초록 ▼

연구목표 :
본 연구 계획에서는 줄기세포의 성장, 분화, 분화된 신경세포의 기능 획득, 신경회로형성등에 관한 세포생물학.분자생물학적 이해를 통해 손상된 신경조직을 복구하거나 기억등의 신경 기능을 강화할 수 있는 전략을 확보코자 한다.

연구내용 :
세부연구과제 구성은 제 3, 4세부에서 신경줄기세포의 증식, 분화등에 관한 보다 근본적인 기전연구에, 제5세부에서 신경줄기세포에서 신경 망형성 촉진 기술개발에 치중하며 제1과제에서는 파킨슨병, 제2세부에서는 알쯔하이머병과 같은 특정질환에 집중한다. 각 세부과제 구성은

연구목표 :
본 연구 계획에서는 줄기세포의 성장, 분화, 분화된 신경세포의 기능 획득, 신경회로형성등에 관한 세포생물학.분자생물학적 이해를 통해 손상된 신경조직을 복구하거나 기억등의 신경 기능을 강화할 수 있는 전략을 확보코자 한다.

연구내용 :
세부연구과제 구성은 제 3, 4세부에서 신경줄기세포의 증식, 분화등에 관한 보다 근본적인 기전연구에, 제5세부에서 신경줄기세포에서 신경 망형성 촉진 기술개발에 치중하며 제1과제에서는 파킨슨병, 제2세부에서는 알쯔하이머병과 같은 특정질환에 집중한다. 각 세부과제 구성은 다음과 같다.
제1세부: 중뇌도파민신경세포 분화, 생존, 기능획득기전 이해를 통한 파킨슨병 줄기세포 치료기술 개발
제2세부: 알츠하이머 질환의 줄기세포 이식을 위한 생체 내 상호작용 기전규명
제3세부: Wnt와 Ras-ERK 신호전달계 조절을 통한 신경줄기세포 분화와 증식 제어
제4세부: 인간성체신경줄기세포의 분리 및 분화능 조사 기술 개발
제5세부: 축삭 성장력 강화을 통한 신경줄기세포 신경망형성 촉진기술개발

(출처 : 보고서 요약서 3p)

Abstract ▼

Ⅲ. Contents & Research result
Two stem cell-based strategies for promoting recovery from damaged CNS tissues are (1) cell therapeutic approach using exogenous stem cell transplantation and (2) strategies promoting self-recovery of damaged CNS tissue using endogenous CNS neural stem cells.
To p

Ⅲ. Contents & Research result
Two stem cell-based strategies for promoting recovery from damaged CNS tissues are (1) cell therapeutic approach using exogenous stem cell transplantation and (2) strategies promoting self-recovery of damaged CNS tissue using endogenous CNS neural stem cells.
To provide substantial tips for the CNS recovery, this proposal is designed to perform multidisciplinary approaches for the mechanisms underlying neuronal differentiation of stem cells, survival, functional acquisition, synaptic formation of stem cell-derived neurons, and control of immune response between donor and host cells. Our mechanistic approaches contain cellular, molecular biological, electrophysiological, and epigenetic gene control studies.
In order to achive our final goal, we emphasize strong collaboratory networks among the researchers participating in this grant proposal. To facilitate the collaborations, we will run a core facility in which all the researchers share the experimental systems such as various types of stem cells, antibodies, genetic materials, disease models, and related techniques.

Project 1 :
1. Study for efficient generation of induced pluripotent stem cells(iPSCs)-dervied dopamine neuron
- Analyzing various types iPSCs(-gene transduced by different ways) differentiation toward dopamine neuron
- Analyzing properties for different types iPSCs-derived dopmaine neurons
- Analyzing the behavior of hemi-parkinsonian model and DA neuron viability and properties in the grafts after iPSCs-derived cells transplantation
- iPSCs, we use in our exp, are differentiated to DA neuron.
- Viability of protein-based iPSCs, among 7-8 types of iPSCs, was higher than another types of iPSCs, senescence was not shown in protein-based iPSCs, but another iPSCs were finally undergone apoptosis for doing several passages.
- In hemi-parkinson rat, rotational behavior tests were improved in rat-transplanted protein-based iPSCs comparing non-treated rat.

2. Mechanism study for Nurr1·Foxa2 collaborative roles in midbrain development
- Analyzing induction of A9 dopaminergic neuron after transduction of Foxa2 with Nurr1
- Analyzing changes of chromatin and epigenetic control after Nurr1·Foxa2 transduction
- Foxa2 is required for efficient induction of DA neuron as a epigenetic regulator interacting with Nurr1
- CoREST, repressor of neuronal differentiation, is repelled from Nurr1 interaction by Foxa2 induction in neural precursors, finally enhanced A9 type DA neuronal differentiation, especially TH and DAT promoters.

3. Mechanism study for dopaminergic neuron differentiation by KCl depolarization in ventral midbrain neural precursors(VM-NPCs)
- Defining phenomenon for DA neuron induction by depolarization in VM-NPCs
- Analyzing epigenetic regulation for chromatin of TH by depolarization
- Mature DA neuronal differentiation is enhanced in VM-NPCs by KCl depolarization
- DA neuronal differentiation is caused by the changes of epigenetic regulation, CoREST, HAT etc.

4. Study for enhanced neurogenic potential of neural precursors by secreted molecules from caRAF transduced neural precursors
- Defining various roles of RAF in neural precursors
- Analyzing effects of intrinsic/extrinsic roles in caRAF over-expressed NPCs
- Analyzaing for neurogenic effects of caRAF in adult neural precursor cells
- caRAF overexpression in neural precursors were shown two different regulation of neural lineages, neuronal differentiation and glial differentiation(radial glia), respectably, intrinsically and extrinsically.
- Interestingly, caRAF conditioned media(extrinsically) induced radial glia-like cells, which has neurogenic potential.

5. Study for induction of mature DA neuron by decreasing and delaying exogenous Nurr1 expression
- Defining the proper expression levels and time of Nurrr1 for mature DA neuron
- Analyzing the way for control of Nurr1 expression using Doxycycline inducible expression vector
- Control for expression level and time of Nurr1 is one of critical factor for generating A9 type DA neuron
- Successed to mimic in vitro DA neuronal differenation culture as a midbrain development using Doxycycline inducible Nurr1 expression vector

6. Mechanism study for the interplays of Ngn2, NeuroD and Nurr1 in midbrain development
- Defining the interaction Nurr1 with Ngn2 and/or NeuroD for DA neuron development
- Culturing for mimicking neural stem cell culture as in vivo environment, is likely expressed in Ngn2 earlier than Nurr1 expression in DA neuronal precursors
- Successed to mimic in vitro DA neuronal differentiation culture as a midbrain development for delaying Nurr1 expression than Ngn2 expression
- Time line of Ngn2/Nurr1 expression in midbrain is important to induce a mature DA neuron

(Part2)
1. Study for proliferation and survival of human embryo stem cells(hESCs) and iPSCs by Doxcycline
- Difining increased proliferation and survival of hESCs and iPSCs by Doxycycline and analyzing change expression level
- Prove for increasing pluripotency gene and stable stem cell characteristic
2. Mechanism study for the interplays of Ngn2, NeuroD and Nurr1 in midbrain development
- Analayzing for expression of Nurr1, Ngn2 and NeuroD1 approaching to developmental and locational part
- Analayzing Changes and interaction of transcription and translation step in dopaminergic neuron by siRNA Neurogenin2, NeuroD1 Mechanism study for the interplays of Ngn2, NeuroD and Nurr1 in midbrain development
- Time line of expression of Nurr1, Ngn2 and NeuroD1 approaching to developmental and locational part
- Interaction of transcription and translation step in dopaminergic neuron by siRNA Neurogenin2, NeuroD1
3. Study for enhanced survival of neural stem cells of astrocyte using gene manipulation
- Set-up viral system for astrocyte about gene manipulation
- Analayzing effect of neural stem cell survival by Nurr1, Foxa2 transduced astrocyte
- Develop various promoter for lenti virus
- Established method of neural stem cell co-culture with astrocytes
- Changing of expreesion levels of transcriptional factor by Nurr1/Foxa2
4. Study for increased survival of dopaminergic neurons in PD model using gene manipulation
- Analayzing inhibition of apoptotic pathway of dopaminergic neuron by Nurr1/Foxa2
- Enhanced survival dopaminergic neuron for inhibition of apoptotic pathway of dopaminergic neuron by Nurr1/Foxa2
5. Study for differentiation to functional dopaminergic neuron using stable expression vector of neural stem cell and gene manipulation considering developmental system
- Defining gene expression difference using Lenti / Retro Viral system
- Set-up for control of Nurr1 and Foxa2 expression same as in vivo development
- Defined lenti/ retro viral system for adapted our gene expression pattern
- Set-up Nurr1 and Foxa2 expression condition as same as in vivo development condition
6. Method of direct differentiation to functional dopaminergic neuron from fibroblast
- Defining candidate factors to direct differentiation to fuctional dopaminergic neuron
- Inducing direct differentiation to functional dopaminergic neuron from mouse fibroblast
- Find candidate for related cell proliferation factor in neurogenesis
- We detected Nestin-GFP levels as made various candidate factor retro virus, BCLxL, AKT1, Raf, Wnt1, Dlx2, Otx2
- When BCLxL retro virus express with BAM, cell proliferation increased compare only BAM.
- Co-labeled TH positive neuron with neuron maker as TuJ1, MAP2, HuCD, related dopamine synthesis factors, DAT, VMAT2, and midbrain specific marker as Foxa2, Pitx3, Nurr1. Finally A9 type midbrain marker, Girk2 too.

Project 2 :
1. Clarifying the mechanism of endogenous stem cell activation by BM-derived stem cell factor
2. Developing the AD patient's neurons using the AD-iPSC
3. Examination the AD pathologies in AD patient's-derived neuron
4. Identify the therapeutic mechanism in AD patient's-derived neuron after ASM regulation

Project 3 :
◉ Characterization of CXXC5, a novel protein plays roles both as a Wnt/β-catenin signaling regulator and transcription factor, in the nervous system
- Find out the roles of CXXC5, a regulatory protein known to controlling the Wnt/β-catenin signaling, in the differentiation and proliferation of NSCs.
◉ Elucidation the roles of CXXC5 in the differentiation, proliferation of NSCs and development of nervous system.
- Investigation of the CXXC5 role in the oligodendrocyte differentiation in NSCs using CXXC5 knockout mice system
◉ Elucidation of the Roles of the Ras/ERK signaling on the differentiation and proliferation of NSCs.
- Find out the roles of Ras/ERK signaling in differentiation of NSCs using various factors which are related with this pathway
◉ Development of the methodologies that regulate the NSC differentiation and proliferation by regulating the Ras-ERK and Wnt/β-catenin signallings.
- Establishment the system for controlling differentiation and proliferation of NSCs base on signaling

Project 4 :
- Differentiation potential of adult neural stem cells
- Isolation and differentiation potential of adult neural stem cells
- Traumatic brain injury model
- Heterogeneity of gene expression in neural stem cell
- caRAF CM induced differentiation of neural stem cell
We developed and observed (1) human-derived stem cell differentiation system using chick embryo, (2) characteristics of adult neural stem cells derived from the subcallosal zone and subventricular zone, (3) set up the traumatic brain injury model, (4) the genetic difference of neural stem cells, and (5) the effect of caRAF CM on neural stem cell. We also obtained gene candidates that may modulate characteristics of stem cells s using microarray analysis

Project 5 :
- Differentiation potential of adult neural stem cells.
- Isolation and differentiation potential of adult neural stem cells.
- Traumatic brain injury model.
- Developed and observed differentiation system using chick embryo, characteristics of adult neural stem cells derived from the subcallosal zone and subventricular zone, and traumatici brain injury model. We also obtained gene candidates that may modulate characteristics of stem cells s using microarray analysis.

(출처 : SUMMARY 16p)

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 보고서 요약서 ... 3
• 요 약 문 ... 4
• SUMMARY ... 16
• 목차 ... 22
• Contents ... 23
• 제1장 연구개발과제의 개요 ... 25
• 제1절. 제 1 세부과제 ... 25
• 제2절. 제 2 세부과제 ... 28
• 제 3절. 제 3 세부과제 ... 35
• 제 4절. 제 4 세부과제 ... 39
• 제 5절. 제 5 세부과제 ... 41
• 제2장 국내외 기술개발 현황 ... 42
• 제 1절. 제 1 세부과제 ... 42
• 제 2절. 제 2 세부과제 ... 43
• 제 3절. 제 3 세부과제 ... 45
• 제 4절. 제 4 세부과제 ... 47
• 제 5절. 제 5 세부과제 ... 49
• 제3장 연구개발수행 내용 및 결과 ... 50
• 제 1절. 제 1 세부과제 ... 50
• 제 2절. 제 2 세부과제 ... 71
• 제 3절. 제 3 세부과제 ... 91
• 제 4절. 제 4 세부과제 ... 107
• 제 5절. 제 5 세부과제 ... 120
• 제4장 목표달성도 및 관련분야에의 기여도 ... 127
• 제 1절. 제 1 세부과제 ... 127
• 제 2절. 제 2 세부과제 ... 135
• 제 3절. 제 3 세부과제 ... 138
• 제 4절. 제 4 세부과제 ... 151
• 제 5절. 제 5 세부과제 ... 154
• 제5장 연구개발결과의 활용계획 ... 157
• 제 1절. 제 1 세부과제 ... 157
• 제 2절. 제 2 세부과제 ... 158
• 제 3절. 제 3 세부과제 ... 160
• 제 4절. 제 4 세부과제 ... 161
• 제 5절. 제 5 세부과제 ... 162
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 164
• 제 1절. 제 1 세부과제 ... 164
• 제 2절. 제 2 세부과제 ... 164
• 제 3절. 제 3 세부과제 ... 165
• 제 4절. 제 4 세부과제 ... 165
• 제 5절. 제 5 세부과제 ... 165
• 제7장 연구시설ㆍ장비 현황 ... 166
• 제8장 참고문헌 ... 167
• 끝페이지 ... 175