe-shape type and modified cap-like shape, respectively. The spermatozoon is approximately $40-45{\mu}m$ in length including the sperm nucleus length (about $1.35{\mu}m$), acrosome length (about $1.50{\mu}m$) and tail flagellum. The axoneme of the sperm tail flagellum consists of nine pairs of microtubules at the periphery and a pair at the center. The axoneme of the sperm tail shows a 9+2 structure. The spawning period of these species lasts from June to July, and the main spawning occurs in July when seawater temperatures are greater than $20^{\circ}C$. The male reproductive cycle of this species can be categorized into five successive stages: early active stage (October to January), late active stage (February to April), ripe stage (April to June), partially spawned stage (June and July), and spent/inactive stage (August to September).

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.46-46
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• 2003

Interleukin-10 (IL-10) is a homodimeric protein with a wide spectrum of anti-inflammatory and immune activities. It inhibits cytokine production and expression of immune surface molecules in various cell types. The transgenic mice carrying the human IL-10 gene in conjunction with the bovine $\beta$-casein promoter produced the human IL-10 in milk during lactation. Transgenic mice were generated using a standard method as described previously. To screen transgenic mice, PCR was carried out using chromosomal DNA extracted from tail or toe tissues with a primer set. In this study, stability of germ line transmission and expression of IL-10 gene integrated into host chromosome were monitored up to generation F15 of a transgenic line. When female mouse of generation F9 was crossbred with normal male, generation F9 to F15 mice showed similar transmission rates (66.0$\pm$20.13%, 61.5$\pm$16.66%, 41.1$\pm$8.40%, 40.7$\pm$20.34%, 61.3$\pm$10.75%, 49.2$\pm$18.82%, and 43.8$\pm$25.91%, respectively), implying that the IL-10 gene can be transmitted stably up to long term generation in the transgenic mice. For ELISA analysis, IL-10 expression levels were determined with an hIL-10 ELISA and a mIL-10 ELISA kit in accordance with the supplier's protocol. Expression levels of human IL-10 from milk of generation F9 to F13 mice were 3.6$\pm$1.20 mg/ml, 4.2$\pm$0.93 mg/ml, 5.7$\pm$1.46 mg/ml, 6.3$\pm$3.46 mg/ml, and 6.8$\pm$4.52 mg/ml, respectively. These expression levels are higher than in generation F1 (1.6 mg/ml) mice. We concluded that transgenic mice faithfully passed the transgene on their progeny and successively secreted target proteins into their milk through several generations, although there was a little fluctuation in the transmission frequency and expression level between the generations.

• 한국수정란이식학회지
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• 제18권3호
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• pp.179-186
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• 2003

Human embryonic stem (hES) cell lines have been derived from human blastocysts and are expected to have far-reaching applications in regenerative medicine. The objective of this study is to improve freezing method with less cryo-injuries and best survival rates in hES cells by comparing various vitrification conditions. For the vitrifications, ES cells are exposed to the 4 different cryoprotectants, ethylene glycol (EG), 1,2-propanediol (PROH), EG with dime-thylsulfoxide (DMSO) and EG with PROH. We compared to types of vehicles, such as open pulled straw (OPS) or electron microscopic cooper grids (EM grids). Thawed hES cells were dipped into sequentially holding media with 0.2 M sucrose for 1 min, 0.1 M sucrose for 5 min and holding media for 5 min twice and plated onto a fresh feeder layer. Survival rates of vitrified hES cells were assessed by counting of undifferentiated colonies. It shows high survival rates of hES cells frozen with EG and DMSO (60.8％), or EG and PROH(65.8％) on EM grids better than those of OPS, compared to those frozen with EG alone (2.4％) or PROH alone (0％) alone. The hES cells vitrified with EM grid showed relatively constant colony forming efficiency and survival rates, compared to those of unverified hES cells. The vitrified hES cells retained the normal morphology, alkaline phosphates activity, and the expression of SSEA-3 and 4. Through RT-PCR analysis showed Oct-4 gene expression was down-regulated and embryonic germ layer markers were up-regulated in the vitrified hES cells during spontaneous differentiation. These results show that vitrification method by using EM grid supplemented with EG and PROH in hES cells may be most efficient at present to minimize cyto-toxicity and cellular damage derived by ice crystal formation and furthermore may be employed for clinical application.

• Asian-Australasian Journal of Animal Sciences
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• 제22권1호
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• pp.26-34
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• 2009

Porcine embryonic stem (ES) cells have a great potential as tools for transgenic animal production and studies of regulation of differentiation genes. Although several studies showed successful derivation of porcine ES-like cells, these cells were not maintained long-term in culture. Therefore, this study was conducted to establish porcine pluripotent ES-like cells using in vivo fertilized embryos and to maintain these cells in long term culture. Porcine ES-like cells from in vivo embryos obtained by immunosurgery or whole explant culture were successfully cultured for over 56 passages. Morphology of porcine ES-like cells was flat-shaped with a monolayer type colony. These cells stained for alkaline phosphatase throughout the culture. Furthermore, porcine ES-like cells reacted with antibodies against Oct-4, SSEA-1, SSEA-4, Tra-1-60, and Tra-1-81, which are typical markers of undifferentiated stem cells. To characterize the ability of porcine ES-like cells to differentiate into three germ layers, embryoid body formation was induced. After plating of these cells, porcine ES-like cells were spontaneously differentiated into various cell types of all three germ layers. In addition, porcine ES-like cells were successfully derived from IVF blastocysts in media containing human recombinant basic fibroblast growth factor.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.891-900
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• 2004

UDP-N-Acetylglucosamine(GlcNAc):$\beta$1,4-D-mannoside$\beta$-l ,4N-acetylglucosaminyltransferase-III (GnT-III) and UDP-N-GlcNAc:$\alpha$-6-D-mannosid$\beta$-1,6N-acetylglucosaminyltransferase-V(GnT - V) activities were determined in human hepatoma cell lines and metastatic colon cancer cells, and their activities were compared with those of normal liver cells and fetal hepatocytes. GnT-III activities were higher than those of GnT-V in hepatic carcinoma cells. When the two enzyme activities were assayed in highly metastatic colon cancer cells, GnT - V activities were much higher than those of GnT-III. When GlcN, GlcN-biant-PA and UDP-GlcNAc were used as substrates, the enzymes displayed different kinetic properties between hepatic and colon cancer cells, depending on their metastatic potentials. Normal cells of two origins had characteristically very low levels of GnT-III and -V activities, whereas hepatoma and colon cancer cells contained high levels of activities. These data were supported by RT-PCR and Northern blot analyses, showing that the expression of GnT-III and -V mRNAs were increased in proportion to the enzymatic activities. The increased GnT-III, md -V activities were also correlated with increased glycosylation of the cellular glycoproteins in hepatoma and colon cancer cells, as examined by lectin blotting analysis by using wheat germ glutinin (WGA), erythroagglutinating phytohemagglutinin (E-PHA), leukoagglutinating phytohemagglutinin (L-PHA), and concanavalin A (Con A). Treatment with retinoic acid, a differentiation agent, resulted in decreases of both GnT-III and -V activities of HepG2 and HepG3 cells. In colon carcinoma cells, however, treatment with retinoic acid resulted in a reduction of GnT-V activity, but not with GnT-III activity. Although the mechanism underlying the induction of these mzymes is unclear, oligosaccharides in many glycoproteins have been observed of cancer cells.

• International Journal of Oral Biology
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• 제41권2호
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• pp.89-96
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• 2016

Tooth development shows dynamic morphological changes from the stages of cap to hard tissue formation and is strictly regulated during development. In the present study, we compared expression and localization of 3 major enamel matrix proteins in rats: amelogenin, enamel and ameloblastin. DD-PCR and RT-PCR revealed differential expression of the major proteins from the cap stage to root stage. Immunofluorescence staining results indicated that amelogenin was not detected in either inner enamel epithelium or reduced enamel epithelium, but highly immunoreactive in preameloblasts and ameloblasts; in addition, it was sporadically expressed in preodontoblasts abutting preameloblasts. Ameloblastin expression was also observed in not only differentiated ameloblasts but also osteoblasts. Immunoreactivity to ameloblastin in ameloblasts was strong in Tomes' processes. Enamelin was exclusively localized along the entire newly formed and maturing enamel. Enamelin was largely localized in near Tomes' processes and enamel rods in maturing enamel. Alendronate treatment resulted in down-regulation of amelogenin and ameloblastin at both transcription and translation levels; whereas, enamelin expression was unchanged in response to the treatment. These results suggested that amelogenin, ameloblastin and enamelin might be implicated in cell differentiation, adhesion of ameloblasts to enamel and enamel crystallization during enamel matrix formation, respectively.

• 한국어류학회지
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• 제19권2호
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• pp.107-111
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• 2007

부화부터 부화 후 120일까지 민어, Miichthys miiuy (Basilewsky)의 성분화를 조사하였다. 시원세포는 부화 후 20일(전장 10.4 mm, 체중 0.14 g)에 출현하여 부화 후 40일(전장 19.4 mm, 체중 0.39 g)에 복강으로 이동하기 시작하였다. 부화 후 65일(전장 31.3 mm, 체중 0.93 g, $1,560D^{\circ}$ (적산온도))에 응축된 염색질 상태인 시원세포는 감수분열을 보여 난소로의 분화가 확인되었다. 부화후 120일(전장 4.60 mm, 체중 1.38 g, $2,880D^{\circ}$)에 난모세포는 주변인기 단계로 직경이 $20{\sim}40{\mu}m$로 증가하였다. 성분화후 난모세포는 크기 증가를 보인 반면, 정소는 부화 후 65일부터 증식하기 시작하였다. 부화 후 80일(전장 37.9 mm, 체중 1.39 g, $1,920D^{\circ}$)에 정소 체세포에 싸여진 정원세포 낭포의 출현과 정소 소엽 형성이 시작되었다. 부화 후 120일에 정소 소엽에는 시원세포와 정모세포가 존재하였다. 본 연구 결과 민어의 성분화 양상은 분화형자웅이체이다.