人類多能幹細胞在寡肽接枝水凝膠上的培養和分化為間葉幹細胞

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研究生：	張育騰 Yu-Tang Chang
論文名稱：	人類多能幹細胞在寡肽接枝水凝膠上的培養和分化為間葉幹細胞 Culture and Differentiation of Human Pluripotent Stem Cells into Mesenchymal Stem Cells on Oligopeptide-grafted Hydrogels
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：	2022
畢業學年度：	110
語文別：	中文
論文頁數：	147
中文關鍵詞：	幹細胞、分化、水凝膠、寡肽、界達電位
外文關鍵詞：	stem cell, differentiation, hydrogel, oligopeptide, zeta potential
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人類多能幹細胞 (hPSCs) 包括人類胚胎幹細胞 (hESCs) 和人類誘導多能幹細胞 (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。hPSCs 充分分化為特定的細胞譜係可以通過細胞培養生物材料進行調節，因為 hPSCs 受細胞培養生物材料的物理和生物學信號的調節。在本研究中，已開發出嫁接了幾種細胞外基質 (ECM) 衍生肽的水凝膠，可用於 hPSC 培養和分化為間充質乾細胞。選擇具有最佳彈性（25.3 kPa）的聚乙烯醇-衣康酸PVA-IA、水凝膠作為基礎細胞培養生物材料。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和玻連蛋白衍生的寡肽移植到 PVA-IA 水凝膠上。人類 iPSC 可以在一些具有 25.3 kPa 彈性（24 小時交聯時間）的寡肽接枝水凝膠上很好地繁殖且繼代超過 10 次。當研究每個代數中每個寡肽接枝水凝膠上 hiPSCs 的膨脹倍數時，發現接枝有 LB2CKKK (GCGGKKKPMQKMRGDVFSP) 和 KKLB2CK (KKGCGGKGGPMQKMRGDVFSP) 寡肽的水凝膠對於 hPSC 增殖是最優選的，其中水凝膠的彈性是 25.3 千帕。發現在寡肽上插入賴氨酸 (K) 的正氨基酸對於 hiPSC 在寡肽接枝水凝膠上的最佳增殖至關重要，這有助於提高水凝膠的 zeta 電位。尤其是，隨著接枝有來自層粘連蛋白β4鏈的幾種寡肽的水凝膠的zeta電位的增加，發現在接枝有幾種寡肽的水凝膠上培養的hiPSCs的增殖更好（更高的膨脹倍數），其中賴氨酸的正氨基酸插入到寡肽促進了與寡肽接枝的水凝膠的高 zeta 電位。人類胚胎幹細胞在移植有 (a) 層粘連蛋白衍生寡肽和 (b) 玻連蛋白衍生寡肽的水凝膠上分化為間葉幹細胞 (MSC) 。LB2CK (KGCGGKGGPMQKMRGDVFSP) 移植水凝膠上的人類 ESC 衍生 MSCs 表現出最佳形態、極好的倍增時間以及最高的 MSCs 表面標誌物表達。人類 iPSC 可以在移植有 LB2CKKK 寡肽的基於樹枝狀大分子的水凝膠上廣泛增殖並形成一個菌落，這是另一種有前途的無異種細胞培養生物材料。我希望這項研究中開發的水凝膠將來可以用於臨床治療。

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. The pluripotency maintenance and adequate differentiation of hPSCs into specific lineage of the cells can be regulated by cell culture biomaterials, because hPSCs are regulated by physical and biological cues of the cell culture biomaterials. The hydrogels grafted with several extracellular matrix (ECM)-derived peptides, which have optimal elasticity have been developed for hPSC culture and differentiation into mesenchymal stem cells in this study. Poly (vinyl alcohol-co-itaconic acid), PVA-IA, hydrogels having optimal elasticity (25.3 kPa) were selected as base cell culture biomaterials. Several types of laminin- and vitronectin-derived oligopeptides were grafted on PVA-IA hydrogels using N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry. Human PSCs could proliferate well on some oligopeptides-grafted hydrogels having 25.3 kPa elasticity (24h crosslinking time) for more than 10 passages. When the expansion fold of hiPSCs was investigated on each oligopeptide-grafted hydrogel at each passage, the hydrogels grafted with oligopeptides of LB2CKKK (GCGGKKKPMQKMRGDVFSP) and KKLB2CK (KKGCGGKGGPMQKMRGDVFSP) were found to be the most preferable for hPSC proliferation where the elasticity of the hydrogels was 25.3 kPa. Positive amino acid of lysine (K) insertion on the oligopeptide was found to be critical for optimal proliferation of hPSCs on the oligopeptide-grafted hydrogels, which contributed to enhance zeta potential of the hydrogels. Especially, better proliferation (higher expansion fold) of hPSCs cultured on the hydrogels grafted with several oligopeptides was found with the increase of the zeta potential of the hydrogels grafted with several oligopeptides derived from laminin β4 chain, where positive amino acid of lysine insertion into the oligopeptides promoted high zeta potential of the hydrogels grafted with oligopeptides. Human ESCs were differentiated into MSCs on hydrogels grafted with (a) laminin derived oligopeptide and (b) vitronectin derived oligopeptide. Human ESC-derived MSCs on LB2CK (GCGGKGGPMQKMRGDVFSP)-grafted hydrogels showed the best morphology, excellent doubling time and also the highest MSCs surface marker expression. Human iPSCs could proliferate and form into a colony extensively on dendrimer-based hydrogels grafted with LB2CKKK oligopeptides, which were another promising xeno-free cell culture biomaterials. I hope that the hydrogels developed in this study can be used in clinical therapy in the future.

Abstract    I
Index of content    IV
Index of figure    VII
Index of table    X
Chapter 1    Introduction    1
1-1    Stem cells    1
1-1-1    Human pluripotent stem cells    1
1-1-2    Human embryonic stem cells    2
1-1-3    Human induced pluripotent stem cells    3
1-1-4    Characterization of human pluripotent stem cells    4
1-1-5    hPSCs for therapeutic application    7
1-2    Mesenchymal stem cells    8
1-2-1    Characterization of human mesenchymal stem cells    9
1-2-2    Differentiation ability of human mesenchymal stem cells    11
1-2-3    Efficient methods for mesenchymal stem cells differentiation    11
1-3    The biomaterial substrates for hPSCs cultivation    12
1-3-1    The feeder cell layers for hPSCs cultivation    12
1-3-2    The maintenance of hPSCs on feeder free and xeno-contained proteins    13
1-3-3    The maintenance of hPSCs on feeder free and xeno-free proteins    14
1-3-4    Oligopeptides for hPSC adhesion, cultivation and differentiation    16
1-4    Dendrimers    18
1-4-1    Poly(amidoamine) dendrimer    18
1-5    The goal of this study    21
Chapter 2    Materials and Method    22
2-1    Materials    22
2-1-1    Cell lines    22
2-1-2    Commercial Culture Dishes    22
2-1-3    Commercial Coated Substrates    22
2-1-4    Medium for hPSCs    22
2-1-5    Medium and chemicals for differentiation of MSCs from hPSCs    22
2-1-6    Medium and chemicals for cell passages    23
2-1-7    Phosphate buffer saline solution (PBS)    23
2-1-8    The chemicals of Oligopeptide-grafted Hydrogels    24
2-1-9    Chemicals for immunostaining    29
2-1-10    Chemicals for flow cytometry    29
2-2    Experimental instruments    30
2-3    Experimental methods    31
2-3-1    Human pluripotent stem cells maintenance    31
2-3-2    The passage method of human pluripotent stem cells    31
2-3-3    Expansion fold and differentiation ration of hPSCs    31
2-3-4    Immunostaining of hPSCs    33
2-3-5    Embryoid body (EB) formation in vitro    36
2-3-6    Flow cytometry measurements    36
2-3-7    Freezing of human pluripotent stem cells    37
2-3-8    Thawing of human pluripotent stem cells    37
2-4    Preparation of oligopeptide-grafted hydrogels    38
2-4-1    Preparation of PVA-IA coated surface    38
2-4-2    Preparation of different oligopeptide-grafted PVA-IA hydrogels    39
2-4-3    Preparation of dendrimer-based oligopeptide-grafted PVA-IA hydrogels    39
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    42
2-6    Differentiation 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
Chapter 3    Results and discussions    45
3-1    Cultivation of hiPSCs (HPS0077) on different peptide-grafted hydrogels    45
3-1-1    Morphologies of hiPSCs (HPS0077) on different peptide-grafted hydrogels in long-term cultivation    46
3-1-2    Expansion fold of hiPSCs (HPS0077) on different peptide-grafted hydrogels in long-term cultivation    48
3-1-3    Pluripotency analysis of hiPSCs (HPS0077) on different peptide-grafted hydrogels in long-term cultivation    53
3-2    Cultivation of hiPSCs (Mix5) on different peptide-grafted hydrogels    57
3-2-1    Morphology of hiPSCs (Mix5) on different peptide-grafted hydrogels in long-term cultivation    57
3-2-2    Expansion fold of hiPSCs (Mix5) on different peptide-grafted hydrogels in long-term cultivation    59
3-2-3    Pluripotency analysis of hiPSCs (Mix5) on different peptide-grafted hydrogels in long-term cultivation    64
3-3    Correlation coefficient of expansion fold of hiPSCs and zeta potential of peptide-grafted hydrogels    68
3-4    Differentiation of human pluripotent stem cells into mesenchymal stem cells on different peptide-grafted hydrogels    70
3-4-1    Differentiation of hiPSCs-HPS0077 and Mix5 into MSCs on different peptide-grafted hydrogels    70
3-4-2    Differentiation of hESCs-H9 into MSCs on different peptide-grafted hydrogels    74
3-4-3    MSCs surface marker expression of H9-derived hMSCs on different peptide-grafted hydrogels    75
3-5    Cultivation of hiPSCs (HPS0077) on dendrimer-based peptide-grafted hydrogels    76
3-5-1    The optimal grafting method of oligopeptide onto dendrimer-based hydrogels    77
3-5-2    The optimal concentration ratio of dendrimer and crosslinker on dendrimer-based peptide-grafted hydrogels    78
3-5-3    The expansion fold of hiPSCs (HPS0077) on dendrimer-based peptide-grafted hydrogels with different concentration ratio of dendrimer and crosslinker    80
3-5-4    The optimal concentration of oligopeptide on dendrimer-based peptide-grafted hydrogels    81
3-5-5    The expansion fold of hiPSCs (HPS0077) on dendrimer-based peptide-grafted hydrogels with different concentration of oligopeptide    83
3-6    Characterization of different peptide-grafted hydrogels    85
3-6-1    X-ray photoelectron spectroscopy analysis of different peptide-grafted hydrogels    85
3-6-2    Zeta potential analysis of the surface electrical potential on different peptide-grafted hydrogels    88
3-6-3    PrimosCR 45 analysis of the surface roughness on different peptide-grafted hydrogels    90
3-7    Characterization of dendrimer-based peptide-grafted hydrogels    93
3-7-1    X-ray photoelectron spectroscopy analysis of different peptide-grafted hydrogels    93
3-7-2    PrimosCR 45 analysis of the surface roughness on different peptide-grafted hydrogels    98
Chapter 4    Conclusion    101
Reference    104

                                

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