羊水間葉幹細胞培養於細胞外間質寡?嫁接具有硬度/彈性表面的材料,其分化能力及多能性之研究

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王品毓(Pin-Yu Wang) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

羊水間葉幹細胞培養於細胞外間質寡?嫁接具有硬度/彈性表面的材料,其分化能力及多能性之研究
(Proliferation and Differentiation of Amniotic Fluid-Derived Stem Cells on Oligopeptide-Grafted Surface Having Different Elasticity)

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摘要(中)

從人類羊水來源的幹細胞是一種多能性幹細胞，因為其具備分化多種譜系的能力，包括代表性的三個胚層。因此，羊水來源幹細胞可能成為一個更適合的幹細胞在再生醫學和組織工程。此外，細胞培養基材的物理特性會影響幹細胞分化的命運。然而，在過去研究報告上沒有探討出最佳的組合以結合物理線索中的彈性及生物線索中的細胞培養基質，以保持幹細胞的多能性和長時間細胞的培養。
因此，在我的研究中，為了探討維持羊水幹細胞的多能性以及如何使羊水幹細胞有效地分化。因為我將羊水來源幹細胞培養於具有不同軟硬度的水凝膠且固定細胞外基質衍生的寡?以提升羊水幹細胞的多能性基因且有效調節細胞的分化能力。一開始，準備polyvinylalcohol-co-itaconic acid (PVA-IA)塗層於細胞培養層上，藉由不同交聯時間已達成控制PVA-IA具有不同的軟硬度，經過活化的程序，便能將細胞外基質衍生的寡?固定在PVA-IA塗層上，最後將羊水幹細胞培養於這些盤子上，以便後續的分析及探索。經過細胞經過數天的培養後，將種於不同條件的羊水幹細胞取其檢測多能性基因：Oct4, Nanog, 和Sox2，和分化基因:Nestin, Sox17, 和Runx2。在最後的檢測結果指出vitronectin來源寡?固定於低彈性的PVA-IA塗層具有較大的潛能以提升羊水幹細胞的多能性；而且在不同軟硬度的PVA-IA塗層能使羊水分化成相似於基材軟硬度的細胞類型，例如：當羊水幹細胞培養於較低彈性的PVA-IA嫁接塗層，會使的羊水幹細胞偏好分化成神經類型細胞；然而，當細胞培養於較高彈性的PVA-IA嫁接途層，將使的細胞分化成成骨幹細胞型態，這項研究結果報導出與先前研究一樣的結果。
因為這項研究中指出，結合培養材料的鋼度以及細胞外基質成份的生物線索可以引導和決定幹細胞多能細和分化的譜系。另一方面，我們進一步發現，不同軟印度和細胞外基質寡?之間的合作將有效地調節羊水幹細胞分化成不同譜系的承諾。

摘要(英)

Stem cells derived from human amniotic fluid (AFSCs) are pluripotent fetal cells capable of differentiating into multiple lineages, including representatives of the three embryonic germ layers. Therefore, 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 notregulated only by the stem cells themselves but also by their microenvironment. Furthermore, physical characteristics of cell culture substrate influence the fate of stem cell differentiation. However, there have been no reports from our database study that investigates the optimal elasticity to keep pluripotency of stem cells for a long time and studies the optimal combination of physical cue and biological cue on cell culture substrates. Here I report pluripotent maintenance and differentiation efficiency of AFSCs cultured on cell culture substrates immobilized extracellular matrix-derived oligopeptides, which have different elasticity. We prepared dishes coated with polyvinylalcohol-co-itaconic acid (PVA-IA) films having different elasticity by controlling the crosslinking time in crosslinking solution containing glutaraldehyde, and grafted with several ECM-derived cell-adhesion peptides though N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride(EDC) and N-hydroxysuccinimide (NHS) chemistry in an aqueous solution. qRT-PCR measurements suggest that pluripotent genes, Nanog, Oct4 and Sox2, were kept on PVA-IA hydrogels grafted with oligopeptide derived from vitronectin (oligoVN) and fibronectin (oligoFN) having moderate elasticity around 12-25kPacompared toAFSCs on conventional tissue culture dishes. I found that there is an optimal elasticity of cell culture matrix to promote pluripotency of AFSCs for their culture. Furthermore, early differentiation marker of osteoblasts (Runx2) were found on AFSCs cultured on stiffer PVA-IA hydrogels grafted with oligopeptides derived from Vitronectin (oligoVN) in expansion medium without any induction (differentiation) components, suggesting stiffer culture substrates grafted with oligoVNguide AFSCs into osteoblast lineage, whereas early neural differentiation marker of nestin were more expressed on softer PVA-IA hydrogels grafted with oligoFN in the expansion medium, suggesting softer culture substrates grafted with oligoFN and oligoVN promote differentiation into neural cell lineages. 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 pluripotency and differentiation lineages of stem cells. On the other hands, I further discoveredthatAFSCs cultured on thecooperation between ECM-ligands and stiffness matrices have been engaged into different lineages commitments.

關鍵字(中)

★ 多能性幹細胞
★ 羊水幹細胞

關鍵字(英)

★ pluripotent stem cell
★ Amniotic Fluid-Derived Stem Cells

論文目次

CHAPTER ONE INTRODUCTION 1
1-1 Stem cell 1
1-1-1 Potency of stem cells 2
1-1-2 Totipotency 2
1-1-3 Pluripotency 2
1-1-4 Multipotency 2
1-2 Souces of stem cells 2
1-2-1 Embryonic stem cells (ESCs) 3
1-2-2 Induced Pluripotent Stem Cells 4
1-2-3 Mesenchymal stem cells (MSCs) 4
1-3 Amniotic fluid-derived stem Cells 6
1-3-2 Amniotic fluid cell type 7
1-3-3 Isolation of amniotic fluid stem cell (AFSCs) 10
1-3-4 Characterization of amniotic fluid stem cell 10
1-3-5 Pluripotency of amniotic fluid stem cells 12
1-4 Niches of stem cells 15
1-4-1 Soluble factors 16
1-4-2 Cell-cell interactions 16
1-4-3 Physical cues affect ex vivo expansion 16
1-4-4 Cell-biomaterials interactions 20
1-5 Extracellular-matrix (ECM) and ECM-mimicking oligopeptides 20
1-5-1 Type and classification of artificial ECMs 23
1-6 The effect of extracellular matrix (ECM) to stem cells 25
1-7 Markers of pluripotent gene 26
1-8 Markers of differentiation lineages of MSCs 28
1-9 Polymerase chain reaction (PCR) 30
1-9-1 Procedure of PCR 30
1-9-2 Reverse transcription polymerase chain reaction (RT-PCR) 32
&Quantitative real time polymerase chain reaction(qRT-PCR) 32
1-9-3 Procedure of RT-PCR 33
CHAPTER TWO MATERIALS AND METHODS 35
2-1 Materials 35
2-1-1 Cultured medium 35
2-1-2 Serum 35
2-1-3 Antibiotics 35
2-1-4 Growth factor 35
2-1-5 PVA-IA film 35
2-1-6 ECM proteins, ECM-derived peptides and CellStart 36
2-1-8 RNA extraction 36
2-1-9 Reverse transcriptase (RT) 36
2-1-10 Real-time PCR (qPCR) 36
2-1-11 Probes for qPCR 37
2-2 Methods and Analysis 37
2-2-1 PVA-IA film preparation 37
2-2-2 Preparation of PVA-IA surfaces grafted with ECM and ECM-derived oligopeptides 39
2-2-3 Elasticity measurement of PVA-IA 40
2-2-4 XPS analysis of dish surface 41
2-2-5 Phosphate buffer saline (PBS) preparation 41
2-2-6 Preparation of FGF-2(β-FGF) protein stock solution 42
2-2-7 Preparation of cell culture medium 42
2-2-8 Cell cultivation 42
2-2-9 Cell density measurement 43
2-2-10 Isolation of total RNA 44
2-2-11 Reverse Transcription of mRNA into cDNA 44
2-2-12 Quantitative real time polymerase chain reaction 45
2-2-13 Imunofluorescence assay 46
CHAPTER THREE RESULTS 49
3-1 The elasticity of the PVA-IA films 49
3-2 XPS measurements of the PVA-IA films (12.2kPa &25.3kPa) grafted with ECM and ECM-derived peptides 52
3-3 Proliferation of AFSCs cultured on soft and stiff PVA-IA films grafted with ECM/ECM-derived peptides 61
3-4 Up-regulating pluripotency of AFSCs cultured on soft and stiff PVA-IA films grafted with and ECM and ECM-derived peptides 70
3-5 Directing AFSC fates on PVA-IA films by controlling matrix elasticity and extracellular adhesion ligand 76
CHAPTER FOUR DISSCUSION 87
CHAPTER FIVE CONCLUSION 91

INDEX OF FIGURES
Figure 1-1 Stem cell pathways [3]. 1
Figure 1-2 Embryonic stem cells are isolation from the inner cell mass of blastocyst [19]. 3
Figure 1-3 Morphology of MSCs derived from amniotic stem fluid. 5
Figure 1-4 MSC differentiation [28]. 5
Figure 1-5 The time line of pregnant period. 7
Figure 1-6 Mature primary colonies of amniotic fluid colony cells after 14-16 days in culture:(a) F-type, (b) AF-type, (c) E-type [30]. 8
Figure 1-7 Extraction of amniotic fluid [37]. 11
Figure 1-8 Surface marker expression of hAFSCs [10]. 12
Figure 1-9 The microenvironment and niches of stem cells and their regulation [26]. 15
Figure 1-10 Tissue elasticity and differentiation of native MSCs [63]. 18
Figure 1-11 Substrate stiffness influenced adhesion structures and dynamics [66]. 19
Figure 1-12 Actin cytoskeleton on cell-on-cell layering [70]. 19
Figure 1-13 PCR mechanism [154]. 31
Figure1-14 Flow chart of the technical steps in qRT-PCR [160]. 33
Figure1-15 Process of qRT-PCR. 34
Figure 2-1 The process of cross linked PVA-IA coating dish grafted with nano-segment. 38
Figure 2-2 Controlling the stiffness of PVA-IA films. 38
Figure 2-3 ECMs and ECM-derived peptides used in this experiment. 39
Figure 2-4 Two major types of immunofluorescence staining. 48
Figure 3-1 The storage modulus of crosslinking PVA-IA films at 1Hz angular frequency. 50
Figure 3-2 The storage modulus of crosslinking PVA-IA films at different angular frequency. 51
Figure 3-3 The storage modulus of crosslinking PVA-IA films at 1Hz of angular frequency. 52
Figure 3-4 The elemental analysis of PVA-IA films (12.2kPa) grafted with ECM and ECM-derived peptides by X-ray photoelectron spectroscopy. 54
Figure 3-5 The elemental analysis of PVA-IA films grafted with ECM and ECM-derived peptides and unmodified PVA-IA film (12.2kPa) by X-ray elemental photoelectron spectroscopy. 55
Figure 3-6 The elemental analysis of PVA-IA films (25.3kPa) grafted with ECM and ECM-derived peptides by X-ray photoelectron spectroscopy. 56
Figure 3-7 The analysis of PVA-IA films grafted with ECM, ECM-derived peptides and unmodified PVA-IA film (25.3kPa) by X-ray photoelectron spectroscopy. 57
Figure 3-8 The elemental analysis of TCPS by X-ray photoelectron spectroscopy. 58
Figure 3-9 O/C ratio of TCPS, PVA-IA films grafted with ECM and ECM-derived peptides and unmodified PVA-IA film. 59
Figure 3-10 N/C ratio of TCPS, PVA-IA films grafted with ECM and ECM-derived peptides and unmodified PVA-IA film. 59
Figure 3-11 The morphologies of AFSCs cultured on PVA-IA films and PVA-IA films (10.8 and 14.6kPa) grafting with COL and COL-derived peptides at day 7 culture. 63
Figure 3-12 The morphologies of AFSCs cultured on PVA-IA films (10.8 and 14.6kPa) grafting with FN, VN and their derived peptides at day 7 culture. 64
Figure 3-13 The morphologies of AFSCs cultured on PVA-IA films and PVA-IA films (12.2 and 25.3kPa) grafting with COL and COL-derived peptides at day 7 culture. 65
Figure 3-14 The morphologies of AFSCs cultured on PVA-IA films (12.2 and 25.3kPa) grafting with FN, VN and their derived peptides at day 7 culture. 66
Figure 3-15 The morphologies of AFSCs cultured on CellStart and TCPS at day 7 culture. 67
Figure 3-16 Growth curve of AFSCs cultured on soft (12.2kPa) PVA-IA films grafted with ECM and ECM-derived peptides at passage 4. 68
Figure 3-17 Growth curve of AFSCs cultured on stiff (25.3kPa) PVA-IA films grafted with ECM and ECM-derived peptides at passage 4. 68
Figure 3-18 Doubling time of AFSCs cultured on PVA-IA films (12.2kPa and 25.3kPa) grafted with ECM and ECM-derived peptides at passage 4. 69
Figure 3-19 Pluripotent genes expression of AFSCs on soft and stiff (12.2 &25.3kPa) PVA-IA films immobilized with ECM and ECM-derived peptides were measured by real-time PCR. 73
Figure 3-20 Pluripotent gene expression of AFSCs on the soft (12.2kPa) PVA-IA films immobilized with ECM and ECM-derived peptides. 74
Figure 3-21 Pluripotent gene expression of AFSCs on the stiff (25.3kPa) PVA-IA films immobilized with ECM and ECM-derived peptides. 75
Figure 3-22 Lineage-specific gene expression of AFSCs cultured on the soft and stiff (12.2 & 25.3 kPa) PVA-IA films immobilized with ECM and ECM-derived peptides by qPCR. 79
Figure 3-23 Immunofluorescences staining for Hochest (blue), Sox2 (green) and β3-tubulin (red) in AFSCs after 7 days of culture on different dishes. 81
Figure 3-24 Immunofluorescences staining for Hochest (blue), Sox2 (green) and β3-tubulin (red) in AFSCs after 7 days of culture on different dishes. 82
Figure 3-25 Immunofluorescences staining for Hochest (blue), Oct4 (red) and α-SMA (green) in AFSCs after 7 days of culture on different dishes. 83
Figure 3-26 Immunofluorescences staining for Hochest (blue), Oct4 (red) and α-SMA (green) in AFSCs after 7 days of culture on different dishes. 84
Figure 3-27 Immunofluorescences staining for Hochest (blue), SSEA4 (green) and AFP (red) in AFSCs after 7 days of culture on different dishes. 85
Figure 3-28 Immunofluorescences staining for Hochest (blue), SSEA4 (green) and AFP (red) in AFSCs after 7 days of culture on different dishes. 86
Figure 4-1 Pluripotency of AFSCs cultured on soft and stiff PVA-IA films (12.2 & 25.3kPa) immobilized with ECM and ECM-derived peptides. 89
Figure 4-2 The correlation between pluripotency and specific lineages of differentiation of AFSCs cultured on soft and stiff PVA-IA films (12.2 & 25.3kPa) immobilized with ECM and ECM-derived peptides. 90

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指導教授

?口亞紺(Akon Higuchi)

審核日期

2014-6-27

推文