• Journal of Embryo Transfer
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• v.14 no.1
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• pp.6-15
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• 1999

ES cell의 수립으로 특히 mouse를 중심으로 한 발생학, 유전학 연구의 획기적 발전과 형질변환 동물의 생산 및 동물 체내에서 유전자 기능의 탐구에 매우 큰 변혁을 가져오게 되었다. 또한 ES cell과 embryoid body는 체외 분화능의 연구에 있어 새로운 cytokine의 발견 및 세포 수준에서의 유전자 기능 해석의 강력한 연구수단으로서 폭 넓게 이용되어 질 수 있는 가능성을 시사하고 있다. 이는 ES cell line이 지닌 두 가지 장점, 즉, 유전자 조작의 용이함과, 거의 모든 종류의 성체 구성세포로 분화할 수 있는 성질 때문이다. 이러한 ES cell technology를 실제로 제반 학문과 특히, 인간에게 적용하기 위해서는 반드시 해결해야 할 중요한 문제점이 있다. 첫째로, ES cell을 대상으로 하는 형질변환 방법의 편의성 및 효율개선이 이루어 wu야 하며, 두 번째로 인간의 유전자 및 세포 이식 치료 등을 비롯한 제반 연구에 직접 적용 가능한 ES cell line의 수립과 체외에서 목적으로 하는 분화 세포를 얻기 위한 배양조건이 확립되어져야 한다. 이러한 목표를 달성하기 위해 ES cell의 발생, 분화과정에 있어서의 분자조절기구, 세포 특이적 promotor, 유도 signal등에 대한 연구가 활발히 진행되어져야 할 것이다.

• The Korean Journal of Physiology and Pharmacology
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• v.71 no.2
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• pp.223-233
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• 2020

The embryoid body test (EBT) is a developmental toxicity test method that measures the size of embryoid bodies (EBs) and the viability of mouse embryonic stem cells (mESCs) and fibroblasts (3T3 cells). The previous pre-validation study confirmed the high accuracy (above 80%) of EBT using 26 coded test chemicals. This second-phase validation study assessed the inter-laboratory reproducibility (5 chemicals in common) and predictive capacity (10 chemicals in each laboratory) test using the coded test chemicals at three laboratories. For the prediction model, the accuracy is increased when more data is accumulated. Therefore, we updated the prediction model and analyzed the results of the second year with the newly created-prediction model. Statistical analysis of the inter-laboratory reproducibility test results indicated that accuracy, sensitivity, and specificity were 87%, 78%, and 100%, respectively. The results of the statistical analysis of the predictive capacity test showed an accuracy of 80%, sensitivity of 78%, and specificity of 81%. In conclusion, the EBT can accurately classify various embryotoxicants within a short period and with relatively little effort. Therefore, EBT can be used as a good way to test developmental toxicity.

• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.221-226
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• 2007

Embryonic stem (ES) cells are known to have an infinite proliferation and pluripotency that are associated with complex processes. The objective of this study was to examine expression of genes differentially regulated during differentiation of human ES cells by suppression subtractive hybridization (SSH). Human ES cells were induced to differentiate into neural precursor cells via embryoid body. Neural precursor cells were isolated physically based on morphological criteria. Immunocytochemical analysis showed expression of pax6 in neural precursor cells, confirming that the isolated cells were neural precursor cells. Undifferentiated human ES cells and neural precursor cells were subject to the SSH. TPX2 (Targeting Protein for Xklp2 (Xenopus centrosomal kinesin-like protein 2)) was identified, cloned and analyzed during differentiation of human ES cells into neural lineages. Expression of TPX2 was gradually down-regulated in embryoid bodies and neural precursor cells relative to undifferentiated ES cells. Targeting Protein for Xklp2 has been shown to be involved in cell division by interaction with microtubule development in cancer cells. Taken together, result of this study suggests that TPX2 may be involved in proliferation and differentiation of human ES cells.

• Toxicological Research
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• v.29 no.4
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• pp.221-227
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• 2013

Embryonic stem (ES) cells have potential for use in evaluation of developmental toxicity because they are generated in large numbers and differentiate into three germ layers following formation of embryoid bodies (EBs). In earlier study, embryonic stem cell test (EST) was established for assessment of the embryotoxic potential of compounds. Using EBs indicating the onset of differentiation of mouse ES cells, many toxicologists have refined the developmental toxicity of a variety of compounds. However, due to some limitation of the EST method resulting from species-specific differences between humans and mouse, it is an incomplete approach. In this regard, we examined the effects of several developmental toxic chemicals on formation of EBs using human ES cells. Although human ES cells are fastidious in culture and differentiation, we concluded that the relevancy of our experimental method is more accurate than that of EST using mouse ES cells. These types of studies could extend our understanding of how human ES cells could be used for monitoring developmental toxicity and its relevance in relation to its differentiation progress. In addition, this concept will be used as a model system for screening for developmental toxicity of various chemicals. This article might update new information about the usage of embryonic stem cells in the context of their possible ability in the toxicological fields.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.1-8
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• 2011

Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.

• Korean Journal of Animal Reproduction
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• v.25 no.3
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• pp.219-225
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• 2001

To establish rabbit Embryonic Stem (ES) cells, rabbit one-cell embryos were collected and cultured in vitro to blastocysts. Blastocysts were co-cultured with mouse embryonic fibroblasts (MEF), rabbit embryonic fibroblasts (REF) or 570 cells expressing LIF (SNL). Although rabbit ES cells were isolated with low efficiencies, total 8 ES cell lines were kept in vitro with normal colony shape. The MEF was the best feeder for rabbit ES cell isolation in regard to growth rate and undifferentiated morphology. The doubling time of rabbit ES cells in MEF was about 84 hours and the undifferentiated morphology was maintained following passing and freezing processes. These rabbit ES cells were differentiated into embryoid body following the culture in the uncoated dishes, indicating that they were undifferentiated stem cells.

• Development and Reproduction
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• v.3 no.1
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• pp.45-51
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• 1999

It is crucial to remove trophectoderm (TE) cells of blastocysts for an efficient isolation of pluripotent embryonic stem (ES)-like cells from bovine blastocysts. We evaluated the effectiveness of chemosurgery using calcium ionophore A23l87 (CIPA) by investigating the viability and pluripotency of ES-like cell lines isolated from in vitro-produced bovine blastocysts after CIPA treatment. The blastocysts treated with 50 $\mu$M CIPA for 25 min colonized most efficiently (51% of blastocysts) and developed to ES-like cell lines through 10 passages (4.8% of blastocysts) among CIPA-treated groups with different concentration and duration. In comparison with CIPA-untreated blastocysts, the colonization rate and overall viability of the CIPA-treated blastocysts were five times higher, suggesting that CIPA treatment condition defined in this study was highly efficient for establishing ES-like cell lines without apparent toxicity of CIPA. We evaluated in vitro pluripotency of the established three ES-like cell lines by examining alkaline phosphatase (AP) activity, capability of embryoid body formation, and chromosomal euploidity of the cells. Our cells showed a heterogeneous AP activity similarly to other reports. The cells were able to form simple embryoid bodies during suspension culture and majority of them showed a normal chromosome number of 60, the euploid chromosomal complement of bovine Therefore, our data suggest that CIPA treatment can be safely used for an efficient isolation of ES-like cell lines from bovine blastocysts.

• Journal of Life Science
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• v.33 no.1
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• pp.64-72
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• 2023

Despite several advances in identification of cardiac transcription factors, there are still needs to find new bioactive molecules that promote cardiomyogenesis from stem cells to highly efficient myocardial differentiation. We analyzed Illumina expression microarray data of mouse embryonic stem cells (mESCs)-derived cardiomyocytes. 276 genes were upregulated (≥ 4fold) in mESCs-derived cardiomyocytes compared undifferentiated ESCs. Secreted phosphoprotein 2 (Spp2) is one of candidates and is known to inhibit bone morphogenetic protein 2 (BMP2) signal transduction as a pseudoreceptor for BMP2. However, its function in cardiomyogenesis is unknown. We confirmed that Spp2 expression increased during the differentiation into functional cardiomyocytes using mESCs, TC-1/Kh2 and E14. Interestingly, Spp2 secretion transiently increased 3 days after formation of embryoid bodies (EBs), indicating that the extracellular secretion of Spp2 is involved in the differentiation of ESCs into cardiomyocytes. To characterize Spp2, we performed experiments using the C2C12 mouse myoblast cell line, which has the property of shifting the differentiation pathway from myoblastic to osteoblastic by treatment with BMP2. Similar to the differentiation of ESCs, transcription of Spp2 increased as C2C12 myoblasts differentiated into myotubes. In particular, Spp2 secretion increased dramatically in the early stage of differentiation. Furthermore, treatment with Spp2-Flag recombinant protein promoted the differentiation of C2C12 myoblasts into myotubes. Taken together, we suggest a novel bioactive protein Spp2 that differentiates ESCs into cardiomyocytes. This may be useful for understanding the molecular pathways of cardiomyogenesis and for experimental or clinical promotion of stem cell therapy for ischemic heart diseases.

• Molecules and Cells
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• v.23 no.1
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• pp.49-56
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• 2007

One of the goals of stem cell technology is to control the differentiation of human embryonic stem cells (hESCs), thereby generating large numbers of specific cell types for many applications including cell replacement therapy. Although individual hESC lines resemble each other in expressing pluripotency markers and telomerase activity, it is not clear whether they are equivalent in their developmental potential in vitro. We compared the developmental competence of three hESC lines (HSF6, Miz-hES4, and Miz-hES6). All three generated the three embryonic germ layers, extraembryonic tissues, and primordial germ cells during embryoid body (EB) formation. However, HSF6 and Miz-hES6 readily formed neuroectoderm, whereas Miz-hES4 differentiated preferentially into mesoderm and endoderm. Upon terminal differentiation, HSF6 and Miz-hES6 produced mainly neuronal cells whereas Miz-hES4 mainly formed mesendodermal derivatives, including endothelial cells, leukocyte progenitors, hepatocytes, and pancreatic cells. Our observations suggest that independently-derived hESCs may differ in their developmental potential.

• The Korean Journal of Zoology
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• v.39 no.3
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• pp.239-247
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• 1996

An embryonic stem (ES) cell-based in vitro model system was examined to determine whether a simple differentiation of embryoid bodies (EB) in the suspension medium is useful to dissect early erythropoiesis. Characteristics of the differentiating EBs were monitored for their differentiation potential to generate hematopoietic cell types by general morphology, benzidine staining and two-step colony assays, and expressivity of several erythroid marker genes by the RT-PCR analysis for total cellular RNA prepared from the differentiating EBs. Every ematopoietic lineage cells were generated from the differentiating EBs with reproducible frequencies, similar to the other sophisticated differentiation protocols. Furthermore, the globin gene switching in differentiating ES cells paralleled the sequence of events found in the mouse embryo, and such that their expression was activated by at least 12 hrs later than those of erythroid-specific transcription factors, GATA-1 and Tal-1 The erythropoietic differentiation program initiated reproducibly and efficiently in this simple differentiation system in a suspension culture, such that this system may be useful for dissection of the molecular events of early erythropoiesis.