초록 ▼

본 연구는 인간전분화능줄기세포-유래 신경전구세포와 중간엽줄기세포의 분비단백체에 의해 손상된 신경조직 재생을 유도하는 기전을 규명하고, 그 과정에 관여하는 조직재생 활성인자를 단백체 분석을 통해 발굴하고자 하며 세부 목표는 다음과 같이 설정하였음
■ 전분화능 줄기세포 유래 신경전구세포의 분비단백체 (secretome) 데이터 생산
■ 분비단백체의 신경조직 재생 관련 인자의 발굴 및 조직재생 기전 규명
■ 조직 재생인자에 의한 신경조직재생 제어기술 개발

개발한 연구 결과는 다음과 같음
■ 전분화능 줄기

본 연구는 인간전분화능줄기세포-유래 신경전구세포와 중간엽줄기세포의 분비단백체에 의해 손상된 신경조직 재생을 유도하는 기전을 규명하고, 그 과정에 관여하는 조직재생 활성인자를 단백체 분석을 통해 발굴하고자 하며 세부 목표는 다음과 같이 설정하였음
■ 전분화능 줄기세포 유래 신경전구세포의 분비단백체 (secretome) 데이터 생산
■ 분비단백체의 신경조직 재생 관련 인자의 발굴 및 조직재생 기전 규명
■ 조직 재생인자에 의한 신경조직재생 제어기술 개발

개발한 연구 결과는 다음과 같음
■ 전분화능 줄기세포 유래 신경전구세포의 분비단백체 (secretome) 데이터 생산
- 전분화능 줄기세포-유래 신경전구세포로 부터 분비단백체의 수획을 위한 최적화된 배양 조건 확립하였음
- 뇌졸중 동물모델을 구축하고 질환의 치료효과를 분석하기 위한 행동개선 기준과 조직학적 분자생물학적 마커를 설정하였음
- 전분화능 줄기세포 유래 신경전구세포와 중간엽 줄기세포의 신경조직 재생 효과 비교 분석함
- 신경전구세포의 경우 이식 초기 paracrine effect에 의한 염증 제어 및 신경보호가, 장기적인 측면에서는 이식세포의 생존과 신경세포 분화에 의한 신경조직의 재생이 치료 효과의 주요한 기전으로 확인됨
- 중간엽 줄기세포는 초기에 강력한 paracrine effect에 의한 신경보호가 치료효과의 주요 기전으로 확인 되었으나, 장기적인 세포의 생존이나 신경세포로의 분화 등의 효과를 확인 할 수 없었음
- 이러한 연구를 통해 전분화능줄기세포-유래 신경전구세포 및 중간엽 줄기세포의 치료 효과에서 paracrine factor들이 초기 염증제어와 신경세포보호에 작용하여 신경조직 보호와 재생을 유도하는 주요 기전임을 확인함
■ 분비단백체의 신경조직 재생 관련 인자의 발굴 및 조직재생 기전 규명
- 뇌졸중 치료효과를 상승시키기 위한 방안으로 중간엽줄기세포에 허혈성 뇌손상유도에서 추출한 뇌추출액의 처리를 통해 중간엽 줄기세포의 분비단백체 중 미세소포체 대량 수획을 개선하였으며, 이 미세소포체의 주입이 동물모델에서 뇌졸중 치료효과가 있는 지를 분석한 결과 분비단백체 주입이 세포이식과 유사한 양상의 치료 효율과 기전을 발휘함을 확인하였음
- 뇌추출액을 처리한 중간엽줄기세포와 대조군 중간엽줄기세포의 미세소포체의 단백체를 비교 분석하여, 신경조직재생, 항염증, 혈관신생, 신경재생 등과 관련된 단백질 성분의 동정 및 신경재생 연관성을 분석함
- 신경전구세포 분비단백체를 배아줄기세포-유래 신경전구세포와 역분화줄기세포-유래 신경전구세포에서 단백체 분석을 진행하였으며, 분비단백체의 동물 뇌졸중 치료 효과 분석을 완료 한 결과 전분화능줄기세포의 이식과 유사한 양상의 뇌졸중 치료 효과를 발휘함을 확인함
- 단백체로부터 신경 조직 재생 유효인자 발굴 및 동정을 진행하고 있음

이들 연구성과를 기반으로 하여 2단계에서는 다음의 연구를 진행하고자 함
■ 조직 재생인자에 의한 신경조직재생 제어기술 개발
- 신경전구세포 분비단백체-특이적 유효 성분을 발굴하고 이들에 의한 신경세포 증식, 분화 유도, 혈관 신생 및 세포 사멸 억제 기능성을 동물 및 시험관 모델에서 검증
- 허혈성 뇌졸중 모델에서의 유효 성분 단백질 투여 후 조직재생의 기전 확인
- 투여 경로, 투여 수 및 함량 조절에 의한 신경재생 검증 및 기전 연구
- 세포치료 및 secretome을 이용한 비세포 치료의 융합치료 효과 검증 및 기전 연구

최종 연구 성과는 뇌졸중을 포함하는 퇴행성 뇌질환의 치료에 활용할 수 있는 비세포성치료제이며, 이 치료제는 타가/동종세포 이식에 따르는 조직이식 거부, 줄기세포 이식에 의한 종양형성의 위험성 등 부작용이 없는 새로운 개념의 안전한 치료기술로 다양한 퇴행성 및 난치성 질환에 대한 효과적인 재생 치료제의 적용이 가능한 고부가 가치의 새로운 줄기세포 기반의 치료제로 개발이 가능할 것임

(출처 : 보고서 요약서 3p)

Abstract ▼

Ⅳ. Results and Discussion

1. Establishement of rat ishcemic storke model with permanent middle cerebral artery occlusion
- Male Sprague-Dawley rats weighing 250-300 g were anesthetized with 3% isoflurane in a 70-30% mixture of N2O to O2. The left common carotid artery and external carotid

Ⅳ. Results and Discussion

1. Establishement of rat ishcemic storke model with permanent middle cerebral artery occlusion
- Male Sprague-Dawley rats weighing 250-300 g were anesthetized with 3% isoflurane in a 70-30% mixture of N2O to O2. The left common carotid artery and external carotid artery were isolated and ligated with a 4-0 surgical suture. A nylon thread was inserted into the left internal carotid artery and advanced to the Circle of Willis. The thread was left in place until the rats were killed.
- Rats with pMCAo showed severe neurologic deficits after 24 h without significant differences between experimental groups with brain ischemia. Sham-operated control surgery was performed in an identical manner without insertion of nylon thread.
- Behaviroal deficits were determined by composite analysis of elevated body swing, beam balance, and prehensile traction.

2. Establishment of optimal culture condition for the collection of NPC secretome
- Expanded human ES and iPS cell-derived neural rosette cells at 80-90 % confluence were subjected to the magnetic activated cell sorting (MACS) procedure for PSA-NCAM-positive neural precursor cells.
- Isolated PSA-NCAM NPC were cultured in N2B27 medium or 1× N2, 0.5× B27, and 0.5× G21 supplement plus 20 ng/ml of bFGF and culture medium was changed every day and the cells were passaged every 2-3 days.
- Serum-free conditioned medium for later proteomic and functional studies was collected from NPCs at 90% confluency.
- For conditioned medium collection DMEM-LG supplemented with bFGF and ITS without antibiotics was the chosen medium.

3. Neural transdifferentiation of human MSCs on hydrophobic polymer-modified surface and therapeutic effects in an animal model of ischemic stroke
- MSCs were induced into neurogenesis using a modified protocol for efficient neural transdifferentiation.
- The therapeutic potency of the resulting neural progenitor cells in a rat model of ischemic stroke was analyzed.
- Using a highly hydrophobic diphenylamino-s-triazine-bridged p-phenylene (DTOPV)-coated surface and adopting a procedure for propagation of neural stem cells, MSCs were efficiently converted into neurosphere-like cellular aggregates (NS-MSCs).
- The spherical cells were subsequently induced to differentiate into neural cells expressing neuroectodermal markers.
- To determine whether these cells had neuronal fates and induced neuro-protective effects in vivo, NS-MSCs were intra-cerebrally administered to rats 48 h after permanent middle cerebral artery occlusion (pMCAo).
- The results showed a remarkable attenuation of ischemic damage with significant functional recovery, although the cells were not fully incorporated into the damaged tissues on post-operative day 26.
- Improvement in the NS-MSC-transplanted rats was faster than in the MSC group and suppression of inflammation was likely the key factor.
- NS-MSCs are an ideal source of therapeutic cells for replenishing/replacing large numbers of different neural cell types that have been damaged or destroyed in the brain.
- Thus, our culture system using the hydrophobic surface of a biocompatible DTOPV coating efficiently supported neural cell differentiation from MSCs. Neural-primed MSCs exhibited stronger therapeutic effects than MSCs in rat brains with pMCAo

4. Comparative analysis of therapeutic effectiveness of neural precursor cells and mesenhcymal stem cells in rat ischemic stroke model
- Therapeutic potential of polysialic acid-neural cell adhesion molecule (PSANCAM)-positive neural precursor cells (PSA-NCAM NPC) derived from human embryonic stem cells (hESCs) in a rat stroke model with permanent middle cerebral artery occlusion.
- Data showed that rats transplanted with PSA-NCAM NPC are superior to those treated with phosphate buffered saline (PBS) or mesenchymal stem cells (MSCs) in behavioral performance throughout time points.
- From day 3 post-transplantation, PSA-NCAM NPC and MSC-transplanted groups started to show behavior improvement and neuroprotection.
- PSA-NCAM NPC evidently lowered the positivity of microglial ED-1 and astrocytic GFAP and elevated α-SMA immunoreactivity and the expression of angiopoietin-1, suggesting a suppression of adverse glial activation in the host brain and angiogenic stimulation in the host brain, respectively.
- Unlike MSCs, PSA-NCAM NPC demonstrated a potent immunoreactivity against human specific nuclei, doublecortin, and Tuj1 at day 26 post-transplantation, implying their survival, differentiation, and integration in the host brain.
- Transplanted NPCPSA-NCAM preserve brain tissue with reduced infarct size and improve behavioral performance through actions encompassing anti-reactive glial activation and pro-angiogenic activity in a rat stroke model.
- Transplantation of PSA-NCAM NPC or MSCs showed substantial improvement in neurological function after 3 days of transplantation suggesting that the early beneficial effects of cell transplantation is induced by paracrine mechanisms rather than cell replacement.

5. Mesenchymal stem cell-derived microvesicles treated with ischemic brain extract reduce ischemic stroke damage in rats
- The quantity of MV from conditioned medium of MSCs represented by protein contents was greatly increased by the treatment of stroke brain extracts (>50 mg/8 x 10⁷cells/48hr for SB-MSC-MV). The stimuli derived from stroke brain may play critical roles in enhancing the neuroprotective effects of MSCs by altering the gene and protein expression profiles of MSCs upon in vitro preconditioning and these changes might affect the molecular profiles of MSCs and MSC-derived MV.
- Intracarotid administration of SB-MSC-MVs ameliorated ischemic brain injury by reducing inflammation and modulating endogenous neurogenesis, resulting in functional improvements that mimic the therapeutic effect of MSCs at an early stage of pMACo.
- The treatment of MSCs with stroke brain extracts induced a number of crucial genes related to the neurogenesis, neuroprotection and anti-inflammation, such as NGF, BDNF, angiopoietin-1, FGF-2, TGF-b and TSG-6.
- The datasets of MSC-MV Proteome of SB-MSC-MV (MV isolated from stroke brain extract-treated MSC) and NB-MSC-MV (MV isolated from normal brain extract-treated MSC) are highly enriched for vesicular proteins, validating the therapeutic effects of the MV-derived proteome.
- The MV components of MSCs result in most, if not all, of the benefits of cell transplantation, while minimizing potential adverse events associated with cell transplantation.
- The number of high level of DCX-expressing cells was significantly elevated compared to that of PBS-control and they were only detectable in the lateral ventricles and hippocampal regions of stroke-damaged hemispheres at day 7, but not in the cerebral cortex or other brain regions. In addition, MV greatly attenuated the activation of astrocytes in the ischemic region.
- Network of SB-MSC-MV therapeutic factors for anti-inflammation, angiogenesis, neurogenesis and apoptosis was reconstructed that might be a target proteome system stimulated by brain extract using systems biology approaches.

6. Secretome datasets from NPCs and analysis of their therapeutic effectiveness in rat ischemic stroke model
- Culture conditions was established for conditioned medium collection from human ES and iPS cell-dervied NPCs without altering their viability and functioanal integrity.
- To determine the protein composition of NPC’s secretome, conditioned medium from NPC culture prepared from three independent isolations were subject to proteomics analysis, a combination of 1D SDS-PAGE, in-gel digestion with trypsin, and LC-MS/MS analysis
- Beginning 3 days after treatment, the NPC secretome-treated group exhibited significant functional enhancement for most of the parameters in rat model of ischemic stroke
- Functional enrichment analysis showed that cellular processes represented by the NPC secretome proteins include inflammation, cytokines, ECM, neurogenesis.

(출처 : SUMMARY 13p)

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 보고서 요약서 ... 3
• 요 약 문 ... 5
• SUMMARY ... 11
• CONTENTS ... 19
• 목차 ... 20
• 제1장 연구개발과제의 개요 ... 21
• 1. 연구개발의 필요성 ... 21
• 2. 경제적·산업적 중요성 ... 23
• 제2장 국내외 기술개발 현황 ... 25
• 제3장 연구개발수행 내용 및 결과 ... 31
• 제4장 목표달성도 및 관련분야에의 기여도 ... 50
• 제5장 연구개발결과의 활용계획 ... 52
• 1. 실용화 연구를 위한 추가 연구 방향 ... 52
• 2. 활용계획 ... 52
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 54
• 제7장 연구시설·장비 현황 ... 57
• 제8장 참고문헌 ... 58
• 끝페이지 ... 61