人類羊水間葉幹細胞培養於具有奈米片段與最佳表面硬度的生醫材料，其增殖與成骨分化能力

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沐督利(Saradaprasan Muduli) 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

人類羊水間葉幹細胞培養於具有奈米片段與最佳表面硬度的生醫材料，其增殖與成骨分化能力
(Proliferation and Osteogenic Differentiation of Human Amniotic Fluid derived Stem Cells Cultured on Biomaterials Having Nanosegments and Optimal Elasticity)

相關論文

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★ 利用具有奈米片段的生醫材料進行純化及去除癌症幹細胞	★ 羊水間葉幹細胞培養於細胞外間質及材料硬度/彈性表面,其分化能力及多能性之研究
★ 人類脂肪幹細胞的膜純化法與分化能力研究	★ 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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摘要(中)

人類羊水間葉幹細胞（hAFSCs）為多能性胎兒細胞，具有能夠分化成特定細胞的能力，包括代表性的三個胚層。AFSCs可能成為幹細胞在再生醫學和組織工程更合適的來源。然而，由於幹細胞的特性，如適當的分化和多能性的維持，不只調節幹細胞本身，也可利用細胞的培養環境。此外，像是調整細胞培養基材的物理特性如軟硬度，可能會影響幹細胞分化的命運。在這個研究裡，調配在基材上不同的軟硬度，並固定有細胞外基質衍生的寡肽的細胞培養基材培養hAFSCs的成骨分化的效率。利用調配具有不同的軟硬度的polyvinylalcohol-co-itaconic acid（PVA-IA）在培養皿上，通過控制交聯劑的交聯時間製備。將PVA-IA培養皿通過N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (（EDC）和N-hydroxysuccinimide（NHS）去接枝上細胞外基質（ECM）衍生的寡肽。在這項研究中選擇了5種不同類型的寡肽，維持 AFSCs的多能性，藉由測定qRT-PCR測量，利用多能性基因的表現（Nanog的，Oct4和Sox2的）找出細胞培養基質的最佳軟硬度。為了分析hAFSCs的成骨分化特性，在兩週和四周利用誘導培養液誘導分化後進行alkaline phosphatase（ALP），alizarin Red S staining和von Kossa staining。利用物理特性如培養材料的軟硬度以及細胞外基質成分的生物特性可以誘導並決定hAFSCs分化成成骨細胞。

摘要(英)

Human amniotic fluid-derived stem cells (hAFSCs) are pluripotent fetal cells capable of differentiation into multiple lineages, including representatives of the three embryonic germ layers. AFSCs may become a more suitable source of stem cells in regenerative medicine and tissue engineering. However, stem cell characteristics, such as proper differentiation and maintenance of pluripotency, are regulated not only by the stem cells themselves but also by their microenvironment. Furthermore, physical characteristics of cell culture substrates such as substrate elasticity may influence the fate of stem cell differentiation. I investigated the efficiency of osteogenic differentiation of hAFSCs cultured on cell culture substrates which have different elasticities and are immobilized with extracellular matrix-derived oligopeptides. The dishes coated with polyvinylalcohol-co-itaconic acid (PVA-IA) films having different elasticities were prepared by controlling the crosslinking time in crosslinking solution that contains glutaraldehyde. The PVA-IA dishes were grafted with extracellular matrix (ECM)-derived oligopeptides through N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry in an aqueous solution. Five different types of oligopeptides were selected in this study. Pluripotent gene expressions (Nanog, Oct4 and Sox2) were evaluated by the qRT-PCR measurements. There was an optimal elasticity of cell culture matrix to keep pluripotency of AFSCs for their culture. To characterize osteogenic differentiation of hAFSCs, alkaline phosphatase (ALP) activity, alizarin Red S staining and von Kossa staining were evaluated after two weeks and four weeks of culture in induction media. It is suggested that physical cues such as stiffness of culture materials as well as biological cues of extracellular matrix components can guide and decide differentiation of hAFSCs into osteoblasts.

關鍵字(中)

★ 人類羊水間葉幹細胞
★ 細胞外基質

關鍵字(英)

★ Human amniotic fluid derived stem cells
★ Extracellular matrix

論文目次

Chapter 1: Introduction 1
1-1 Regenerative medicine 1
1-1-1 Use of stem cells in regenerative medicine 1
1-2 What is a stem cell? 2
1-2-1 Self-renewal 2
1-2-2 Potency 3
1-2-3 Where do stem cells come from? 3
1-3 Types of stem cells 4
1-3-1 Embryonic stem cells (ESCs) 4
1-3-2 Induced pluripotent stem cells (iPSCs) 6
1-3-3 Adult stem cells 7
1-4 Amniotic fluid 9
1-4-1 Definition 9
1-4-2 Contents of amniotic fluid 9
1-4-3 Clinical significance of amniotic fluid 10
1-4-4 Isolation of amniotic fluid by amniocentesis 10
1-5 Amniotic fluid-derived stem cells 11
1-5-1 Cells present in amniotic fluid 11
1-5-1 Cells present in amniotic fluid 12
1-5-2 Isolation of amniotic fluid derived stem cells (AFSCs) 15
1-5-3 Characterization of amniotic fluid derived stem cells 15
1-6 Stem cell microenvironments 19
1-6-1 Cell-soluble factor interactions 19
1-6-2 Cell-cell interactions 20
1-6-3 Cell-biomaterial interactions 20
1-7 ECM and ECM-mimicking oligopeptides 22
1-7-1 Type and classification of artificial ECMs 24
1-7-2 ECM protein-derived peptides 26
1-7-3 The effect of extracellular matrix (ECM) on stem cells 27
1-8 Osteogenic differentiation 28
1-8-1 The process of bone development in situ 28
1-8-2 Developmental pathways for bone formation 29
1-8-3 The marker of osteogenic differentiation 33
1-9 Cardiomyocyte differentiation 34
1-9-1 Developmental pathways for Heart formation 34
1-9-2 Transcription factor programs in the myocardium 35
1-9-3 Meta-analysis of protocols for cardiomyocyte differentiation of MSCs 36
Chapter 2: Materials and methods 41
2-1 Materials 41
2-1-1 Cell culture medium 41
2-1-3 Serum 41
2-1-4 Antibiotics 41
2-1-5 Growth factor 42
2-1-6 PVA-IA film 42
2-1-7 ECM-derived peptides 42
2-1-9 RNA extraction 43
2-1-10 Reverse transcription (RT) 43
2-1-11 Quantitative PCR (qPCR) 43
2-1-12 qPCR Probes 43
2-2 Methods and Analysis 44
2-2-1 PVA-IA film preparation 44
2-2-2 Preparation of PVA-IA surfaces grafted with ECM-derived oligopeptides 45
2-2-5 Phosphate buffered saline (PBS) preparation 46
2-2-6 Preparation of FGF-2 (b-FGF) protein stock solution 46
2-2-7 Preparation of 5-Azacytidine stock solution 46
2-2-8 Preparation of cell culture medium 46
2-2-9 Cell cultivation 47
2-2-10 Counting of total cell number 48
2-2-11 Isolation of total RNA 49
2-2-12 Reverse Transcription of mRNA into cDNA 50
2-2-12 Quantitative real time polymerase chain reaction 51
2-2-13 Imunofluorescence assay 53
2-2-14 Alkaline phosphatase activity 54
2-2-15 Alizarin Red staining 54
2-2-15 von Kossa staining 55
2-2-16 Quantitative analysis of osteogenesis 55
Chapter 3: Results and Discussion 56
3-1 Isolation of hAFSC from human amniotic fluid on surface modified biomaterials 56
3-2 Proliferation of hAFSCs on PVAIA hydrogels grafted with different oligopeptides having different elasticities 61
3-3 Pluripotency of hAFSCs cultured on PVA-IA hydrogels grafted with and without oligopeptides, which have different elasticities 72
3-4 Osteogenic differentiation of hAFSCs cultured on PVA-IA hydrogels grafted with and without oligopeptides, which have different elasticities 77
3-5 Cardiomyogenic differentiation of hAFSCs in xeno-free condition 88
Chapter 4: Conclusion 93
References 95

Index of Figures
Figure1-1 Bone marrow transplant. 2
Figure1-2 Isolation and culture of ESCs from blastocysts. 5
Figure1-3 Generation of induced pluripotent stem cells (iPSCs) [31]. 6
Figure1-4 Morphology of MSCs derived from human amniotic fluid. 7
Figure1- 5 MSC differentiation 8
Figure1-6 Human fetus surrounded by amniotic fluid. 9
Figure1-7 Isolation of amniotic fluid by amniocentesis. 11
Figure1-9 Mature primary colonies of amniotic fluid cells after 14-16 days in culture: (a) F-type, (b) AF-type, (c) E-type [37]. 13
Figure1-10 Surface markers and pluripotent markers expression of hAFSCs [16]. 16
Figure1-11 ECM proteins guide mesenchymal stem cell fate [30]. 18
Figure1-12 The microenvironment of stem cells [30]. 19
Figure1- 13 Tissue elasticity and differentiation of native MSCs [75]. 22
Figure1-14 Osteogenic differentiation of MSCs. 29
Figure1-15 Runx2 is a mediator of molecular switches for bone development [150]. 32
Figure1-16 Temporal prolife of homeodomain proteins during the BMP2 induced 32
Figure1-17 Model of a molecular pathway for cardiac development [203]. 36
Figure1-18 Meta analysis of protocols for cardiomyocyte differentiation of MSCs. 38
Figure 2-1 Preparation of surface modified dishes with different elasticities and grafted with different oligopeptides. 45
Figure 2-2 Hemocytometer with 18 grids to count total cell number. 49
Fig 3-2 Growth curve of primary (passage 0) hAFSCs on ECM-coating and non-coating TCPS dishes in DMEM medium with 20% FBS. 59
Fig 3-3 Doubling time of primary (passage 0) hAFSCs cultured on ECM-coating and non-coating TCPS dishes in DMEM medium with 20% FBS. 59
Fig 3-4 Detachment of primary (passage 0) hAFSCs from ECM-coating and non-coating TCPS dishes by treating trypsin/EDTA for 5 minutes. The hAFSCs attachment on the Synthemax-coated dish is the strongest compared to that of other ECM-coating and non-coating TCPS dishes. 60
Figure 3-5 Morphology of hAFSCs cultured on PVA-IA hydrogels grafted with and without oligovitronectin (oligoVN) which have different elasticities (10.6, 11.1, 12.2 and 18.3 kPa), in DMEM with 20% FBS on day 3 of passage 4. 63
Figure 3-6 Morphology of hAFSCs cultured on PVA-IA hydrogels grafted with and without oligovitronectin (oligo-VN) which have different elasticities (25.3 and 30.4 kPa) and on TCPS (3700 kPa), in DMEM with 20% FBS on day 3 of passage 4. 64
Figure 3-7 Morphology of hAFSCs cultured on PVA-IA hydrogels grafted with and without different oligopeptides which have elasticity of 12.2 kPa, in DMEM with 20% FBS on day 3 of passage 4. 65
Figure 3-8 Morphology of hAFSCs cultured on PVA-IA hydrogels grafted with and without different oligopeptides which have elasticity of 18.3 kPa, in DMEM with 20% FBS on day 3 of passage 4. 66
Figure 3-9 Morphology of hAFSCs cultured on PVA-IA hydrogels grafted with and without different oligopeptides which have elasticity of 25.3 kPa, in DMEM with 20% FBS on day 3 of passage 4. 67
Figure 3-10 Morphology of hAFSCs cultured on PVA-IA hydrogels grafted with and without different oligopeptides which have elasticity of 30.4 kPa, in DMEM with 20% FBS on day 3 of passage 4. 68
Figure 3-11 Growth curve of hAFSCs cultured on TCPS dishes and PVA-IA hydrogels grafted with and without oligopeptides having elasticity of 12.2 kPa in DMEM with 20% FBS at passage 4. 69
Figure 3-13 Growth curve of hAFSCs cultured on PVA-IA hydrogels grafted with and without oligopeptides which have elasticity of 25.3 kPa, in DMEM with 20% FBS at passage 4. 70
Figure 3-14 Growth curve of hAFSCs cultured on PVA-IA hydrogels grafted with and without different oligopeptides which have elasticity of 30.4 kPa, in DMEM with 20% FBS at passage 4.. 70
Figure 3-15 Doubling time of hAFSCs cultured on TCPS, PVA-IA hydrogels grafted with and without oligopeptides which have different elasticities (12.2, 19.6, 25.3 and 30.4kPa), in DMEM with 20% FBS at passage 4. 71
Figure 3-16 SOX2 gene expression of hAFSCs on PVA-IA hydrogels grafted with and without oligopeptides, which have different elasticities (12.2, 19.6, 25.3, and 30.4kPa). 73
Figure 3-17 OCT4 gene expression of hAFSCs on PVA-IA hydrogels grafted with and without oligopeptides, which have different elasticity (12.2, 19.6, 25.3, and 30.4kPa). 75
Figure 3-18 Timeline and characterization for osteogenic differentiation of hAFSCs. 79
Figure 3-19 ALP activity of hAFSCs on PVA-IA hydrogels grafted with and without oligo-VN, which have elasticity of 10.6 kPa (2h) to 30.4 kPa (48h) after culturing in osteogenic induction medium for 14 days. 79
Figure 3-20 ALP activity of hAFSCs on PVA-IA hydrogels grafted with different oligopeptides, which have different elasticities after culturing in osteogenic induction media for 14 days. 80
Figure 3-21 Alizarin Red S staining of hAFSCs cultured on PVA-IA hydrogels grafted with and without oligopeptides, which have different elasticities after culturing in osteogenic induction medium for 28 days. 83
Figure 3-22 Percentage of Alizarin Red S staining cells analyzed by using Image J system (NIH). 83
Figure 3-23 von-Kossa staining (calcium phosphate deposition) of hAFSCs cultured on PVA-IA hydrogels grafted with and without oligo-peptides, which have different elasticities after culturing in osteogenic induction media for 28 days. 86
Figure 3-24 Percentage of von-Kossa staining cells analyzed by using image J system (NIH). 86
Figure 3-25 Differentiation protocol for chemically defined generation of cardiomyocytes from hAFSCs. 89
Figure 3-26 Morphology of hAFSCs cultured on TCPS dishes in growth medium (MesenCult-xenofree medium) for 5 days (day -5 to day 0) before differentiating into cardiomyocytes and then cultured in differentiation medium (CDM3) for 10 days (day 0 to day 10). 90
Figure 3-27 Immunostaining of sarcomeric proteins (α-SMA & cTnT) on hAFSCs at day 4, day 8, day 10 and day 12 of cardiomyocyte differentiation in CDM3 medium. 92

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指導教授

樋口亞绀(Akon Higuchi)

審核日期

2016-7-18

推文