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系統識別號 U0026-2407201514324800
論文名稱(中文) 由誘導型多潛能幹細胞分化成血球前驅細胞所需的必要因子
論文名稱(英文) Essential Drivers for the Differentiation of Bloods Precursor Cells from Human Induced Pluripotent Stem Cells
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 103
學期 2
出版年 104
研究生(中文) 石健佑
研究生(英文) Chien-Yu Shih
學號 S96014051
學位類別 碩士
語文別 英文
論文頁數 103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 誘導型多潛能幹細胞  血球前驅細胞  分化  細胞外因子  轉分化 
英文關鍵字 iPS  blood precursor  differentiate  extracellular factor  transdifferentiation 
學科別分類
中文摘要 近幾年在將胚胎幹細胞分化為成熟的血球前驅細胞時大多使用 feeder cells, 血清或是特殊培養液。但是這些物質往往無法應用在臨床上,因為可能會產生感染或是過敏原交叉污染。因此我們需要一個乾淨的系統來將胚胎幹細胞分化成血球前驅細胞。更進一步為了避免排斥反應,使用誘導型多潛能幹細胞來進行分化是一個很好的選擇,因此我們將會建立一個使用已知化合物來分化誘導型多潛能幹細胞成為血球前驅細胞的系統,而且會試著了解在這個系統中影響血球分化的關鍵因子是如何運作的。
為了能夠建立一個將誘導型多潛能幹細胞分化成為血球前驅細胞的系統,我們測試了許多外源性因子來增加血球前驅細胞的生成並且優化這個系統。在成功建立了分化系統之後我們經由standard colony-forming assay與免疫缺陷小鼠的移植去證明這些血球前驅細胞擁有分化成不同血球細胞的潛力。最後我們還使用shRNA knockdown 與RT-qPCR的方式證明了有哪些細胞外或細胞內因子對於血球前驅細胞的生成是重要的。
我們所建立的分化系統可能會提供一個乾淨無污染且能將誘導型多潛能幹細胞分化成為血球前驅細胞的環境。再加上我們發現的那些能誘發血球前驅細胞形成的關鍵細胞外因子,也許在未來能夠讓我們實現直接將其他體細胞轉分化為血球細胞的夢想。
英文摘要 Current approaches to differentiate embryonic stem cells to mature blood precursors in vitro rely on feeder cells, sera, or conditioned culture media. However, these substances are not compatible with clinical applications for issues of infection or allergen carryovers. Thus, a clean system to differentiate human embryonic stem cells into blood cells is needed. Further, to avoid allogeneic rejection, a way to differentiate induced pluripotent stem (iPS) cells into blood would be highly useful. Here we propose to establish a system to differentiate iPS cells into blood precursors in a chemically-defined system and to understand how key factors determine hematopoietic differentiation in the defined system.
In order to establish a system to differentiate blood precursor cells from iPS cells, we tested several exogenous factors that enhanced the formation of blood precursors and further refined the system. After establishing the culture system, we also tried to show that the derived blood precursors performed multilineage- differentiation potential by standard colony-forming assay and by transplantation into immunodeficient mice. Finally, shRNA knockdown and RT-qPCR were used to identify key extracellular or intracellular factors importance for the formation of blood precursors.
The culture system potentially offers a clean, confounder-free culture environment to differentiate human iPS/ES cells into blood precursors. Moreover, the discovery of key extracellular factors that drive hematopoietic differentiation would allow us to directly transdifferentiate other somatic cell types into blood cells in the future.
論文目次 CONTENTS

INTRODUCTION………………………………………………………………………....1
At the present stage clinical application of regenerative medicine…........................1
Embryonic stem cell (ESC) -The key to regenerative medicine……………………...3
Induced pluripotent stem cell (iPS) ¬- The miracle of reprogramming………………..4
The stem cell differentiation technique.……………………………………………..….6
What are blood precursor cells and blood cells?......................................................8
The application of blood precursor cells……………………………………………....10
The differentiation of blood precursor cells…………………………………………...12
The bone morphogenetic protein 4 (BMP4) signaling pathway plays an important role in differentiation of blood precursors……………………………………………..14
The vascular endothelial growth factor (VEGF) signaling pathway induces blood precursor formation…………….……………………………………………………….16
Transcription factors - The master driver of hematopoietic cell fate…………..…...18
ETS family, GATA2 and TAL1 the mediator in early stage of hematopoietic formation……………………………………………...................................................19
RUNX1 and MYB the important driver of blood precursor formation…………….22
Summary…………………………………………………………………………………24
Methods and Material……………………………….……………….........................26
List of materials……………………………………………………………………….…26
Culture of human iPS/ES cells…………………………………………………………26
Differentiation of iPS/ES cells into human blood precursor cells…………………..26
Colony-forming unit (CFU) assay……...………………………………………………27
Immunofluorescence stain…………………………………………………………......27
Flow cytometry…………………………………………………………………………..28
Cytospin…………………………………………………………………………………..28
Wright-Giemsa stain…………………………………………………………………….28
Immunohistochemistry for CD34 and CD235a of CFU assay……………………..29
Quantitative reverse transcription PCR (RT-qPCR)………………………………...29
Blood precursor cell transplantation….……………………………………………….30
Transfection for virus production……………………………………………………....30
Virus concentration…………………………………………………............................31
Virus transduction and antibiotic selection……………………………………………31
Small molecule inhibition and shRNA knockdown…………………………………...31
Statistical analysis……………………………………………………………………….32
RESULTS……………………………………………………………………….…….....33
Y27632 and VTN are essential for the survival of replated ES/iPS cells……….....33
CHIR99021 and Activin A triggered the formation of mesodermal cells………..….33
The requirement of BMP4, VEGF and FGF for blood precursor cell formation and ALKi for purity………………………………………………………………………........36
The other markers of the blood precursor cells such as CD117, CD34, CD45 and CD235a………………………………………………………………………………......37
The blood precursor cells can differentiate into multilineage blood cells but fail in transplantation……………………………………………………………….................38
BMP4 and VEGF signaling pathways were specific for blood precursor cell formation………………………………………………………………………………....39
BMP4 and VEGF could induce GATA2 and TAL1 expression, respectively, which had a relationship with RUNX1 expression…………………………………………..40
DISSCUSSION…………………………………………….........................................42
The microenvironment during differentiation plays an important role in blood precursor formation……………………………………………………………………..42
GATA2, TAL1 and RUNX1 have important correlations with blood precursor cell formation………………………………………………………………………………….43
In a colony formation assay, we can only obtain CFU-myeloid, CFU-erythroid and CFU-mix but not CFU-lymphoid…………………....................................................44
The blood precursor cells can’t perform their function in vivo……………………....46
MicroRNA probably plays an important role in blood precursor cell formation.......47
Histone modification could determine hematopoietic fate at the epigenetic level...49
FIGURES……………………………………………………………………………..….51
Figure 1. Differentiate iPS/ES cells into mesodermal cells………………………….51
Figure 2. Demonstrate the formation of KDR+ hemangioblast……………………..56
Figure 3. Inducing blood precursor cell formation using several extracellular factors…………………………………………………………………………………….59
Figure 4. In vitro and in vivo functional assays for blood precursor cells…………..62
Figure 5. BMP4 and VEGF signaling pathways plays an important role for blood precursor cell formation………………………………………………………………...65
Figure 6. BMP4 and VEGF can induce transcription factors GATA2 and TAL1 to regulate blood precursor cell formation…………………………………………….....67
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