含RGD序列之胜肽對人類多能幹細胞培養的比較研究

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姓名

呂明威(Ming-Wei Lu) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

含RGD序列之胜肽對人類多能幹細胞培養的比較研究
(Comparative Study on RGD-Containing Motifs of Synthetic Peptides for Human Pluripotent Stem Cells Cultivation)

相關論文

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★ 羊水間葉幹細胞培養於接枝細胞外間質寡肽與環狀肽具有最佳表面硬度的生醫材料,其增殖能力及多能性之研究	★ 人類體細胞從組成誘導型多能性幹細胞培養在無飼養層上

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至系統瀏覽論文 (2025-9-1以後開放)

摘要(中)

人類多能幹細胞，包括人類胚胎幹細胞和人類誘導多能幹細胞，在再生組織工程領域裡是相當有前景的來源。起初，人類多能幹細胞需要培養在動物飼養層細胞或動物源蛋白塗佈的培養皿上進行培養，例如小鼠胚胎成纖維細胞（MEF）或Matrigel，這些異種源細胞所分泌細胞外間質（ECM）有利於幹細胞進行附著進而增長，但因為這些異種源細胞與蛋白質含有未知的化學組成，對於外來的臨床試驗增添許多不確定性。因此，可靠的、無異源的生物材料──合成胜肽，不僅提供確定的化學成分，還提供了可重複培養的條件。
在我們之前的研究中已成功的將人類多能幹細胞培養在寡玻連蛋白（KGGPQVTRGDVFTMP）嫁接的聚乙烯醇-衣康酸水膠上。但是，一旦我們將誘導人類多能幹細胞進一步分化，與培養在其他細胞外間質相比，人類多能幹細胞較容易從寡玻連蛋白胜肽嫁接的聚乙烯醇-衣康酸水膠上脫附。因此本研究之目的，希望能設計出新胜肽不僅提供早期的細胞增殖，還可以協助後期的細胞分化。所以本實驗在層粘連蛋白（LN）發現了可能的新胜肽，並將新衍生的胜肽嫁接在聚乙烯醇-衣康酸水膠上來研究其中關鍵的細胞與基材結合機制，此次新設計的層粘連蛋白衍生之胜肽（PASYRGDSC和PMQKMRGDVFSP），其中序列裡的RGD基序提供細胞貼附的主要識別系統且有望用於進一步的支持細胞分化。一方面，本研究同時製造幾種不同的設計的層粘連蛋白衍生胜肽，設計不同長度的節鏈結來仿生原來的細胞外間質，評估鏈接的長度和結構效應。另一方面，在胜肽的末端使用或不使用酰胺修飾（-CONH2），用以比較細胞附著的效率。此外，在多能幹細胞培養於合成胜肽嫁接表面上後，評估其四種多能基因（Oct4，Sox2，SSEA-4和Nanog）的表現量。最後檢驗人類胚胎幹細在不同設計的胜肽上對分化能力的影響，從幹細胞分化成間業幹細胞（MSC）和心肌細胞（CM）。
我們的結果將有助於理解現階段培養人類多能幹細胞中細胞與基材結合機制，進而針對不同的使用條件客製專屬的、有利的增長環境。

摘要(英)

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are attractive source for regenerative tissue engineering. Typically, hPSCs are used to culture on animal feeder cells or secreted protein-coated dishes, such as mouse embryonic fibroblast (MEF) or Matrigel, respectively which secrete extracellular matrices (ECMs) and growth factors for cell attachment but provide uncertain chemical compositions for cell growth. On the other hand, reliable biomaterials such as xeno-free synthetic peptides-immobilized surface, offer not only a chemically defined composition but also reproducible conditions for hPSC cultivation.
In previous studies in our laboratory, hPSCs were cultured on polyvinyl alcohol-co-itaconic acid (PVA-IA) hydrogels, which were conjugated with oligo-vitronectin (KGGPQVTRGDVFTMP) stably. However, once I induced the hPSCs for further differentiation, hPSCs detached much easier comparing to hPSCs cultured on other ECM-coated surface. In this study, I designed new peptides from Laminin (LN), which was a remarkable ECM for cell differentiation in previous studies where the key cell binding mechanism was investigated by using PVA-IA hydrogels. Several new designs of laminin-derived peptide (PASYRGDSC and PMQKMRGDVFSP) were grafted on PVA-IA hydrogels, which provided RGD binding motifs and constituted the major recognition system for hPSC adhesion. These synthetic peptides were investigated by comparing the ability to support long term hPSCs cultivation and differentiation into mesenchymal stem cells (MSCs) and cardiomyocytes (CMs). The different lengths and sequences of joint segment, mimicking the original ECM, were evaluated to investigate the length effect and the effect of structures on hPSC culture and differentiation. Moreover, the C-terminus of peptides were treated with and without amide modification (-CONH2) to compare the efficiency of hPSC attachment. Furthermore, after hPSC cultivation on those synthetic peptides-grafted surface, hPSCs were evaluated for the expression level of four pluripotent protein (Oct4, Sox2, SSEA-4 and Nanog) expression. These results will help understanding of the cell-substrate binding mechanisms in sustaining hPSC culture and differentiation .

關鍵字(中)

★ 幹細胞
★ 生醫材料

關鍵字(英)

★ human pluripotent stem cells
★ biomaterials

論文目次

Abstract I
Index of content IV
Index of Figure VIII
Index of table XII
Chapter 1. Introduction 1
1-1. Pluripotent stem cells 1
1-1-1. Embryonic stem cells 2
1-1-2. Induced pluripotent stem cells 3
1-1-3. The limitations of human embryonic stem cells and induced pluripotent stem cells 4
1-1-4. hPSCs for therapeutic application in future 6
1-2. Characterization of hPSCs 6
1-2-1. Colony formation of hPSCs 8
1-2-2. pluripotent gene expression 8
1-2-3. Immunofluorescence 9
1-2-3. Differentiation ability 9
1-3. The substrates for hPSCs cultivation 12
1-3-1. The feeder cell layers for hPSCs maintenance 12
1-3-2. The feeder-free and xeno-contained proteins for hPSCs cultivation 13
1-3-3. Feeder-free and xeno-free human extracellular matrices 14
1-3-4. Oligopeptides 15
1-3-5. Cell-substrate binding mechanism 17
1-4. Differentiation of hPSCs into mesenchymal stem cells 19
1-4-1. Efficient methods for mesenchymal stem cells differentiation 20
1-4-2. Characterization of mesenchymal stem cells 21
1-5. The goal of this study 21
Chapter 2 Materials and Method 23
2-1. Materials 23
2-1-1. Cell Line 23
2-1-2. Commercial Culture Dishes 23
2-1-3. Commercial Coated Substrates 23
2-1-4. Medium and Others 23
2-1-5. The chemicals in oligopeptide-grafted dish 24
2-1-6. Chemical for immunostaining 31
2-1-7. Chemical for flowcytometry 32
2-2. Cell culture 32
2-2-1. human pluripotent stem maintenance 32
2-2-2. Passage method of human pluripotent stem cells 32
2-2-3. Preparation of PVA-IA hydrogel coated dish 33
2-2-4. Preparation of oligopeptides grafted PVA-IA hydrogel 34
2-2-5. Passage method of hPSCs cultured on peptide-grafted PVA-IA dish 34
2-2-6. Cryopreservation of human pluripotent stem cells 35
2-2-7. Thawing of human pluripotent stem cells 35
2-3. Characterization of Dish Surface 36
2-3-1. Atomic Force Microscope (AFM) 36
2-3-2. X-ray Photoelectron Spectroscopy (XPS) 36
2-3-3. Zeta potential 37
2-4. Characterization of hPSCs 37
2-4-1. Expansion Fold and Doubling Time of hPSCs 37
2-4-2. Differentiation Ratio of hPSCs 38
2-4-3. Immunostaining of Cells 38
2-4-4. Embryoid Body Formation 40
2-5. Mesenchymal stem cells differentiation 40
2-5-1. The protocol of MSCs differentiation 40
2-5-2. Flow-cytometry measurement for MSCs differentiation 42
Chapter 3 Results and Discussion 43
3-1. Cultivation of hPSCs on PVA-IA hydrogels grafted with oligopeptides 43
3-1-1. Determination of key domains derived from laminin-111 43
3-1-2-1. Cultivation of hPSCs on PVA-IA hydrogels grafted with oligopeptides derived from several modification of laminin α5 chain 45
3-1-2-2. Expansion fold and differentiation of hiPSCs cultured on PVA-IA hydrogels conjugated with LN-α5 derived oligopeptides 48
3-1-3. Cultivation of hPSCs on PVA-IA hydrogels grafted with different modification of laminin β4 chain derived oligopeptides 52
3-1-4. Expansion fold and differentiation of hiPSCs cultured on PVA-IA conjugated with LN-β4 derived oligopeptides 56
3-1-5. The long-term cultivation of hPSCs on PVA-IA hydrogels grafted with oligopeptides 60
3-1-6. Immunostaining of hESCs cultured on PVA-IA hydrogels grafted with LN and VN derived oligopeptides having different designs 63
3-1-7. Embryoid body formation of hPSCs on PVA-IA hydrogels grafted with VN and LN derived oligopeptides. 68
3-1-8. MSC differentiation from adherent hESCs on oligopeptides grafted surface 72
3-2. Characterization of PVA-IA hydrogels conjugated with design of oligopeptides 74
3-2-1. Atomic force microscope scanning measurements of PVA-IA hydrogels conjugated with design of oligopeptides 75
3-2-2. X-ray photoelectron spectroscopy analysis of PVA-IA hydrogels conjugated with design of oligopeptides 78
3-2-3. Zeta potential analysis of the effect of surface charge on PVA-IA hydrogels conjugated with design of oligopeptides 83
Chapter 4 Conclusion 85
Reference 87
Appendix 97

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指導教授

樋口亞紺(Akon Higuchi)

審核日期

2020-8-20

推文