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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 劉逸軒 | |
dc.contributor.author | Ming-Kang Lee | en |
dc.contributor.author | 李明剛 | zh_TW |
dc.date.accessioned | 2021-07-11T14:44:22Z | - |
dc.date.available | 2021-10-14 | |
dc.date.copyright | 2016-10-14 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-04 | |
dc.identifier.citation | Aggarwal, S., and Pittenger, M.F. (2005). Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105, 1815-1822.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78164 | - |
dc.description.abstract | Mesenchymal stem cells (MSCs) hold great potential in cell therapies by virtue of the regenerative effects and immunomodulatory properties, but the scarce nature of MSCs makes ex vivo expansion indispensable prior to transplantation purposes. However, potential loss of stemness ensuing culture expansion has hindered the advancements in MSCs-based treatments. In principles, stemness can be preserved by reconstructing the stem cell niche, but the physiological nature including the endogenous stem cell niche of MSCs remains elusive. Emerging hypotheses suggested that pericytes residing subendothelium might be one of the primitive origins of MSCs, and accordingly, we speculated that endothelial cells (ECs) might participate in the constitution of the stem cell niche for MSCs. In this study, ECs derivatives including extracellular matrix (ECM) and paracrine factors collected from conditioned medium (CM) of aortic endothelial cells (AECs) and Mile Sven 1 endothelial cell line (MS1) were investigated for the potential to maintain MSCs stemness. When compared with MSCs cultured alone, on MSCECM and in endothelial CMs, MSCs on endothelial ECMs especially on MS1ECM possessed the morphology of more juvenile cells, showed quiescence in proliferation, in agreement with general description of primitive MSCs. Besides, MSCs expanded on MS1ECM possess greater osteogenic, adipogenic and chondrogenic potential under proper stimuli. These results indicated that MS1ECM preserved the stemness of MSCs. We further discovered that the possible mechanism resulted from MSCs expanded on MS1ECM had significantly higher H3K27me3 mark with significantly lower expression of Kdm6b, a specific H3K27 demethylase. Higher H3K27me3 mark in MS1ECM can be interpreted as transcriptional inactivation, and it probably explained proliferative quiescence and better differentiation plasticity. Taken together, MS1ECM retained MSCs stemness by shaping an inhibitory chromatin signature via maintaining lower expression of Kdm6b. Our work provided not only a supportive evidence that MSCs can reside in perivascular niche, but also a feasible novel approach for MSCs ex vivo expansion. The detailed signal between extracellular environment and intracellular chromatin signature requires further investigation. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T14:44:22Z (GMT). No. of bitstreams: 1 ntu-105-R03626001-1.pdf: 2604912 bytes, checksum: 5b7c116563a2b1b21ba064adca909fbc (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 致謝 i
中文摘要 ii ABSTRACT iii CONTENTS v LIST OF FIGURES ix LIST OF TABLES x Chapter 1 Introduction 1 1.1 Mesenchymal stem cells: the essence and the definition 1 1.2 Reappraisal of the definition of MSCs 2 1.3 A rising star in cell therapy: therapeutic effects of MSCs 3 1.3.1 Bioactive factors 4 1.3.2 Immunomodulatory properties 5 1.3.3 MSCs-based cell therapies 7 1.4 Stemness loss of MSCs: a mountain to climb 8 1.5 The stem cell niche: how to keep stem cells as stem cells? 10 1.6 A perivascular origin of MSCs 11 1.7 Pericytes and endothelial cells: intimacy shared 12 1.8 Home sweet home: large or small vessels endothelial cells? 13 1.9 Extracellular matrix: a cozy bedding for stem cells 14 1.10 Endothelial microenvironment: guidance for MSCs quiescence 15 1.11 Histone modification on MSCs identity 16 1.12 The pericyte hypothesis 17 Chapter 2 Specific aim 19 Chapter 3 Material and Methods 22 3.1 Animals 22 3.2 Isolation of bone marrow mesenchymal stem cells 22 3.3 Establishment of mouse aortic endothelial cells (AECs) 23 3.4 Culture of Mile Sven 1 (MS1) cell line 24 3.5 Cell passaging 24 3.6 Cryopreservation and thawing of cells 25 3.7 Preparation for collagen I-coated 6 well plate 25 3.8 Preparation of cell-free extracellular matrix-coated plate 26 3.9 Harvest of conditioned medium 27 3.10 Quantification of long/short axis ratio 27 3.11 MTT assay 27 3.12 Osteogenic differentiation 28 3.13 Adipogenic differentiation 28 3.14 Chondrogenic differentiation 29 3.15 Total RNA extraction 30 3.16 Reverse transcription PCR 31 3.17 Quantitative PCR 31 3.18 Protein quantification 33 3.19 Dot blot 33 3.20 Western blot 34 3.20.1 Buffer preparation 34 3.20.2 Preparation of SDS-PAGE gel 35 3.20.3 Sample loading 35 3.20.4 Electrophoresis 35 3.20.5 Transferring protein samples 36 3.20.6 Antibody staining 36 3.21 Statistical analysis 37 Chapter 4 Results 38 4.1 MSCs maintained on endothelial ECM appeared morphologically juvenile 38 4.2 MSCs on endothelial ECM remained quiescent 42 4.3 Endothelial ECM aggrandized the harvest of MSCs 45 4.4 MS1ECM exerted osteogenic potential of MSCs 46 4.5 Endothelial derivatives enhanced osteogenic genes expression of MSCs after induction 49 4.6 MSCs on MS1ECM retained adipogenic plasticity 54 4.7 Endothelial derivatives altered chondrogenic commitment 56 4.8 MSCs on MS1ECM preserved stemness 59 4.9 MSCs on MS1ECM had higher H3K27me3 61 4.10 Higher H3K27me3 mark was conducted by lower KDM6B expression 62 Chapter 5 Discussion 65 5.1 MSCs-derived ECM only partially construct their own stem cell niche 65 5.2 Contradiction of endothelial ECM on MSCs proliferation 66 5.3 Endothelial ECM and CM expressed diverse influences on MSCs proliferation. 67 5.4 Small vessels-derived ECs conserved MSCs lineage plasticity 69 5.5 Aortic endothelial cells triggered MSCs osteogenesis 70 5.6 Other histone modifications on MSCs identity 71 5.7 Consecutive passaging downregulated enzymatic dynamic on H3K27 72 5.8 ECM as a critical effector on MSCs stemness 73 5.9 Is ratio of axis a sufficient indicator for juvenility? 74 5.10 What if the improved performance was conducted by pre-coated collagen I rather than cell-derived ECM? 74 5.11 Is Kdm6b/ H3k27 mark the key to MSCs stemness? 75 5.12 Epigenetic changes before and after MSCs commitment 76 5.13 Epigenetic changes of MSCs from quiescence to excessive cell cycle. 77 Chapter 6 Conclusion 78 REFERENCE 79 | |
dc.language.iso | en | |
dc.title | 內皮細胞胞外基質維持骨髓間葉幹細胞之幹細胞特性 | zh_TW |
dc.title | Endothelial-derived Extracellular Matrix Preserves the Stemness of Bone Marrow-derived Mesenchymal Stem Cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林劭品,陳逸然,黃效民 | |
dc.subject.keyword | 間葉幹細胞,內皮細胞,周圍細胞,幹細胞龕,細胞外基質, | zh_TW |
dc.subject.keyword | mesenchymal stem cells,endothelial cells,pericytes,stem cell niche,extracellular matrix, | en |
dc.relation.page | 93 | |
dc.identifier.doi | 10.6342/NTU201601819 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-05 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
顯示於系所單位: | 動物科學技術學系 |
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