• Clinical and Experimental Reproductive Medicine
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• 제37권1호
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• pp.13-23
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• 2010

목 적: 본 연구는 인간 배아줄기세포를 생식세포로의 분화를 유도하고 효소에 의해 분리된 단일 배아줄기세포의 배양조건을 확립하기 위해 수행하였다. 연구방법: Embryonic body (EB)는 배아줄기세포 (hESCs) colony를 떼어내어 3일간 hanging drop culture 방법으로 작성하였고, 이러한 EB를 retionic acid (RA)와 bone morphogenetic protein-4 (BMP4)를 단독 또는 함께 배양액에 첨가하여 14일간 배양함으로써 생식세포로의 분화를 유도하였다. 분화를 유도한 EB는 생식세포 발현유전자인 c-kit과 VASA의 표지인자를 이용한 면역조직형광법으로 분화여부를 조사하였다. 줄기세포는 Collagenase, Tryple 그리고 Accutase 등의 효소로 각각 분리하였고 분리된 단일세포들의 colony formation rate를 조사하였다. 한편 Rho-associated kinase inhibitor (Y-27632)를 단일세포 배양액에 첨가하여 단일세포 분리과정 중에 발생하는 apoptotic damage를 감소시키고자 하였다. 결 과: Tryple 또는 Accutase를 이용하여 분리한 단일세포가 Collagenase에 의해 분리된 세포에 비해 높은 colony formation rate를 나타내었다. 단일세포를 $5{\times}10^3$ cells/well (4 well dish) 농도로 지지세포 위에 seeding하였을 때 다른 농도의 세포를 seeding한 것에 비해 높은 colony formation rate를 확보하는데 효과적이었다. Y27632의 첨가는 단일세포의 colony formation rate를 유의하게 향상시켰으며 특히 Tryple로 분리한 단일세포에 보다 효과적이었다. EB의 분화유도후 c-kit과 VASA의 표지인자를 이용한 면역조직형광염색은 대조군인 정소조직에 비해 약한 형광염색을 나타내었다. 결 론: Tryple을 이용한 단일세포 분리가 건강한 단일세포를 얻는데 가장 효율적이었으며 Y27632 의 첨가는 단일 세포의 생존 및 colony formation에 유익하다는 것을 확인하였다. 다른 연구와는 달리 본 연구에서는 단지 생식세포 표지인자의 희미한 형광염색만을 관찰하였는데 이러한 결과는 본 연구의 분화유도기간이 상대적으로 짧았던 것이 원인이었을 것으로 생각된다.

• BMB Reports
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• 제54권10호
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• pp.505-515
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• 2021

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) derived from blastocysts and human induced pluripotent stem cells (hiPSCs) generated from somatic cell reprogramming. Due to their self-renewal ability and pluripotent differentiation potential, hPSCs serve as an excellent experimental platform for human development, disease modeling, drug screening, and cell therapy. Traditionally, hPSCs were considered to form a homogenous population. However, recent advances in single cell technologies revealed a high degree of variability between individual cells within a hPSC population. Different types of heterogeneity can arise by genetic and epigenetic abnormalities associated with long-term in vitro culture and somatic cell reprogramming. These variations initially appear in a rare population of cells. However, some cancer-related variations can confer growth advantages to the affected cells and alter cellular phenotypes, which raises significant concerns in hPSC applications. In contrast, other types of heterogeneity are related to intrinsic features of hPSCs such as asynchronous cell cycle and spatial asymmetry in cell adhesion. A growing body of evidence suggests that hPSCs exploit the intrinsic heterogeneity to produce multiple lineages during differentiation. This idea offers a new concept of pluripotency with single cell heterogeneity as an integral element. Collectively, single cell heterogeneity is Janus-faced in hPSC function and application. Harmful heterogeneity has to be minimized by improving culture conditions and screening methods. However, other heterogeneity that is integral for pluripotency can be utilized to control hPSC proliferation and differentiation.

• Clinical and Experimental Reproductive Medicine
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• 제31권4호
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• pp.261-271
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• 2004

Objective: This study was performed to evaluate the possibility of prolonged culture of human embryonic stem cells (hESC; SNUhES2) on human amniotic fluid cells (hAFC), which had been storaged after karyotyping. Method: The hAFC was prepared for feeder layer in the presence of Chang's medium and STO medium (90% DMEM, 10% FBS) at $37^{circ}C$ in a 5% $CO_2$ in air atmosphere. Prior to use as a feeder layer, hAFC was mitotically inactivated by mitomycin C. The hESCs on hAFC were passaged mechanically every seven days with ES culture medium (80% DMEM/F12, 20% SR, bFGF). Results: The hAFC feeder layer support the growth of undifferentiated state of SNUhES2 for at least 59 passages thus far. SNUhES2 colonies on hAFC feeder appeared slightly angular and flatter shape as compared with circular and thicker colonies observed with STO feeder layer and showed higher level with complete undifferentiation in seven days. Like hESC cultured on STO feeders, SNUhES2 grown on hAFC expressed normal karyotype, positive for alkaline phosphatase activity, high telomerase activity, Oct-4, SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81 and formed embryoid bodies (EBs). Conclusion: The hAFC supports undifferentiated growth of hESC. Therefore, these results may help to provide a clinically practicable method for expansion of hESC for cell therapies.

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.143-151
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• 2010

Pluripotency of human embryonic stem cell (hESC) is one of the most valuable ability of hESCs for applying cell therapy field, but also showing side effect, for example teratoma formation. When transplant multipotent stem cell, such as mesnchymal stem cell (MSC) which retains similar differentiation ability, they do not form teratoma in vivo, but there exist limitation of cellular source supply. Accordingly, differentiation of hESC into MSC will be promising cellular source with strong points of both hESC and MSC line. In this study, we described the derivation of MSC like cell population from feeder free cultured hESC (hESC-MSC) using direct differentiation system. Cells population, hESC-MSC and bone marrow derived MSC (BM-MSC) retained similar characteristics in vitro, such as morphology, MSC specific marker expression and differentiation capacity. At the point of differentiation of both cell populations, differentiation rate was slower in hESC-MSC than BM-MSC. As these reason, to verify differentially expressed molecular condition of both cell population which bring out different differentiation rate, we compare the molecular condition of hESC-MSC and BM-MSC using 2-D proteomic analysis tool. In the proteomic analysis, we identified 49 differentially expressed proteins in hESC-MSC and BM-MSC, and they involved in different biological process such as positive regulation of molecular function, biological process, cellular metabolic process, nitrogen compound metabolic process, macromolecule metabolic process, metabolic process, molecular function, and positive regulation of molecular function and regulation of ubiquitin protein ligase activity during mitotic cell cycle, cellular response to stress, and RNA localization. As the related function of differentially expressed proteins, we sought to these proteins were key regulators which contribute to their differentiation rate, developmental process and cell proliferation. Our results suggest that the expressions of these proteins between the hESC-MSC and BM-MSC, could give to us further evidence for hESC differentiation into the mesenchymal stem cell is associated with a differentiation factor. As the initial step to understand fundamental difference of hESC-MSC and BM-MSC, we sought to investigate different protein expression profile. And the grafting of hESC differentiation into MSC and their comparative proteomic analysis will be positively contribute to cell therapy without cellular source limitation, also with exact background of their molecular condition.

• Molecules and Cells
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• 제43권9호
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• pp.804-812
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• 2020

In cells, proteins form macromolecular complexes to execute their own unique roles in biological processes. Conventional structural biology methods adopt a bottom-up approach starting from defined sets of proteins to investigate the structures and interactions of protein complexes. However, this approach does not reflect the diverse and complex landscape of endogenous molecular architectures. Here, we introduce a top-down approach called Electron Microscopy screening for endogenous Protein ArchitectureS (EMPAS) to investigate the diverse and complex landscape of endogenous macromolecular architectures in an unbiased manner. By applying EMPAS, we discovered a spiral architecture and identified it as AdhE. Furthermore, we performed screening to examine endogenous molecular architectures of human embryonic stem cells (hESCs), mouse brains, cyanobacteria and plant leaves, revealing their diverse repertoires of molecular architectures. This study suggests that EMPAS may serve as a tool to investigate the molecular architectures of endogenous macromolecular proteins.