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系統識別號 U0026-2009201613403600
論文名稱(中文) 定義最低基本必需因子在無動物性物質培養系統中從iPS / ES細胞得到高純度的人類內皮細胞
論文名稱(英文) Defining Minimum Essential Factors to Derive Highly Pure Human Endothelial Cells from iPS/ES Cells in an Animal Substance-Free System
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 105
學期 1
出版年 105
研究生(中文) 吳育庭
研究生(英文) Yu-Ting Wu
學號 S96021040
學位類別 碩士
語文別 英文
論文頁數 49頁
口試委員 指導教授-江伯敏
口試委員-蔡坤哲
口試委員-陳芃潔
中文關鍵字 誘導型多功能幹細胞  內皮細胞  分化  細胞外因子 
英文關鍵字 Induced pluripotent stem cells  Endothelial cells  Differentiation  Extracellular factors 
學科別分類
中文摘要 對於無限獲得內皮細胞並用於研究與治療上是非常需要的。然而,目前從人類身上獲取內皮細胞的方法存在著一些問題,例如內皮細胞供應限制,遭受動物性物質的汙染,以及冗長且複雜的提取過程。這裡,我們可在五天之內將胚胎幹細胞與誘導幹細胞分化出高純度的內皮細胞。化學成分確定的系統是一個穩固耐用、容易操作且不含動物性物質的系統。使用此系統,我們證實同時結合TGFβ與Wnt促效劑足以將iPS/ES轉換成中胚層。此外,在高細胞密度時內皮細胞分化需要VEGF-KDR訊號,同時補充FGF可使集群的內皮細胞分化。最後,抗吸附劑可以使內皮前驅細胞選擇性貼附以增加內皮細胞的產出。此系統被證實在iPS/ES細胞可製造具有形成體外類微血管結構以及在體內結合到宿主中形成血管結構之能力的內皮細胞。總之,簡單而穩固的分化系統允許人類內皮細胞獲得無限供應。驗證此成分確定且無動物性物質的分化系統適合於內皮細胞的臨床應用以及其特性描述。
英文摘要 It is desirable to obtain unlimited supplies of endothelial cells for research and therapeutics. However, current methods of deriving endothelial cells from humans suffer from issues, such as limited supplies, contamination from animal substances, and lengthy/complicated procedures. In this article we developed a way to differentiate human iPS and ES cells to highly pure endothelial cells in 5 days. The chemically defined system is robust, easy to perform, and free of animal substances. Using the system, we verified that combined TGFβ and canonical Wnt agonists are essential and sufficient for iPS/ES cell-to-mesoderm transition. Besides, VEGF-KDR signaling alone is required for endothelial formation at high density while supplementation with FGF allows for colonial endothelial differentiation. Finally, anti-adsorptive agents could enrich the endothelial output by allowing selective attachment of the endothelial precursors. The system was validated to work on multiple iPS/ES cells lines to produce endothelial cells capable of forming capillary-like structures in vitro and integrating into host vasculature in vivo. In sum, the simple yet robust differentiation system permits the unlimited supply of human endothelial cells. The defined and animal substance-free nature of the system is compatible with clinical applications and characterization of endothelial differentiation in an unbiased manner.
論文目次 Contents
Introduction 1
Materials and Methods 3
List of materials 3
Culture of human ES cells and iPS cells 3
Differentiation of ES/iPS cells into endothelium 3
Flow cytometry and cell sorting 4
Immunofluorescence 4
Matrigel capillary assay 5
Matrigel plug assay 5
Immunohistochemistry for PECAM1 on Matrigel plugs 5
RT-qPCR 6
Statistical analysis 7

Results 8
The treatment of a glycogen synthase kinase inhibitor and a TGFβ agonist triggered
mesoderm formation 8
Anti-adsorptives dramatically improved the purity of endothelial cells by selectively
allowing the attachment of their precursors 10
VEGF-KDR and basic FGF pathways allowed for colonial differentiation from
mesoderm to endothelial cells 12
In vitro and in vivo characterizations confirmed the generality of the method and the
identity of the differentiated human endothelium 14

Discussion 15
References 20
Tables 24
Figures 25


Tables Contents
Table 1. Quantification of endothelial formation with three independent ES/iPS cell lines 24






Figures Contents
Figure 1. The requirement of TGFβ and Wnt agonists for the formation of mesoderm 25
Figure 2. The effect of anti-adsorptives on the yield and purity of the differentiated endothelial cells 33
Figure 3. The requirement of VEGF and FGF pathways for mesoderm-to-endothelium transition 40
Figure 4. The validation of endothelial identity across multiple iPS/ES cell lines 46
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