通用人類誘導多能幹細胞在多胜肽接枝水凝膠上的培養和分化

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洪兆俞(Zhao-Yu Hong) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

通用人類誘導多能幹細胞在多胜肽接枝水凝膠上的培養和分化
(Proliferation and Differentiation of Universal Human Induced Pluripotent Stem Cells on Hydrogels Grafted with Mixed ECM-derived Peptides)

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至系統瀏覽論文 (2026-8-20以後開放)

摘要(中)

幹細胞是應用於細胞治療中重要的細胞來源。胚胎幹細胞（ESC）和人類誘導多能幹細胞（hiPSC）都屬於人類多能幹細胞（hPSC）的範疇內，而這些幹細胞皆具有分化為內胚層、中胚層和外胚層, 這些胚胎層的能力。 Matrigel是含異種基質材料的細胞外基質 (ECM) 用來培育和分化 hPSC 最常用的外基質之一。 Matrigel是一種從 Engelbreth-Holm-Swarm (EHS) 小鼠肉瘤中提取的溶解基底膜萃取物，由多種分子組成其中包含有膠原蛋白IV、層粘連蛋白-111等組成的。大多數研究中，在接枝ECM衍生的胜肽水凝膠已可以用於PSC的培育和分化。而有鑑於Matrigel由多種成分組成，我設計了接枝多種 ECM 混和衍生的胜肽水凝膠。我利用NHS和EDC 這兩種藥品並使用的化學方法來製備聚乙烯醇-衣康酸、PVA-IA、來接枝有玻連蛋白衍生胜肽 (VN2CK) 和層黏連蛋白β4 衍生胜肽 (LB2CK) 的水凝膠，經過測試後這水凝膠能控制在25.3kPa的彈性應力。與單一胜肽接枝水凝膠相比，人類 PSC在混合的胜肽上接枝水凝膠有更高的效益。接枝混合的胜肽 (1000 : 1000μg/mL) 水凝膠可以更有效率的促進 hPSC 的黏附並幫助其多能性的維持。我還在胜肽的第一個位置上添加了氨基酸（賴氨酸，K）。在氨基酸添加後，及為KKLB2CK的氨基，比未添加的LB2CK表現出更高的反應活性。從中發現，在胜肽的第一個位置上插入的帶正電氨基酸 (K) 顯著增強了胜肽接枝水凝膠的表面接枝密度，並且還增加了水凝膠的zeta電位。在這項研究中，我展示了由三種來自不同人體的人類羊水中並在沒有基因編輯的情況下衍生出的通用 hiPSCs。這種通用 hiPSC 在有混合玻連蛋白和層黏連蛋白 β4 衍生胜肽的水凝膠上進行培育和分化，且最後用人類週邊血單核細胞 (hPBMCs)來共同培養並驗證hiPSCs的免疫原性。

摘要(英)

Stem cells are attractive source of cells for cell therapy. Human pluripotent stem cells (hPSCs) including embryonic stem cells (ESCs) and human induced pluripotent stem cells (hiPSCs) have the potential to differentiate into any cell types derived from endoderm, mesoderm and ectoderm lineages. Xeno-containing Matrigel is one of the most commonly used substrates for culturing and differentiating hPSCs. Matrigel is a solubilized basement membrane extracts from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma composed of multiple molecules, including collagen IV and laminin-111. Instead of Matrigel-coated dishes, hydrogels grafted with single ECM-derived peptides have been utilized for the culture and differentiation of PSCs by most of researches. Considering that Matrigel is comprised of various components, hydrogels grafted with mixed ECM-derived peptides have been designed in this study. Poly(vinyl alcohol-co-itaconic), PVA-IA, hydrogels grafted with vitronectin-derived peptide (VN2CK) and laminin β4-derived peptide (LB2CK) were prepared using EDC/NHS chemistry, where the elasticity of the hydrogels was fixed at 25.3 kPa. Compared to the hydrogels grafted with single peptides, hPSCs demonstrated more efficient proliferation on the hydrogels grafted with mixed peptides. Hydrogels grafted with a combination of mixed peptides (1000:1000 μg/mL) were effective in promoting adhesion and preserving pluripotency of hPSCs, where positive amino acids (Lysine, K) was added on the first of the peptide sequences. The hydrogels grafted with KKLB2CK having positively charged amino acids on the first peptide sequence exhibited higher reactivity than those grafted with LB2CK. Interestingly, the insertion of positively charged amino acids on the peptides significantly enhanced both the surface grafting density and the zeta potential of the peptide-grafted hydrogels. The universal hiPSCs were prepared from the reprogramming of human amniotic fluids after mixing three different source of amniotic fluid without gene editing. This universal hiPSCs were cultured and differentiated on the hydrogels grafted with mixed vitronectin- and laminin β4-derived peptides. Immunotolerance of the universal hiPSCs were evaluated from the treatment of human peripheral blood-derived mononuclear cells (hPBMCs).

關鍵字(中)

★ 寡肽
★ 重編程
★ 人類誘導多能幹細胞
★ 羊水幹細胞
★ 人類白血球抗原
★ 心肌細胞

關鍵字(英)

★ oligopeptides
★ reprogramming
★ humam induced pluripotent stem cells
★ amniotic stem cells
★ HLA
★ cardiomyocytes

論文目次

Abstract I
摘要 III
Index of Content IV
Index of Figures V
Index of Tables XI
Chapter 1. Introduction 1
1-1 Stem cells 1
1-1-1 Human embryonic stem cells (hESCs) 2
1-1-2 Human induced pluripotent stem cells (hiPSCs) 4
1-1-3 The potential risks associated with human pluripotent stem cells (hPSCs) 5
1-1-4 Characterization of human pluripotent stem cells (hPSCs) 6
1-1-5 Human pluripotent stem cells (hPSCs) for therapeutic application 9
1-2 Human amniotic fluid stem cells (hAFSCs) 10
1-3 Cardiomyocytes 10
1-3-1 Characterization of human cardiomyocytes 11
1-3-2 Effective method for inducing cardiomyocyte differentiation 12
1-4 Biomaterials used to cultivate hPSCs 13
1-4-1 Feeder free and xeno-contained culture for hPSCs 14
1-4-2 Feeder free and xeno-free ECMs for hPSCculture 15
1-4-2-1 Cellular receptors for ECM 16
1-4-3 Creation of peptides with a joint-chain and a double-chain design 18
1-4-4 ECM-derived peptides for hPSCs adhesion, cultivation, and differentiation 18
1-5 Human peripheral blood mononuclear cells (hPBMCs) 20
1-6 Development of universal hiPSCs 20
1-7 The goal of this study 22
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 Culture medium 24
2-1-4 Passage processing medium 24
2-1-5 Human induced pluripotent stem cells (hiPSCs) generation kit 25
2-1-6 Isolation of allogenic mononuclear cells 25
2-1-7 Phosphate buffer saline solution (PBS) 25
2-1-8 The chemicals for preparation of peptide-grafted hydrogels 26
2-1-9 Differentiation medium 29
2-1-10 Characterization of stem cells 29
2-2 Instruments 31
2-3 Methods 32
2-3-1 Preparation of culture medium 32
2-3-2 Generation and isolation of stem cell lines 33
2-3-3 Cultivation and passage method of stem cells 36
2-3-4 Cell number measurements 39
2-3-5 Characterization of hPSCs 40
2-3-5-1 Expansion fold evaluation of hPSCs 40
2-3-5-2 Differentiation ratio of hPSCs 40
2-3-5-3 Immunofluorescence staining 40
2-3-5-4 Flow cytometry measurements 43
2-3-5-5 Embryoid body (EB) formation assay in vitro 46
2-3-6 Isolation of allogenic mononuclear cells 46
2-3-7 Live and dead staining of the cells 47
2-4 Preparation of peptide-grafted hydrogels 48
2-4-1 Preparation of PVA-IA coated surface 48
2-4-2 Preparation of different peptide-grafted PVA-IA hydrogels 49
2-5 Characterization of peptide-grafted hydrogels 50
2-5-1 X-ray photoelectron spectroscopy (XPS) measurements 50
2-5-2 Zeta potential measurements 51
2-6 Differentiation method of hPSCs into cardiomyocytes 52
2-6-1 The protocol for differentiation of hPSCs into cardiomyocytes 52
2-6-2 Characterization of hPSC-derived cardiomyocytes 53
Chapter 3. Results and Discussion 55
3-1 Reprogramming and cultivation of hAFSCs-derived hiPSCs 55
3-1-1 The morphologies of mixed hAFSCs 55
3-1-2 Generation and isolation of hAFSCs-derived hiPSCs 56
3-1-3 Immune marker expression of universal hiPSCs and several human stem cells 59
3-2 Cultivation of universal hiPSCs on different peptide-grafted hydrogels 63
3-2-1 The morphologies of universal hiPSCs on different peptide-grafted hydrogels during long-term cultivation 65
3-2-2 Expansion fold of hiPSCs (HPS0077) on different peptide-grafted hydrogels 69
3-2-3 Pluripotency analysis of hiPSCs (HPS0077) cultured on different peptide-grafted hydrogels 71
3-2-4 EB formation of universal hiPSCs (mix-3-4) on different peptide-grafted hydrogels 74
3-3 Cultivation of hiPSCs (HPS0077) on different peptide-grafted hydrogels 76
3-3-1 The morphologies of hiPSCs (HPS0077) on different peptide-grafted hydrogels during long-term cultivation 76
3-3-2 Expansion fold of hiPSCs (HPS0077) on different peptide-grafted hydrogels 79
3-3-3 Pluripotency analysis of hiPSCs (HPS0077) cultured on different peptide-grafted hydrogels 81
3-3-4 EB formation of hiPSCs (HPS0077) on different peptide-grafted hydrogels 84
3-4 Characterization of different peptide-grafted hydrogels 86
3-4-1 X-ray photoelectron spectroscopy analysis of different peptide-grafted hydrogels 86
3-4-2 Zeta potential analysis of the surface electrical potential on different peptide-grafted hydrogels 92
3-5 Differentiation of human pluripotent stem cells into cardiomyocytes on different peptide-grafted hydrogels 95
3-5-1 Differentiation of hPSCs into cardiomyocytes on different peptide-grafted hydrogels 95
3-5-2 Specific marker expression of cardiomyocytes derived from universal hPSCs (mix-3-4) cultured on different peptide-grafted hydrogels 97
Chapter 4. Conclusion 100
References 102
Supplementary Data 110

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指導教授

樋口亞紺(Akon Higuchi)

審核日期

2024-7-25

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