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Mesenchymal Stem Cell-derived Therapeutic Nanovesicles for Treatment of Central Nervous System Injuries
중간엽 줄기세포 유래 치료용 나노베지클을 이용한 중추신경계 손상 치료

DC Field Value Language
dc.contributor.advisor김병수-
dc.contributor.author김한영-
dc.date.accessioned2019-10-21T02:35:20Z-
dc.date.available2020-10-06T05:03:33Z-
dc.date.issued2019-08-
dc.identifier.other000000157482-
dc.identifier.urihttp://hdl.handle.net/10371/162048-
dc.identifier.urihttp://dcollection.snu.ac.kr/common/orgView/000000157482ko_KR
dc.description학위논문(박사)--서울대학교 대학원 :공과대학 화학생물공학부,2019. 8. 김병수.-
dc.description.abstract뇌졸중, 뇌출혈, 외상성 뇌 손상, 그리고 척수 손상과 같은 중추신경계 손상 질환은 장기적인 신체 장애, 더 나아가 죽음까지 이르게 하는 원인이다. 새로운 조직 재생 치료 전략으로서, 중간엽줄기세포 유래 엑소좀을 이용한 중추신경계 손상 치료는 최근 각광을 받고 있다. 중간엽줄기세포 유래 엑소좀은 그 만의 독특한 성질 때문에 중추 신경계 손상을 비롯한 다양한 질병에서 활발히 연구되어 왔다. 그러나 중추신경계 손상 치료를 위해 엑소좀을 사용하는 방식에는 여전히 한계가 남아 있다. 극소량의 엑소좀만이 중간엽줄기세포에서 분비되며, 엑소좀을 전신 주사 (정맥 주사) 하여도 표적 조직에 잘 도달하지 않는다. 따라서 본 주제에서는 산화철 나노입자를 중간엽줄기세포에 처리하였고 해당 세포로부터 엑소좀-모방 나노베지클을 분리하였다. 산화철 나노입자는 중간엽 줄기세포 내에서 철 이온으로 이온화 되며, 해당 이온들은 세포의 JNK/c-JUN 세포 신호전달 체계를 자극하여 다양한 성장 인자의 발현을 촉진시킨다. 따라서 해당 세포로부터 분리한 나노베지클은 자성을 띄는 산화철 나노입자 뿐만 아니라 고용량의 성장 인자가 탑재되어 있다. 본 연구에서는, 산화철 나노입자가 탑재된 나노베지클을 중추 신경계 병변으로 체내 전달하였고 그에 따른 치료효과를 제시한다.
먼저, 제 3장에서는 철나노입자가 처리된 중간엽줄기세포에서 철나노입자가 탑재된 철-나노베지클을 분리하였고, 이를 마우스 척수손상 모델에 전신 주사하여 외부 자장의 도움으로 손상된 척수에 전달되었다. 철나노입자가 처리된 중간엽줄기세포는 세포 자체의 큰 직경 때문에 대부분 폐에 걸렸지만, 나노미터 크기의 철-나노베지클은 폐 모세혈관을 통과하여 자기장이 있는 상태에서 손상된 척수에 상당 부분 축적되었다. 척수 병변에 축적 된 철-나노베시클은 혈관 재건에 기여하였고, 염증성 대식세포를 항염증성 아형으로 분화시켰으며, 성상교세포증을 감쇠하고 신경 세포 사멸을 억제 하였다. 결과적으로 동물 행동 검사에 의해 평가 된 바와 같이, 손상된 척수에 축적된 철-나노베지클은 치료 효과를 발휘하였고 척수 기능을 개선시켰다.
제 4 장에서는 중간엽줄기세포에서 유래하였고 철나노입자가 함유 된 자성 철-나노베지클을 랫드 허혈성 뇌졸중 모델에 정맥 주사 하였다. 동물 모델은 과도 중간 대뇌 동맥 폐색 수술을 통해 허혈성 뇌졸중을 유발했다. 세포 실험을 통해, 철-나노베지클은 대조군 나노베지클과 비교하여 다양한 유형의 세포에서 증진된 혈관 생성 효과, 세포 사멸 억제 효과, 그리고 항염증 효과를 나타내었다. 동물 실험에서는, 철-나노베지클의 정맥 주사 직후에 외부 자기장으로 작용하는 자석 헬멧을 동물 모델에 씌웠다. 그리고 동물 형광 촬영을 통해 좌뇌 반구 (뇌졸중 병변)에 철-나노베지클이 축적됨을 관찰하였다. 주사 후 3 일째에 경색 부위 크기를 정량화 한 결과, 외부 자기장 존재 아래 철-나노베지클을 투여 한 실험군에서 경색 부위가 대조군에 비하여 현저하게 감소되었다. 또한, 동물 행동 실험을 통하여 철-나노베지클 (자기장 O) 의 주입 및 치료가 동물 모델의 운동 기능을 향상 시킨다는 것을 입증하였다.
따라서 본 연구에서는 산화철 나노입자가 처리된 중간엽줄기세포로부터 분리한 나노베지클은 고용량의 성장 인자뿐만 자석 항법 도구로 쓰일 수 있는 산화철 나노입자를 함유하며, 외부 자기장을 통해 손상된 척수 또는 대뇌 병변에 표적 전달되는 것을 확인했다. 이러한 철-나노베지클은 중추신경계 손상 치료를 위한 우수한 치료제로서 활용될 수 있으며, 통상적인 중간엽줄기세포 기반 또는 중간엽줄기세포 유래 엑소좀 기반의 치료 기법을 대체할 수 있다. 본 연구의 기술은 미래에 중추신경계 손상의 성공적인 치료를 위해 사용 될 수 있을 것이다.
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dc.description.abstractCentral nervous system (CNS) injuries such as ischemic stroke, hemorrhage, traumatic brain injury, and spinal cord injury (SCI) are leading cause of long-term disability or death. As a novel tissue regenerative strategy, application of mesenchymal stem cell-derived exosomes (MSC-exosomes) for treatment of CNS injuries has drawn much attention. Owing to their unique properties, MSC-exosomes have been actively studied for treatment of wide variety of diseases including CNS injuries. However, it still remains challenging to improve the therapeutic outcomes of using exosomes to treat CNS injuries. Very small quantities of exosomes are released from MSC, and MSC-exosomes poorly accumulate in target tissue after systemic administration. Thus, iron oxide nanoparticles (IONP) were introduced to MSC and exosome-mimetic nanovesicles were isolated from those IONP-treated MSC. Treatment of IONP contribute to enhanced expression of therapeutic growth factors in MSC, which are attributed to IONP that are slowly ionized to iron ions which activate the JNK and c-Jun signaling cascades in MSC. Isolated nanovesicles incorporate not only IONP which act as magnet-navigating tool, but also large amount of therapeutic growth factors. In this thesis, in vivo systemic delivery of IONP-harboring nanovesicles and their therapeutic effects in CNS lesions are presented.
First, IONP-haboring nanovesicles (NV-IONP) were fabricated by serial extrusion of IONP-treated MSC (MSC-IONP), and systemically delivered to injured spinal cord in mouse SCI model with help of external magnetic field (MF). While intravenous injection of MSC-IONP resulted in severe lung accumulation due to their large size, nano-sized NV-IONP evaded lung entrapment and significantly accumulated in injured spinal cord in presence of MF. NV-IONP accumulated in SCI lesion contributed to blood vessel reconstruction, polarized inflammatory M1 macrophages to anti-inflammatory M2 subtype, attenuated astrogliosis, inhibited neuronal death. Consequently, increased amount of NV-IONP accumulated in injured spinal cord exerted therapeutic effects and improved the spinal cord function, as evaluated by Basso Mouse Scale (BMS) behavioral test.
Second, IONP-incorporated magnetic nanovesicles (MNV) derived from MSC were intravenously administered to rat ischemic stroke models. Animals received middle cerebral artery occlusion (MCAO) to induce ischemic stroke. In vitro, MNV exerted enhanced angiogenic, anti-apoptotic, and anti-inflammatory effect in various types of cells, as compared to control nanovesicles (NV). In vivo, we applied magnet helmet which act as an external MF, to rat MCAO models immediately after intravenous injection of MNV. Fluorescence imaging showed successful accumulation of MNV specifically in left hemisphere of brain (ischemic lesion). Quantification of infarcted area at 3 days after treatment revealed that administration of MNV in presence of MF resulted in significant reduction of infarcted area compared to control group. Limb placement test (LPT) also demonstrated that treatment of MNV (MF+) improves the functional behavior of MCAO-received animals.
Thus, we observed that the nanovesicles synthesized from IONP-laden MSC contain both greater amounts of reparative growth factors and IONP which act as a magnet-guided navigation tool toward injured spinal cord or cerebral lesion. These IONP-harboring nanovesicles derived from MSC can serve as excellent therapeutic agent for treatment of CNS injuries, and can be a preferable replacement of conventional MSC or MSC-exosome therapy. This technology can be used in the future for successful therapy of CNS injuries.
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dc.description.tableofcontentsChapter 1. Research background and objectives 1
1.1. Challenges and limitations of MSC-based therapeutics 3
1.2. Extracellular vesicles 5
1.3. Therapeutic potency of MSC-derived EVs. 8
1.3.1. In vitro effects on target cells 8
1.3.2. Pre-clinical studies in various disease models 9
1.4. Exosome-mimetic nanovesicles 10
1.5. Magnetic drug delivery 12
1.6. Research objectives of the thesis 13

Chapter 2. Experimental procedures 15
2.1. Cell culture 17
2.2. Preparation and characterization of materials 18
2.2.1. IONP synthesis 18
2.2.2. Fabrication of NV-IONP derived from hMSC-IONP 19
2.2.3. Characterziation of NV-IONP 20
2.3. In vitro assays 21
2.3.1. IONP uptake by hMSC 21
2.3.2. Angiogenic effect of NV-IONP 22
2.3.3. Evaluation of anti-apoptosis in neuronal cells. 23
2.3.4. Assessment of astrocyte modulation 24
2.3.5. Macrophage polarization 25
2.3.5.1 Preparation of rBMDM 25
2.3.5.2 Evaluation of macrophage phenotype 26
2.3.6. qRT-PCR, western Blot, ELISA, and protein array assay 27
2.4. In vivo studies 28
2.4.1. Experimental animal models 28
2.4.1.1. SCI models 28
2.4.1.2. MCAO ischemia-reperfusion models 29
2.4.2. In vivo biodistribution 30
2.4.2.1. Biodistribution of MSC-IONP and NV-IONP in SCI mouse model 30
2.4.2.2. MNV biodistribution in MCAO rat model 31
2.4.3. Animal group assignment 32
2.4.3.1. SCI mouse model 32
2.4.3.2. MCAO rat model 33
2.4.4. Therapeutic mechanism of NV-IONP 34
2.4.4.1. Evaluation of apoptosis and fibrotic scar formation in SCI model 34
2.4.4.2. Immunohistochemistry and western blot analysis of extracted spinal cord 35
2.4.4.3. Western blot and IHC analysis of extracted brain tissue of MCAO model 36
2.4.5. Evaluation of therapeutic effects from accumulated NV-IONP 37
2.4.5.1. Hindlimb locomotor score of SCI model 37
2.4.5.2. Measurement of cerebral infarct volume of MCAO model 38
2.4.5.3. Functional behavior test of MCAO model 39
2.5. Statistical analysis 40

Chapter 3. Therapeutic efficacy-potentiated and diseased organ-targeting nanovesicles derived from mesenchymal stem cells for spinal cord injury treatment 41
3.1. Introduction 43
3.1. Result and discussion 47
3.2.1. IONP internalization into hMSCs and up-regulation of growth factors 47
3.2.2. Characterization of nanovesicles isolated from hMSC-IONP 51
3.2.3. Enhanced angiogenesis and anti-apoptotic effect of NV-IONP 60
3.2.4. Stimulation of therapeutic growth factor expression in astrocytes by NV-IONP 64
3.2.5. In Vitro attenuation of inflammatory responses and M2 polarization in macrophages 65
3.2.6. Spinal cord targeting of NV-IONP via external magnetic guidance 70
3.2.7. Enhanced angiogenesis and M2 polarization in injured spinal cord by magnet-guided NV-IONP 74
3.2.8. Attenuation of glial scar formation and enhanced functional recovery by magnet-guided NV-IONP 79

Chapter 4. Stem Cell-derived Magnetic Nanovesicles Target and Attenuate Ischemic Stroke 81
4.1. Introduction 83
4.2. Result and discussion 86
4.2.1. IONP uptake and enhanced therapeutic growth factors in MSC 86
4.2.2. IONP and higher level of therapeutic growth factors in MNV 92
4.2.3. In vitro therapeutic effects of MNV 96
4.2.4. Magnet-assisted ischemic lesion targeting of MNV 100
4.2.5. In vivo therapeutic effect and mechanism of MNV for treatment of ischemic stroke 105

Chapter 5. Conclusion 107

References 111

요약 (국문 초록) 132
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dc.language.isoeng-
dc.publisher서울대학교 대학원-
dc.subjectmesenchymal stem cells-
dc.subjectexosomes-
dc.subjectnanovesicles-
dc.subjectiron oxide nanoparticles-
dc.subjectspinal cord injury-
dc.subjectischemic stroke-
dc.subjectcentral nervous system injury-
dc.subject.ddc660.6-
dc.titleMesenchymal Stem Cell-derived Therapeutic Nanovesicles for Treatment of Central Nervous System Injuries-
dc.title.alternative중간엽 줄기세포 유래 치료용 나노베지클을 이용한 중추신경계 손상 치료-
dc.typeThesis-
dc.typeDissertation-
dc.contributor.AlternativeAuthorHan Young Kim-
dc.contributor.department공과대학 화학생물공학부-
dc.description.degreeDoctor-
dc.date.awarded2019-08-
dc.identifier.uciI804:11032-000000157482-
dc.identifier.holdings000000000040▲000000000041▲000000157482▲-
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College of Engineering/Engineering Practice School (공과대학/대학원)Dept. of Chemical and Biological Engineering (화학생물공학부)Theses (Ph.D. / Sc.D._화학생물공학부)
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