多能幹細胞在無異種條件下分化為間充質幹細 胞的生物材料比較研究

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王君閣(Chun-Ko Wang) 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

多能幹細胞在無異種條件下分化為間充質幹細胞的生物材料比較研究
(Comparative study of biomaterials for hPSCs differentiation into MSCs in xeno-free conditions)

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摘要(中)

移植物抗宿主病(GVHD)是現今器官和造血幹細胞移植的主要受到限制因素之一。許多研究發現，在組織移植時人類間充質幹細胞(hMSCs)可以保護移植組織免受 GVHD 的影響。然而，由於從組織中萃取出的hMSCs受到傳代數量有限和從骨髓中提取過程困難與痛苦的限制。這些限制使hMSCs治療有許多障礙。因此，人類多能幹細胞(hPSCs)衍生的hMSCs (hPSC-derived MSCs)被認為是一種有前途且可無限提供hMSCs的來源。
在這項研究中，我將Xu等人發表的分化方案(Int. J. Biol. Sci., 14 (2018) 1901) 進行一些修改，其中將原始方案中使用的 Matrigel 更改為無異種生物材料。在這研究中我使用人細胞外基質 (ECM) 塗層培養皿作為無異種生物材料，其中包括重組玻連蛋白、層粘連蛋白-511、層粘連蛋白-521、纖連蛋白和膠原蛋白。利用添加骨塑型蛋白-4 (BMP4) 和A83-01抑製劑的方法將細胞從hPSCs分化成hMSCs。最後，為了檢測hPSC-derived MSCs的性質，將使用流式細胞儀分析MSC標誌物（CD44、CD73、CD90 和 CD105）的表達和評估hPSC-derived MSCs分化為成骨細胞、軟骨細胞和脂肪細胞的能力。根據結果，Laminin-521 和 I 型膠原蛋白是hPSCs分化為hMSCs的兩種最佳生物材料。儘管在所有條件下分化的hPSCs都顯示出類似的hMSCs特徵，例如細胞形態，但在這兩種條件下培養的細胞顯示出更好的倍增時間和更高的 MSC 標記表達。因此，Laminin-521 塗層表面被認為是用於hPSC-derived MSCs製備的最可靠和無異種細胞培養生物材料。
另外，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)和層粘連蛋白-β4衍生肽 (PMQKMRGDVFSP) 嫁接的聚乙烯醇-衣康酸水膠上。他們發現添加正電荷序列 (KGG) 可能有助於細胞附著的效率。為了研究正電荷序列 (KGG) 是否可增進hESCs的增殖且同時hESCs在無異種細胞培養條件下能保持其多能性和分化為hMSCs的能力，因此我用不同數量的正電荷序列修改了層粘連蛋白衍生肽的新寡肽（PMQKMRGDVFSP) 。通過比較hESCs長期培養和分化為間充質乾細胞 (hMSCs) 的能力來評估其在新設計的寡肽環境下的生醫領域之應用特性。根據結果，在hESC培養中，我發現KLBC2K（在linker前面加了正電荷氨基酸）和LB2CKKK（在linker和主鏈之間有更多的正電荷關節段）能提供hESCs穩定的長期培養，且可以成功分化為hMSCs。除此之外，在分化成hMSC方法下，hESC-derived MSCs培養在LB2CK和KLB2CK的PVA-IA水凝膠上能顯示出更好的倍增時間和較高的 MSC 標記表達。因此，整合hESC培養與分化成hMSCs的結果，嫁接在PVA-IA水凝膠表面的KLB2CK是一種可靠的新設計的用於 hPSC-derived MSCs分化的寡肽。
未來，將評估hPSC-derived MSCs向成骨細胞、軟骨細胞和脂肪細胞的分化能力。在用同種異體單核細胞處理hPSC-derived MSCs後，利用活死細胞染色法和炎性細胞因子的分泌，以評估hPSC-derived MSCs對 GVHD 的保護作用。預計目前的hPSC-derived MSCs可以在未來廣泛應用於再生醫學。

摘要(英)

Graft-versus-host disease (GVHD) is one of the major limiting factors of organ transplantation and hematopoietic stem cell transplantation. The transplantation of human mesenchymal stem cells (hMSCs) has shown great effects to protect transplanted tissues from GVHD. However, hMSC treatment has the main barrier because of limited passage numbers of hMSCs and painful extraction procedures from bone marrow. Human pluripotent stem cells (hPSCs)-derived hMSCs (hPSC-MSCs) should be a promising and limitless source of hMSCs for patient treatment of GVHD. hMSCs were generated from differentiation of hPSCs using the modified protocol reported by Xu et al. (Int. J. Biol. Sci., 14 (2018) 1901) where Matrigel used in the original protocol was changed to xeno-free biomaterials. hPSCs were differentiated into hMSCs by treating bone morphogenetic protein 4 (BMP-4) and A83-01 inhibitor. The xeno-free protocol for hMSC differentiation was developed using different human extracellular matrix proteins (hECMs)-coated dishes, including recombinant vitronectin, laminin-511, laminin-521, fibronectin, and collagen I. The flow cytometry was used to evaluate the expression of MSC markers (CD44, CD73, CD90, and CD105) on hPSC-MSCs, and the differentiation abilities of hPSC-MSCs into osteoblasts, chondrocytes, and adipocytes were evaluated. Laminin-521 and Collagen type I were found to be the top two best cell culture biomaterials for hPSCs differentiation into hMSCs. Although hPSCs differentiated on all ECM protein-coated dishes showed similar hMSC characteristics such as cell morphology, the cells cultured on Laminin-521-coated and Collagen type I-coated dishes showed good (short) doubling time and the highest MSC marker expression in this study. Therefore, Laminin-521 coated surface is considered to be the most reliable and xeno-free cell culture biomaterial for hPSC-MSCs preparation. Besides, 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) and laminin beta 4-derived peptide (PMQKMRGDVFSP). They found that insertion of the positive joint segment (KGG) on the oligopeptides could improve the hPSC cultivation. Several molecular designs of oligopeptide-grafted hydrogels having an optimal number of positive joint segments were developed in this study, which supported the proliferation of hESCs while hESCs maintained their pluripotency and differentiation ability into hMSCs under xeno-free cell culture conditions. Especially, new oligopeptides of laminin-derived peptides (PMQKMRGDVFSP) with different numbers of positive joint segments were grafted on PVA-IA hydrogels. PVA-IA hydrogels grafted with these synthetic peptides were investigated to support long-term hESCs cultivation and differentiation into hMSCs. PVA-IA hydrogels grafted with KLBC2K (adding the positive amino acid in front of the linker) and LB2CKKK (which has more positive joint segments between the linker and the main chain) were stable for hESC long-term cultivation and can promote differentiation of hESCs into hMSC. After hESC cultivation on those synthetic peptides-grafted PVA_IA hydrogels, hESCs were evaluated for the expression level of four pluripotent protein (Oct4, Sox2, and Nanog) expression. Furthermore, hESC-MSCs cultured on PVA-IA hydrogels grafted with LB2CK and KLB2CK showed better doubling time and higher MSC marker expression than those cultured on PVA-IA hydrogels grafted with another design of peptides in this study. Therefore, the KLB2CK grafted on the PVA-IA hydrogels surface is a reliable new design of oligopeptide for hPSC-MSCs differentiation. Live and dead staining and secretion of inflammatory cytokines were also evaluated after hPSC-MSCs were treated with allogeneic mononuclear cells to evaluate the protective effect of hPSC-MSCs from GVHD. It is expected that the present hPSC-MSCs can be widely applied for regenerative medicine in the future.

關鍵字(中)

★ 幹細胞
★ 生物材料
★ 無異種
★ 間充質幹細胞
★ 多能幹細胞

關鍵字(英)

★ stem cell
★ biomaterial
★ xeno-free
★ MSC
★ PSC

論文目次

Abstract I
摘要 IV
Index of Content VI
Index of Figures XII
Index of Table XX
Chapter 1. Introduction 1
1-1 Stem cell application in clinical trials 1
1-2 Stem cells 2
1-3 Human pluripotent stem cells (hPSC) 4
1-3-1 Human embryonic stem cells (hESCs) 5
1-3-2 Human induced pluripotent stem cells (hiPSC) 6
1-3-3 Characterization of hPSCs 7
1-3-4 The limitations of human pluripotent stem cells 9
1-4 Human mesenchymal stem cells (hMSCs) 10
1-4-1 Characterization of human mesenchymal stem cells 11
1-4-2 Differentiation abilities of human mesenchymal stem cells 13
1-4-2-1 Osteogenic differentiation of stem cells 13
1-4-2-2 Adipogenic differentiation of stem cells 15
1-4-2-3 Chondrogenic differentiation 15
1-4-3 The therapeutic application in future and the limitations of human mesenchymal stem cells 16
1-5 Biomaterials for hPSC cultivation 19
1-5-1 Human PSC culture on feeder-free and xeno-contained proteins 20
1-5-1-1 Matrigel 21
1-5-1-2 Collagen 21
1-5-2 Human PSC culture on feeder-free and xeno-free hECMs 22
1-5-2-1 Vitronectin 25
1-5-2-2 Laminin 25
1-5-2-3 Fibronectin 26
1-5-3 Oligopeptides 26
1-6 Differentiation of hPSCs into mesenchymal stem cells and modification of the differentiation methods 28
1-7 The goal of this study 29
Chapter 2. Materials and Method 32
2-1 Materials 32
2-1-1 Cell line 32
2-1-2 Commercial culture dishes 32
2-1-3 Commercial coating substrates 32
2-1-4 Medium and others 33
2-1-4-1 Cell culture medium 33
2-1-4-2 Cell passage 34
2-1-4-3 Phosphate buffered saline solution (PBS) 34
2-1-4-4 The chemicals in oligopeptide-grafted dish 34
2-1-5 Chemicals for immunostaining 37
2-1-5-1 Primary antibodies 37
2-1-5-2 Second antibody 38
2-1-5-3 Other 38
2-1-6 Chemical for flow cytometry 38
2-1-7 RNA extraction kit 39
2-1-8 Reverse transcription kit 39
2-1-9 Real-time polymerization chain reaction 39
2-1-10 qRT-PCR probe 39
2-1-11 Osteogenic differentiation analysis 40
2-1-11-1 Homemade medium 40
2-1-11-2 Alkaline phosphate assay 40
2-1-11-3 Alizarin red S staining 40
2-1-11-4 von Kossa staining 40
2-1-12 Chondrogenic differentiation analysis 40
2-1-12-1 Alcian blue staining 40
2-1-13 Adipogenic differentiation analysis 41
2-1-13-1 Oil red O staining 41
2-2 Experimental instruments 41
2-3 Experimental methods 41
2-3-1 The protocol of hPSCs differentiation into hMSCs 41
2-3-2 Preparation of the cell culture medium for human mesenchymal stem cells (hMSCs) 43
2-3-3 Passage method of human mesenchymal stem cells 44
2-3-4 Characterization of hMSCs 45
2-3-4-1 Doubling time of hMSCs 45
2-3-4-2 Flow cytometry measurements 46
2-3-5 Osteogenic differentiation 46
2-3-5-1 Preparation of homemade osteogenic differentiation medium 47
2-3-5-2 Alkaline phosphate (ALP) activity measurement 47
2-3-5-3 Alizarin red S staining assay 48
2-3-5-4 von Kossa staining assay 48
2-3-6 Chondrogenic differentiation 49
2-3-6-1 Alcian blue staining of chondrocytes 50
2-3-7 Isolation of mononuclear cells 50
2-3-8 Live and Dead staining of the cells 52
2-3-9 Maintenance of human pluripotent stem cells and their passage 52
2-3-10 Characterization of hPSCs 53
2-3-10-1 Cell density measurement 53
2-3-10-2 Differentiation rate of hPSCs colonies 55
2-3-10-3 Expansion fold change of hPSCs 55
2-3-10-4 Immunofluorescence staining of the cells 55
2-3-10-5 Embryoid body (EB) formation in vitro 59
2-3-11 Characterization of biomaterial surfaces 60
2-3-11-1 X-ray photoelectron spectroscopy (XPS) measurements 60
2-3-11-2 Zeta potential measurements 61
2-3-12 Preparation of PVA-IA hydrogel-coated dish 61
2-3-13 Preparation of oligopeptides grafted PVA-IA hydrogels 63
Chapter 3 Results and Discussion 64
3-1 Differentiation method of hiPSCs into hMSCs 64
3-1-1 The morphology and doubling time of hMSCs differentiation from hPSCs on different ECM protein-coated dishes 66
3-1-2 The morphology and doubling time of hPSCs-derived MSCs on different ECM protein-coated dishes for long-term cultivation 75
3-1-3 Identification of the hESC-derived MSCs cultured on different ECM protein-coated dishes from MSC surface marker expression analysis using flow cytometry 78
3-1-4 Identification of the hiPSC-derived MSCs cultured on different ECM protein-coated dishes from MSC surface marker expressions analysis using flow cytometry 82
3-1-5 Morphology of osteogenic differentiation of hPSC-derived MSCs on different ECM protein-coated dishes at passage 10 88
3-1-6 The early-stage analysis of osteogenic differentiation of hiPSC-derived MSCs on different ECM protein-coated dishes at passage 10 91
3-1-7 The late-stage analysis of osteogenic differentiation of hiPSC-derived MSCs on different ECM protein-coated dishes at passage 10 93
3-2 Cultivation of hPSCs on PVA-IA hydrogels grafted with oligopeptides 97
3-2-1 Cultivation of hPSCs on PVA-IA hydrogels grafted with different oligopeptides derived from laminin β4 chain and vitronectin 97
3-2-2 Expansion fold of hESCs cultured on PVA-IA hydrogels grafted with LN-β4 derived oligopeptides and VN derived oligopeptides for 10 passages 100
3-2-3 Differentiation ratio of hESCs cultured on PVA-IA hydrogels grafted with LN-β4 derived oligopeptides and VN derived oligopeptides for 10 passages 102
3-2-4 Immunostaining of hESCs cultured on PVA-IA hydrogels grafted with LN-β4 derived oligopeptides and VN derived oligopeptides for 5 and 10 passages 103
3-2-5 SSEA4 surface marker expression of hESCs cultured on PVA-IA hydrogels grafted with LN-β4 derived oligopeptides and VN derived oligopeptides for 10 passages via flow cytometry 107
3-2-6 Embryoid body formation of hESCs cultured on PVA-IA hydrogels grafted with LN-β4 derived oligopeptides and VN derived oligopeptides for 5 passages 108
3-3 Characterization of PVA-IA hydrogels grafted with previous and new designs of oligopeptides 111
3-3-1 Zeta potential analysis of the effect of surface charge on different biomaterials 111
3-3-2 X-ray photoelectron spectroscopy (XPS) analysis for different biomaterials 113
3-4 hESCs differentiation into hMSCs on PVA-IA hydrogels grafted with LN-β4 derived oligopeptides and VN derived oligopeptides 117
3-4-1 The morphology and doubling time of hESC differentiation into hMSCs on different PVA-IA hydrogels grafted with LN-β4 derived oligopeptides and VN derived oligopeptides 118
3-4-2 Flow cytometry of the hPSC-MSCs cultured on different PVA-IA hydrogels grafted with LN-β4 derived oligopeptides for MSC surface marker analysis 121
Chapter 4. Conclusions 124
References 127
Supplementary Data 140

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指導教授

樋口亞绀(Akon Higuchi)

審核日期

2021-8-9

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