초록 ▼

인간배아줄기세포 표면분자를 인식하는 57-C11항체가 인식하는 인산화 된 E1B-AP5은 새로운 인간만능줄기세포의 세포표면분자이며 분화 유도시 표면에서 그 발현이 급격히 감소하며, 세포표면에서 GRP78과 complex를 이루고 있다. in vivo에서는 생쥐 정소의 정원줄기세포와 그 niche에서 niche 마커들과 같은 장소에서 발현한다. E1B-AP5 knockdown, chemical inhibitor 처리, 57-C11 처리 등을 통해 E1B-AP5가 인간배아줄기세포의 재생과 전분화능 조절에 젓요한 인자임을 밝혔으며, 구체

인간배아줄기세포 표면분자를 인식하는 57-C11항체가 인식하는 인산화 된 E1B-AP5은 새로운 인간만능줄기세포의 세포표면분자이며 분화 유도시 표면에서 그 발현이 급격히 감소하며, 세포표면에서 GRP78과 complex를 이루고 있다. in vivo에서는 생쥐 정소의 정원줄기세포와 그 niche에서 niche 마커들과 같은 장소에서 발현한다. E1B-AP5 knockdown, chemical inhibitor 처리, 57-C11 처리 등을 통해 E1B-AP5가 인간배아줄기세포의 재생과 전분화능 조절에 젓요한 인자임을 밝혔으며, 구체적으로 E1B-AP5는 세포성장을 촉진하는데 Cyclin E, Cdc25A 단백질 분해 조절로 세포주기의 G1-S transition을 조절하며 knockdown시 외부적 DNA damage 유도 없이도 ATR-ChK1-p53-γH2AX경로를 활성화시켜 인간배아줄기세포 DNA replication 개시에 젓요한 역할을 수행하는 것으로 추정된다. 인간배아줄기세포에서 E1B-AP5의 재생과 전분화능 관련 신호전달 경로 분석에서 E1B-AP5가 MAPK/ERK, PI3K/AKT, GSK-3β신호전달경로를 이용하는 것을 알았으며 이젓에서 MAPK/ERK 경로는 세포표면으로 이동한 E1B-AP5가 이용하는 경로임을 밝혔으며 E1B-AP5 knockdown H9 세포는 teratoma 형성을 저해함을 관찰하였다. 이와 같이 E1B-AP5는 핵내와 표면에서 줄기세포의 재생과 전분화능을 조절하며 만능줄기세포에서만 표면으로 노출되는 새로운 표면마커이다.
(출처: 보고소 요약서 3p)

Abstract ▼

Pluripotent human embryonic stem cells (hESCs) provide appropriate systems for developmental studies and prospective donor cell sources for regenerative medicine. Identification of surface markers specific to hESCs is a prerequisite for studying hESC biology and can be used to generate clinical-leve

Pluripotent human embryonic stem cells (hESCs) provide appropriate systems for developmental studies and prospective donor cell sources for regenerative medicine. Identification of surface markers specific to hESCs is a prerequisite for studying hESC biology and can be used to generate clinical-level donor cell preparations that are free from tumorigenic undifferentiated hESCs. We previously reported the generation of monoclonal antibodies that specifically recognize hESC surface antigens using a decoy immunization strategy. In this study, we show that monoclonal antibody 57-C11 recognizes a phosphorylated form of adenovirus early region 1B-associated protein 5 (E1B-AP5). E1B-AP5 is a nuclear RNA-binding protein, but we report that 57-C11-reactive E1B-AP5 is expressed on the surface of undifferentiated hESCs. In undifferentiated hESCs, 57-C11-reactive E1B-AP5 is localized to SSEA3-, SSEA4-, TRA-1-60-, TRA-1-81-, OCT4-, SOX2-, and NANOG-positive hESCs. In mixtures of undifferentiated hESCs and hESC-derived neurons, 57-C11 exclusively recognizes undifferentiated hESCs but not hESC-derived neuronal cells. Further, the expression of 57-C11-reactive E1B-AP5 decreases upon differentiation. Our results demonstrate that 57-C11-reactive E1B-AP5 is a novel surface molecule that is involved in the undifferentiated state of hESCs. As far as we know, this is the first report demonstrating that heterogeneous nuclear RNA-binding protein is expressed on the surface of undifferentiated hESCs.
In the subsequent studies, we found that 57-C11-reactive E1B-AP5 is expressed on the surface of all human pluripotent stem cells (hPSCs) tested and also on the surface of mouse spermatogonial stem cells and their niches, suggesting that cell surface-expressed E1B-AP5 may be closely associated with hPSCs, some adult stem cells, and their niches. Therefore, we investigated the functional roles of E1B-AP5 in hESCs by siRNA technology, chemical inhibitor and antibody treatments. E1B-AP5 knockdown impaired hESC self-renewal and pluripotency via defective proliferation and preferential differentiation into α-fetoprotein-positive cells. E1B-AP5 knockdown blocked the cell cycle progression into S-phase through degradation of cyclin E and CDC25A. E1B-AP5 interacted with the ATR-interacting protein and single stranded DNA binding protein RPA32 in hESCs, and E1B-AP5 knockdown activated ATR/ChK1-dependent DNA damage checkpoint signaling without any exogenous DNA damage agents, suggesting that E1B-AP5 may play a role in the firing of replication origin during the G1/S transition in hESCs. E1B-AP5 knockdown caused a marked decrease in the levels of activating phosphorylation of ERK and AKT and inhibitory phosphorylation of GSK-3β, indicating that E1B-AP5 regulates hESC self-renewal and pluripotency via the PI3K/AKT, Wnt/GSK-3β/β-catenin, and MAPK/ERK signaling pathways. Chemical inhibitor studies showed that cell surface-expressed E1B-AP5 was under the control of the MAPK/ERK signaling pathways among above three signaling pathways. Treatment of hESCs with 57-C11, a monoclonal antibody specific to cell surface-expressed E1B-AP5, further showed that cell surface-expressed E1B-AP5 regulates hESC self-renewal and pluripotency via the MAPK/ERK signaling pathway. These findings provide for the first time mechanistic insights into how E1B-AP5 regulates hESC self-renewal and pluripotency in the nucleus and cell surface
(출처: Summary 7p)

목차 Contents

• 표지 ... 1제 출 문 ... 2보고서 요약서 ... 3요 약 문 ... 4SUMMARY ... 7CONTENTS ... 8목차 ... 9제1장 연구개발과제의 개요 ... 10제2장 국내외 기술개발 현황 ... 14제3장 연구 개발수행 내용 및 결과 ... 16 1. 실험 방법 ... 16 2. 연구 내용 및 결과 ... 23제4장 목표달성도 및 관련분야에의 기여도 ... 48제5장 연구개발결과의 활용계획 ... 50제6장 연구개발과정에서 수집한 해외과학기술정보 ... 51제7장 연구시설·장비 현황 ... 53제8장 참고문헌 ... 54끝페이지 ... 55