人類多能幹細胞的長期培養和分化在混合寡肽接枝的水凝膠表面

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研究生：	陳諺泓 Yen-Hung Chen
論文名稱：	人類多能幹細胞的長期培養和分化在混合寡肽接枝的水凝膠表面 Hydrogel Surface Grafted with Mixed Oligopeptides for Long-Term Cultivation and Differentiation of Human Pluripotent Stem Cells
指導教授：	樋口亞紺 Akon Higuchi
口試委員:
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程與材料工程學系 Department of Chemical & Materials Engineering
論文出版年：	2023
畢業學年度：	111
語文別：	英文
論文頁數：	139
中文關鍵詞：	混和寡肽接枝水凝膠、生醫材料、無異種條件培養、人類多能幹細胞、多能幹細胞分化
外文關鍵詞：	Mixed oligopeptide grafted hydrogel, biomaterials, Xeno free condition, human pluripotent stem cells, cell differentiation
相關次數：	點閱：9 下載：0
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人多能幹細胞（hPSCs）包括人胚胎幹細胞（hESCs）和人誘導多能幹細胞（hiPSCs），具有分化成內胚層、外胚層和中胚層等三個胚層的細胞的能力。近年含異種的Matrigels被用於培養及分化 hPSC，其中Matrigels由含異種的膠原蛋白 IV、層粘連蛋白-111 等組成。此外，含異種的水凝膠的臨床應用受到限制，因為它們源自動物且化學成分不確定。根據先前的研究單一種細胞外基質 (ECM) 衍生的寡肽接枝水凝膠常被使用於 hPSC 培養和分化。考量到 Matrigels 中包含的多種成分，我們開發混合肽接枝水凝膠，它可以利用細胞上不同種類的整合素來改善 hPSC 增殖和分化。我們使用N-(3-二甲基氨基丙基)-N'-乙基碳二亞胺鹽酸鹽 (EDC) 和 N-羥基琥珀酰亞胺 (NHS) 化學將幾種類型的層粘連蛋白(KKGCGGKGGPMQKMRGDVFSP)和玻連蛋白衍生(GCGGKGGPQVTRGDVFTMP)的寡肽移植到 PVA-IA 水凝膠上。其中將水凝膠的彈性控制在 25.3 kPa（24 小時交聯時間）。與單一肽接枝水凝膠相比，人 PSC 可以在混合肽接枝水凝膠上有效增殖。用低濃度混合肽 (100:100 μg/mL) 製備的水凝膠可以很好地貼附、維持多能性及連續繼代超過10次。此外，在寡肽的第一個序列上插入正氨基酸（K，賴氨酸）有助於提高寡肽接枝水凝膠的表面接枝密度，即 KKGCGGKGGPMQKMRGDVFSP、KGCGGKGGPQVTRGDVFTMP。這解釋了肽第一位側鏈上的氨基（KKGCGGKGGPMQKMRGDVFSP）比肽主鏈上的氨基（GCGGKGGPMQKMRGDVFSP）更具反應性。此外，在寡肽上插入帶正電的賴氨酸 (K) 有助於增強水凝膠的 zeta 電位，此特性有利於細胞的貼附。我們的含有賴氨酸的混合肽接枝水凝膠有望促進 hPSC 的增殖效率和分化為具有低貼附性的分化細胞，例如神經細胞和視網膜色素上皮細胞。

Human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have the ability to differentiate into the cells derived from three germ layers, such as endoderm, ectoderm and mesoderm. Xeno-containing Matrigels are typically used to culture and differentiate hPSCs where Matrigels consist of xeno-containing collagen IV, laminin-111, etc. Moreover, clinical application of the xeno-containing hydrogels is limited since they are derived from animal and not chemically defined. Typically, single extracellular matrix protein (ECM)-derived oligopeptide-grafted hydrogels have been used for hPSC culture and differentiation in xeno-free culture conditions. Considering multiple components contained in Matrigels, I designed mixed peptide-grafted hydrogels, which may improve hPSC proliferation and differentiation using different binding sites of hPSCs. I prepared vitronectin-derived peptide (GCGGKGGPQVTRGDVFTMP) and laminin β4-derived peptide (KKGCGGKGGPMQKMRGDVFSP)-grafted poly(vinylalcohol-co-itaconic) hydrogels using N-hydroxysuccinimide (NHS)/1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (EDC) chemistry where the elasticity of the hydrogels was controlled at 25.3 kPa (24h crosslinking time). Human PSCs could proliferate on the mixed peptide-grafted hydrogels compared to single peptide-grafted hydrogels efficiently. Hydrogels prepared with low concentration of mixed peptides (100:100 μg/mL) could support hPSC adhesion and pluripotency. Furthermore, positive amino acid (K, lysine) insertion on the first sequence of the peptide contributed to the enhancement of surface grafting density of peptide-grafted hydrogels, i.e., KKGCGGKGGPMQKMRGDVFSP and KGCGGKGGPQVTRGDVFTMP. This is explained that the amino group on the side chain of the first position of peptide (KKGCGGKGGPMQKMRGDVFSP) is more reactive than the amino group of the main chain of the peptide (GCGGKGGPMQKMRGDVFSP). Furthermore, insertion of positive lysine (K) on the peptide contributed to enhance zeta potential of the hydrogels, which is favor to adhere hPSCs. Our mixed peptide-grafted hydrogels containing lysine and joint peptide are expected to promote efficient proliferation and differentiation of hPSCs into differentiated cells with low adhesion such as neural cells and retinal pigment epithelium cells.

Abstract    I
摘要    III
Index of content    IV
Index of figure    VIII
Index of Table    XIV
Chapter 1    Introduction    1
1-1    Stem cells    1
1-1-1    Human embryonic stem cells    1
1-1-2    Human induced pluripotent stem cells    3
1-1-3    Characterization of human pluripotent stem cells    3
1-1-4    hPSCs for therapeutic application    5
1-2    Cardiomyocytes    7
1-2-1    Characterization of human cardiomyocytes    7
1-2-2    Efficient methods for cardiomyocytes differentiation    8
1-3    Mesenchymal stem cells    9
1-3-1    Characterization of human mesenchymal stem cells    12
1-3-2    Efficient differentiation methods for mesenchymal stem cells    12
1-4    The biomaterial substrates for stem cells cultivation    13
1-4-1    The feeder cell layers for hPSCs cultivation    13
1-4-2    The maintenance of hPSCs on feeder free and xeno-contained proteins    14
1-4-3    The maintenance of hPSCs on feeder free and xeno-free proteins    15
1-4-4    Development and screening of ECM protein-derived peptides    16
1-4-5    Immobilization method of ECM protein-derived peptides    17
1-4-6    Design of peptides with a joint chain and a dual chain    19
1-4-7    ECM protein-derived peptides for hPSCs adhesion and differentiation    20
1-5    The goal of this study    22
Chapter 2    Materials and Method    24
2-1    Materials    24
2-1-1    Cell lines    24
2-1-2    Commercial culture dishes    24
2-1-3    Commercial coated substrates    24
2-1-4    Medium for hPSCs    24
2-1-5    Medium and chemicals for MSCs differentiated from hPSCs    24
2-1-6    Medium and chemicals for cardiomyocytes differentiated from hPSCs    25
2-1-7    Medium and chemicals for cell passages    25
2-1-8    Phosphate buffer saline solution (PBS)    25
2-1-9    The chemicals of Oligopeptide-grafted Hydrogels    26
2-1-10    Chemicals for immunostaining    30
2-1-11    Chemicals for flow cytometry    31
2-2    Experimental instruments    31
2-3    Experimental methods    33
2-3-1    Human pluripotent stem cells maintenance    33
2-3-2    The passage method of human pluripotent stem cells    33
2-3-3    Expansion fold and differentiation ratio of hPSCs    33
2-3-4    Immunostaining of hPSCs    35
2-3-5    Embryoid body (EB) formation in vitro    38
2-3-6    Flow cytometry measurements    38
2-3-7    Freezing of human pluripotent stem cells    39
2-3-8    Thawing of human pluripotent stem cells    39
2-4    Preparation of oligopeptide-grafted hydrogels    40
2-4-1    Preparation of PVA-IA coated surface    40
2-4-2    Preparation of different oligopeptide-grafted PVA-IA hydrogels    40
2-5    Characterization of oligopeptide-grafted hydrogels    41
2-5-1    X-ray photoelectron spectroscopy (XPS) measurements    41
2-5-2    Zeta potential measurements    42
2-5-3    PrimosCR 45 measurements    43
2-6    Differentiation method of Mesenchymal stem cells    43
2-6-1    The protocol for differentiation of MSCs from hPSCs    43
2-6-2    The passage method for MSCs    44
2-7    Differentiation method of Cardiomyocytes    45
2-7-1    The protocol for differentiation of Cardiomyocytes from hPSCs    45
Chapter 3    Results and discussions    47
3-1    Cultivation of hiPSCs (HPS0077) on different peptide-grafted hydrogels    47
3-1-1    Morphologies of hiPSCs (HPS0077) on hydrogels prepared with different concentration of oligopeptides    48
3-1-2    Expansion fold of hiPSCs (HPS0077) on oligopeptide-grafted hydrogels prepared with different concentration of oligopeptides    53
3-1-3    The morphologies of hiPSCs (HPS0077) on different oligopeptide-grafted hydrogels during long-term cultivation    55
3-1-4    Expansion fold of hiPSCs (HPS0077) on different peptide-grafted hydrogels    59
3-1-5    Pluripotency analysis of hiPSCs (HPS0077) cultured on different oligopeptide-grafted hydrogels    64
3-2    Cultivation of hESCs (WA09) on different oligopeptide-grafted hydrogels    70
3-2-1    Morphologies of hESCs (WA09) on oligopeptide-grafted hydrogels prepared with different concentration of oligopeptides    70
3-2-2    Expansion fold of hESCs (WA09) on oligopeptide-grafted hydrogels prepared with different concentration of oligopeptides    75
3-2-3    Morphologies of hESCs (WA09) on different oligopeptide-grafted hydrogels for Long-term cultivation    77
3-2-4    Expansion fold of hESCs (WA09) on different oligopeptide-grafted hydrogels    81
3-2-5    Pluripotency analysis of hESCs (WA09) on different oligopeptide-grafted hydrogels    82
3-3    Differentiation of human pluripotent stem cells into mesenchymal stem cells on different oligopeptide-grafted hydrogels    88
3-3-1    Differentiation of hPSCs into MSCs on different oligopeptide-grafted hydrogels    88
3-3-2    Surface marker expression of hPSCs derived hMSCs on different oligopeptide-grafted hydrogels    90
3-4    Differentiation of human pluripotent stem cells into cardiomyocytes on different oligopeptide-grafted hydrogels    91
3-4-1    Differentiation of hPSCs into cardiomyocytes on different oligopeptide-grafted hydrogels    91
3-4-2    Specific marker expression of Cardiomyocytes derived from hPSCs cultured on different oligopeptide-grafted hydrogels    94
3-5    Characterization of different oligopeptide-grafted hydrogels    95
3-5-1    X-ray photoelectron spectroscopy analysis of different oligopeptide-grafted hydrogels    95
3-5-2    Zeta potential analysis of the surface electrical potential on different oligopeptide-grafted hydrogels    100
3-5-3    PrimosCR 45 analysis of the surface roughness on different oligopeptide-grafted hydrogels    103
3-6    Correlation between cells expansion fold, concentration, and peptide graft density    106
Chapter 4    Conclusion    108
References    110


                                

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