重組及培養人類誘導型多能性幹細胞在奈米片段接枝表面

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林佳逸(Jia-yi Lin) 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

重組及培養人類誘導型多能性幹細胞在奈米片段接枝表面
(Reprogramming and Culture of Human Induced Pluripotent Stem Cells on a Substrate with Immobilized Cell Adhesion Peptides)

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摘要(中)

人類誘導型多能性幹細胞由於具有分化成三胚層細胞之能力，在臨床上相當具有潛力用於許多疾病的治療。然而人類誘導型多能性幹細胞由於飼養層具有異種來源，在臨床上發展受到許多限制。使用具有奈米片段表面改質之生醫材料在無飼養層之培養環境下，可以提供具有再現性的培養環境、低成本的培養材料並且不會引入異種汙染源。這些條件將可以改善人類誘導型多能性幹細胞在臨床上之運用。在這篇文章中我們在使用老鼠胚胎纖維母細胞作為飼養層環境下，成功利用具有多能性基因之逆轉錄病毒，將人類脂肪幹細胞誘導成人類誘導型多能性幹細胞。人類誘導型多能性幹細胞群落可以在病毒轉染後第八天觀察到，從一開始種植105個人類脂肪幹細胞，經過誘導後可以在10cm飼養層上總共得到484個群落，而其誘導成多能性幹細胞之效率為0.484%。此外，鹼性磷酸?活性及多能性蛋白指標Oc4,Sox2及SSEA-4都清楚的表現在人類誘導型多能性幹細胞之群落上。
我們同時也利用聚乙烯醇-共-衣康酸之薄膜上接枝奈米片段，建立新的無飼養層之培養系統用於培養人類多能性幹細胞。在這篇研究中，隨著增加由玻連蛋白中找到之寡胜?的濃度，人類多能性幹細胞將表現較高之群落貼附率、較高群落數放大率及較低群落分化率。此外也利用控制聚乙烯醇-共-衣康酸薄膜之交聯時間，調控薄膜之表面軟硬度，從10.3千帕斯卡至30.4千帕斯卡。在這裡使用人類胚胎幹細胞(WA09)培養在具有較硬表面之聚乙烯醇-共-衣康酸細胞培養系統，比起較表面具有較好的表現。另外，在經過數代培養在表面接枝500毫克/毫升濃度之寡胜?在25.3千帕斯卡表面軟硬度之聚乙烯醇-共-衣康酸細胞培養系統，人類胚胎幹細胞群落仍然表現鹼性磷酸?活性及多能性蛋白指標Oct4、Sox2、Nanog、SSEA-4、Tra-1-60及Tra-1-81。這些結果顯示人類多能性幹細胞可以培養在我們的聚乙烯醇-共-衣康酸細胞培養系統，並且維持人類幹細胞之多能性表現。在未來，聚乙烯醇-共-衣康酸細胞培養系統可用作具有無異種來源及無飼養層之培養環境來誘導人類組織細胞形成人類多能性幹細胞。

摘要(英)

Human induced pluripotent stem cells (hiPSCs) have significant potential in therapeutic applications for many diseases because they have the specific ability to differentiate into all types of human somatic cells. However, the tentative clinical potential of hiPSCs is restricted by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. The feeder-free cultures using synthetic biomaterials having nanosegments as stem cell culture materials offer more reproducible culture conditions and lower the cost of production without introducing xenogenic contaminants. These improvements will increase the potential clinical applications of differentiated hiPSCs. Here we report that hiPSCs can be successively generated with usage of a feeder layer of MEFs during generation of hiPSCs by transfection of retrovirus containing pluripotent genes into human adipose-derived stem cells (hADSCs). hiPSC colonies were clearly observed for the cells cultured on MEFs at day 8 after transfection The number of colonies generated on MEFs was 484 per 10 cm dishes, when 105 hADSCs were seeded on the dishes. The efficiency of hiPSC generation on MEFs was 0.484%. Furthermore, the hiPSC colony showed alkali phosphatase activity clearly, and immunofluorescence suggested that the hiPSCs were generated on MEFs expressing pluripotent protein of Oct4, Sox2 and SSEA-4.
At same time, polyvinylalcohol-co-itaconic acid (PVA-IA) films grafted with nanosegment (KGGPQVTRGDVFTMP [cell-binding domain derived from vitronectin, oligoVN] was established for cultivation of human pluripotent stem cells. In this study, with increase the concentration of oligo-VN, hPSCs shown the higher colony attachment ratio, colony expansion fold and lower differentiation ratio. Furthermore, the elasticity of PVA-IA films grafted with oligo-VN was regulated from 10.3 kPa to 30.4kPa by control of crosslinking time of PVA-IA. hESCs (WA09) cultured on PVA-IA culture system with stiffer elasticity of surface (25.3kPa) shown the better performance than the soft one (15.8kPa). Moreover, hESCs (WA09) cultured on PVA-IA films grafted with 500μg/ml oligo-VN with 25.3kPa elasticity of surface for 5 passage shown alkali phosphatase activity and pluripotent protein expression such as Oct4, Sox2, Nanog, SSEA-4, Tra-1-60 and Tra-1-81. This result indicates that hPSCs could be cultured on our PVA-IA culture system and maintain pluripotency of hPSCs. In the future, the PVA-IA culture system could be used to generation of hiPSCs from primary human tissue cells on xenogenic-free and feeder-free conditions.

關鍵字(中)

★ 多能性幹細胞
★ 生醫材料

關鍵字(英)

★ pluripotent stem cell
★ Cell Adhesion Peptides

論文目次

Chapter 1: Introduction 1
1-1 Stem Cells 1
1-1-1 Self Renewal and Lineage Plasticity 2
1-1-2 Totipotency and Nuclear Transfer 3
1-1-3 Pluripotent Stem Cells and Induced Pluripotent Stem Cells 5
1-1-4 Adult Stem Cells 7
1-2 Microenvironment Effect on Human Pluripotent Stem Cells 8
1-2-1 Physical Cues 8
1-2-1-1 Stem Cell Fate Determination by Matrix Elasticity 8
1-2-1-2 How Do Stem Cells Feel Their Environment 9
1-2-2 Chemical Effect 10
1-2-2-1 Effect of Extra Cellular Matrix on Stem Cell Pluripotency 10
1-2-2-2 Chemically Defined Materials for Stem Cell Culture 11
1-3 Reprogramming method 44
1-4 Characterization of Pluripotent Stem Cells 46
1-4-1 Colony formation 46
1-4-2 Alkali phosphatase activity 46
1-4-3 Pluripotent gene Expression 48
1-4-4 Pluripotent protein Expression 48
1-4-5 Differentiation ability 48
1-5 Immunofluorescence 51
Chapter 2: Materials and Methods 53
2-1 Material 53
2-1-1 Cell line 53
2-1-2 Chemical 53
2-1-3 Medium 55
2-2 Cell Isolation 56
2-2-1 Human adipose-derive stem cell (hADSCs) 56
2-2-2 Mouse embryonic fibroblasts (MEFs) 57
2-3 Cell Culture 59
2-3-1 Culture of hADSCs and MEFs 59
2-3-2 Culture of 293FT cell line 59
2-3-2-1 Preparation of 293FT cell culture medium 59
2-3-2-2 Thawing 293FT cell line 59
2-3-3 Culture of human pluripotent stem cell lines 60
2-3-3-1 Feeder-layer culture of pluripotent stem cells 60
2-3-3-2 Feeder-layer free culture of pluripotent stem cells 61
2-3-3-3 Passage of human pluripotent stem cells by using dispase 61
2-3-3-4 Storage of hiPS cells and hES cells 62
2-3-3-5 Thaw frozen stock of hiPS cells and hES cells 63
2-4 Amplification of plasmid 64
2-4-1 Bacteria transformation 64
2-4-2 Plasmid Amplification 64
2-4-3 Plasmid purification 64
2-5 Transfection of 293FT cells 65
2-6 The generation of induced pluripotent stem cells 66
2-7 Quantitative RT-PCR 66
2-7-1 Isolation of total RNA 66
2-7-2 Reverse transcription of mRNA into cDNA 67
2-7-3 Quantitative real time polymerase chain reaction 68
2-8 Immunofluorescence 70
2-9 Colony attachment rate 71
2-10 Colony differentiation rate 72
2-11 Alkaline phosphatase live staining assay 74
2-12 Embryoid body formation 74
2-13 Teratoma formation 75
2-14 Preparation of PVA-IA film 76
2-15 Preparation of PVA-IA coating dish grafted with oligopeptide 77
2-16 XPS analysis of dish surface 78
Chapter 3: Result and Discussion 79
3-1 Reprogramming of hADSCs into hiPSCs 79
3-1-1 Transfection of 293FT cells and generation of retrovirus containing pluripotent gene 79
3-1-2 Transduction of hADSCs into hiPSCs 80
3-1-3 Characterization of hiPSCs derived from hADSCs 82
3-2 Cultivation of hPSCs on PVA-IA films grafted with vitronectin derived oligopeptide 85
3-2-1 Transfer of hESCs from feeder-layer culture system to Matrigel coated dish 85
3-2-3 Characterization of hESCs before cultivation on PVA-IA culture system 86
3-2-4 Cultivation of hESCs on PVA-IA films having different stiffness grafted with vitronectin derived oligopeptide with different concentration 89
3-2-5 Characterization of hESCs after cultivation on PVA-IA culture system .98
Chapter 4: Conclusion 101
Supplement Data 102
Reference 111

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指導教授

?口亞紺(Akon Higuchi)

審核日期

2014-6-16

推文