초록 ▼

1. 줄기세포 Niche 최적화
● 골수 내의 줄기세포 Niche 연구;
PPAR-δ 효현제 및 KO mice를 이용하여 PPAR-δ 가 골수내 줄기세포에 미치는 영향을 연구하고, 이를 통한 조혈모세포의 풀 (pool) 증가와 말초 동원 조건 개발.
● 질환장기 내의 줄기세포 Niche 연구;
심근경색 동물모델의 질환부위에 줄기세포 Niche 형성 세포, 형성 물질 분석. 줄기세포 Niche 형성 중간엽 세포 표지자 중 Niche 형성에 필수적인 표지자 확인으로 줄기세포와 질환장기 Niche의 관계 규명. 암의

1. 줄기세포 Niche 최적화
● 골수 내의 줄기세포 Niche 연구;
PPAR-δ 효현제 및 KO mice를 이용하여 PPAR-δ 가 골수내 줄기세포에 미치는 영향을 연구하고, 이를 통한 조혈모세포의 풀 (pool) 증가와 말초 동원 조건 개발.
● 질환장기 내의 줄기세포 Niche 연구;
심근경색 동물모델의 질환부위에 줄기세포 Niche 형성 세포, 형성 물질 분석. 줄기세포 Niche 형성 중간엽 세포 표지자 중 Niche 형성에 필수적인 표지자 확인으로 줄기세포와 질환장기 Niche의 관계 규명. 암의 Niche 연구를 통해 발암 및 전이 조절법 개발
● 인간 체외 줄기세포 Niche 연구;
자가 말초혈액을 이용한 3차원 혈구세포괴(Hematosphere)의 배양 및 심장줄기세포를 이용한 “인간 줄기세포의 체외구현 Niche” 개발, 이를 통한 생물학적 특성규명으로 줄기세포 조절 기법 연구.
● 유전체 프로파일링에 기반한 Niche 최적화 기술 개발
줄기세포 Niche의 형성과 조절에 관여하는 핵심 인자 발굴을 위해 관련 전사조절인자 및 후성유전인자와의 상관관계를 Niche의 변화에 따른 세포의 히스톤 변형 마커 부위 및 small RNA 발현 연구로 밝히고, 선별된 인자를 Niche 최적화 기술 개발에 응용함.

2. 줄기세포의 생체 이식 최적화
● 새로운 줄기세포 증폭, 줄기세포 전달 기법 개발을 통한 전임상 기반 연구
세포 특이 무혈청 배양법 개발, 세포 맞춤형-세포시트 배양법 및 3차원 세포융합체 개발
미세환경조절을 위한 인공 세포조직체 개발
● 중·대 동물모델을 이용한 고효율 줄기세포 이식기술 개발
줄기세포 이식기술 최적화를 위한 중•대 동물 심근경색 모델 및 뇌졸중 모델 작성
3차원 미세소관 이식체를 통한 생체 이식 기술 최적화 기법 개발

(출처 : 보고서 요약서 3p)

Abstract ▼

<1세부과제>
I. Title
Development of Convergent Stem Cell Therapy by Regulation of Stem Cell Niche

II. SUMMARY
The research on embryonic or adult stem cells had become an important topic in the field of biological science. Despite that, the study on the Stem Cell Niche is remained at the

<1세부과제>
I. Title
Development of Convergent Stem Cell Therapy by Regulation of Stem Cell Niche

II. SUMMARY
The research on embryonic or adult stem cells had become an important topic in the field of biological science. Despite that, the study on the Stem Cell Niche is remained at the basic level globally. Hence, it is important to understand its’ Niche which is the optimal environment condition that stem cells reside. Consequently, a study on how to manipulate this stem cell environment is essential and most important for practical use of stem cells in field of regenerative medicine. Although there had been many attempts to use bone marrow-derived endothelial progenitor cells (EPC) as source of cell therapy for the treatment of ischemic diseases, it received negative acclaim and met its limitation due to low amounts of EPCs present in vivo. To overcome this hurdle, there have been many efforts to improve number of EPCs or their function by modifying the genome or using small molecules. However, a genetic modification is not appropriate method for clinical use because of a safety issues including carcinogenicity. Therefore, a new type of research approach is needed in order to ameliorate the number of heart-derived stem cells and EPCs by investigating their niche, which is an environment these stem cells reside and developing a method to manipulate such environments. Traditionally, a concept of stem cell niche was confined to just a bone-marrow. However, since a discovery that other tissues instead of bone-marrow were able to form niche, this research field had received lots of interests. By studying the micro-environment that stem cells reside, our group would like to build a scientific knowledge by investigating the life of stem cells and develop a technology to overcome clinical limitations of cell therapy by directly modulating stem cell niche based on the observation of niche environment and finally trailblazing the field of regenerative medicine by practically applying stem cells in clinic.

<2세부과제>
Ⅰ. Title
Development of optimized techniques to control stem cell niche via genome profiling

Ⅱ. Purpose
Stem cells with self-renewal properties have a potential to differentiate into various types of cells. To therapeutically apply stem cells in a clinical field, it is necessary to develop an experimental technique and optimize the culture condition in order for stem cells to have pluripotency after many repeated cycles of subculture without gradual differentiation to a specific cell type . To accomplish this purpose, here we propose a novel method to optimize the stem cell niche-specific transcriptomic regulation via fine tuning of key genes' expressional regulation by genome-wide profiling. In this study, we will focus on the technical development of fine-tuning of stem cell niche via identification and functional annotation of the whole genome in stem cells and stem cell niche cells. Small RNA, histone modification, and chromatin conformation related with genome activity will be applied to technical improvement of niche-specific gene expression of stem cells.

Ⅲ. Contents
Stem cells are characterized by two properties which are keeping stemness and differentiating into a diverse spectrum of specialized cell types. They can be divided into two primary groups, embryonic stem cells and somatic (adult) stem cells. Unlike embryonic stem cells, adult stem cells are originated from some fully differentiated tissues such as brain, bone marrow, peripheral blood, skin, liver, and etc. and have less differentiation potential than embryonic stem cells. The environmental differences of two distinctive types of stem cells in aspect by stem cell niche are investigated by (1) identification and functional annotation of all small RNAs existing in the whole genome (2) changes of chromatin environment by genome-wide epigenetic modification profiling at histones H3K4 and H3K27, (3) interpretation and comparison of activation of transcription factors, (4) identification and functional assessment of cis-acting elements related with gene regulation, and (5) the differences in gene regulation due to changes of chromatin conformation. Cell type-specific genome network derived after completing above criteria will be applied to develop a technique in order to control stem cell niche-specific gene regulation and to obtain huge amount of stem cells with high quality of stemness through investigation and quantification of the importance of various mechanisms studied in this proposal.

Ⅳ. Results
In this study, we investigated mechanisms that regulate transcriptome of stem cells influenced by stem cell niche using 4 different niche conditions. First, we identified small RNAs that are differentially expressed in mouse embryonic fibroblast (MEF) cells compared to four other different niche. Then we analyzed transcriptomes of mouse embryonic stem cells (ESCs) which were cultured in 4 different niches. These niches were designed to examine effects of cytokine LIF or aging of MEF cell, or both. Results showed that LIF effect was related to regulation of pluripotency genes expression, and ageing effect was related to PRC2. ESCs that were cultured in the old MEF niche decreased expressions of PRC2 components, such as Suz12, Eed, and Ezh2. Also, PRC2 components’ target genes were up-regulated in ESCs cultured in the old MEF niche. The correlation analysis of transcriptome and histone ChIP-seq showed that genes containing H3K27me3 and bivalent modifications (both H3K4me3 & H3K27me3) were up-regulated. These results suggest that de-repression of these genes occurred in ESCs cultured from old MEF niche condition. Also, these genes were associated with general differentiation and development. At the same time, in order to study three dimensional chromatin topology and interactions in genome-wide, we tried to perform ChIA-PET (Chromatin interaction Analysis using Paired-End Tag Sequencing). This study can lead to development of a technique to understand the relationship between chromatin topology and gene regulation, and the mechanism to regulate the stemness of embryonic stem cells according to their niche.

Ⅴ. Expected contribution
The new experimental technique to manipulate and obtain the huge amount of stem cells with high quality of stemness is essential to be applied in a clinical field. A specific transcriptome regulation method of stem cell niche obtained from this study will be useful to develop the technique of stem cell production for clinical use. The current research progress in the field of specific cell-lineage differentiation is behind that of the other leading countries. Our specific technique to regulate stem cell niche-specific gene expression can contribute to higher efficiency of cell differentiation into desired cell type. Although many other groups in this field are also developing a method to control stemness and differentiation of stem cell by identifying a specific niche, this technical limitation can be overcome by examining the genome-wide epigenetic modification based on our group’s previous research experiences and advanced technology source.

<3세부과제>
The purpose of this study is to develop the novel and useful clinical-grade stem cell culture technologies using "novel triggers or modulators of stem cell expansion and differentiation", that is currently developed by other teams affiliated with same research cluster as ours. Our goal is to biologically intervene Niche environment for optimization study made by other our teams (part I-II) for "development of stem cell therapy technologies". To achieve this, we developed three novel stem cell culture systems; 1) Local immunity modulation by hybrid mesenchymal stem cell sphere, 2) Development of optimized on-phage to cell interaction by tissue engineering, 3) Identification of the specific regulator of stem cell niche in hematopoietic stem cell through functional analysis. By using hindlimb ischemia, myocardiac infarction and animal models of stroke, we demonstrate possibilities of vascular repair by transplantation of our functionally up-graded stem cells.

<4세부과제>
Ⅰ. Title
Optimization of stem cell transplantation with endogenous pathophysiological factors

Ⅱ. Purpose
To maximize the therapeutic efficacy of stem cell exposed to diseased niche, pathophysiological factors need to be modulated in order to augment the stem cell function. Stem cell therapy will be studied in the animal model to optimize the protocol for the safe and efficient therapy.

Ⅲ. Contents
1. Characterization of stem cells exposed to niche from a diseased environment
• Establishment of disease model with diabetes/metabolic syndrome/aging
• Establishment of cardiac function assessments of diseased animal models
• Characterization of endogenous stem cells derived from diseased animal models
• Selection of small molecules to restore or enhance stem cell function
2. Identification of the diseased niche factors
• Comparative study of gene expression of stem cells exposed to risk factors
• Mechanism study of the actions of responsible genes and their targets
3. Development of technology to regulate diseased niche environment
• Developing a technology to control diseased niche environments by using selected genes and small molecules.
• Study of functional recovery of endogenous stem cells in diseased animal models
4. Study of therapeutic effect of recovered stem cells
• Investigation of therapeutic effect of functionally recovered stem cells by regulating factors
• Evaluation of efficacy and long-term safety of recoevered stem cells.

Ⅳ. Results
1. From the study, we revealed that mesenchymal stem cells exposed to diseased niche lost their therapeutic effect on cardiac injury due to decrease in proliferation and angiogenesis. We identified that Kruppel-like factor 2 (KLF2), stem cell factor (SCF), miR-132, and miR-34c were responsible factors that induced stem cell dysfunction., We also developed methodologies for functional restoration of dysfunctional stem cells.
2. We identified the regulating mediators of mesenchymal stem cells on pro-inflammatory macrophages.

Ⅴ. Expected Contribution
1. This study is applicable to real clinical situations because most of cardiovascular patients carry various risk factors. We found that pathophysiological factors were responsible for the dysfunction of endogenous mesenchymal stem cells. We also developed the core technologies to restore therapeutic function of attenuated mesenchymal stem cells. We have plans to industrialize our productive technologies in nearby future.
2. The fact that immune tolerance of stem cells remains to be unveiled, we identified critical factors of transplanted mesenchymal stem cells to modulate macrophage phenotypes. Based on our data, this technologic development can contribute to the industrialization of therapeutic modalities on chronic diseases and aging in addition to cardiovascular diseases.

<5세부과제>
Niches represent specialized microenvironments where the quiescent and activated state of resident stem cells is regulated. In this research project, here we propose to study a new niche controlling factor using the mouse genetic systems in the cardiac development. Initially, we identify a new niche factor, LPHN2, in the development of heart stem cells and characterize its functions on the development of heart stem cells. We found that knockout of LPHN2 during the cardiac development lead to molecular fate changes of heart stem cells which finally resulted in abnormal heart differentiation, suggesting that a novel niche factor LPHN2 can control microenvironments of stem cells and opens a new solution for the cell replacement therapy. Thus, these studies provide important information for the optimized stem cell transplantation and the contribution to niche based clinical applications.

(출처 : SUMMARY 9~13p)

목차 Contents

• 표지 ... 1제 출 문 ... 2보고서 요약서 ... 3요 약 문 ... 4SUMMARY ... 9CONTENTS ... 13목차 ... 14제1장 연구개발과제의 개요 ... 16 제1절 연구개발 대상 기술의 경제적·산업적 중요성 및 연구개발의필요성 ... 16 제2절 줄기세포 Niche 연구개발의 필요성 ... 18제2장 국내외 기술개발 현황 ... 20 제1절 국외 기술개발 현황 ... 20 제2절 국내 기술개발 현황 ... 24제3장 연구개발수행 내용 및 결과 ... 32 줄기세포 Niche 조절을 통한 줄기세포치료 융합기술개발 : 1세부 ... 32 유전체 프로파일링에 기반한 줄기세포 niche 제어 최적화기술개발 : 2세부 ... 79 새로운 줄기세포 증폭, 분화 배양법 개발 및 전임상 기반연구 : 3세부 ... 101 체내 유래 pathophysiological factor를 활용한 줄기세포 이식의 최적화 : 4세부 ... 119 유전자 조작 마우스 실험체계를 이용한, 줄기세포 niche 조절 인자의 역할 규명 : 5세부 ... 139제4장 목표달성도 및 관련분야에의 기여도 ... 150 1세부과제 ... 150 2세부과제 ... 152 3세부과제 ... 153 4세부과제 ... 154 5세부과제 ... 1555장. 각 세부과제 간 유기적 공동연구를 통한 목표 결과 창출 ... 155제6장 연구개발결과의 활용계획 ... 166제7장 연구개발과정에서 수집한 해외과학기술정보 ... 175제8장 연구시설·장비 현황 ... 177제9장 참고문헌 ... 181끝페이지 ... 184