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研究生:江逸蓬
研究生(外文):Yi-Peng Jiang
論文名稱:藉由分離及培養初代羊水幹細胞取得並分析通用誘導多能幹細胞的特性
論文名稱(外文):Generation and characterization of universal induced pluripotent stem cells by isolation of primary amniotic fluid stem cells
指導教授:樋口亞紺
學位類別:碩士
校院名稱:國立中央大學
系所名稱:化學工程與材料工程學系
學門:工程學門
學類:化學工程學類
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:114
中文關鍵詞:通用誘導多能幹細胞幹細胞誘導多能幹細胞
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每年數百萬人由於事故、先天缺陷和疾病致使器官和組織的損失及傷害。對於細胞治療而言,幹細胞是相當吸引人的資源。多能幹細胞,如人類胚胎幹細胞(hESCs)和人類誘導多能幹細胞(hiPSCs),具有分化為三個胚層中任何細胞類型的潛力。如今,由於人類誘導多能幹細胞其多能性、無限增殖且沒有倫理問題,已為治療學的研究增加許多可能。人類誘導多能幹細胞(hiPSCs)可以透過成體幹細胞引入轉錄因子(例如Oct3/4, Sox2, c-myc, klf-4)進行重新編程而得。然而過去必須建立特定患者的細胞株或是人類白血球抗原(HLA)的幹細胞庫以避免治療產生免疫反應,這是耗時耗力且昂貴的。在這項研究中,我們成功自人類羊水幹細胞(hAFSCs)中取得通用人類誘導多能幹細胞,即使將此細胞分化為特定譜系,也不會顯示第一類(HLA-A, -B, and -C)和第二類人類白血球抗原(HLA)。此通用人類誘導多能幹細胞將成為幹細胞治療最有淺力的細胞來源。首先,我們利用以下不同的培養基分離初代的人類羊水幹細胞(1)20%胎牛血清(FBS)培養基(2)20%胎牛血清與等體積羊水混合(FBS+AF)之培養基(3)10%人類血小板裂解液(hPL)培養基(4)羊水(AF)。使用20%胎牛血清與等體積羊水混合(FBS+AF)之培養基可以使初代羊水幹細胞貼附最多並成長最快。因此利用此方式分離並建立後續實驗所需之細胞株。我們假設可從某些細胞系(例如胎兒細胞(hAFSCs))取得並產生通用人類誘導多能幹細胞。在室溫下混合兩個至是五個不同個體捐贈之羊水兩天後,置於細胞培養皿(TCPS)中。不同個體的羊水成分以及單核球在混合期間將產生免疫反應,僅有通用人類羊水幹細胞存活。我們成功的利用仙台病毒轉染多能基因(山中伸彌因子)將(a)混合的人類羊水幹細胞以及(b)單一的人類羊水幹細胞轉為人類誘導多能幹細胞。藉由流式細胞儀檢測第一類(HLA-A, -B, and -C)及第二類人類白血球抗原於通用人類誘導多能幹細胞及其分化之心肌細胞之表現,結果為陰性。即使利用不同捐贈者取得之單核球處理通用人類誘導多能幹細胞分化之心肌細胞,此心肌細胞仍可持續跳動。因此,理論上我們取得之通用人類誘導多能幹細胞僅需單一細胞株便可用於所有不同的患者進行幹細胞治療。
Millions of people suffer from loss and damage of organs and tissue every year due to accidents, birth defects and disease. Stem cells are an attractive source for cell therapy. Pluripotent stem cells such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have the potential to differentiate into any types of the cells derived from all three germ layers. Nowadays, hiPSCs have opened up many gateways for the research in therapeutics because of their pluripotency and unlimited proliferation with no ethical concern. hiPSCs can be reprogrammed from adult somatic cells by introducing transcription factors such as Oct3/4, Sox2, c-myc and klf-4. However, it is necessary to establish patient-specific stem cell line or stem cells bank having specific human leukocyte antigen (HLA) to avoid immune response of the patients. The stem cell bank is laborious, time-consuming and extremely expensive. In this study, universal hiPSCs derived from human amniotic fluid stem cells (AFSCs) have been developed; these cells do not or less express human leukocyte antigen (HLA) class Ia (HLA-A, -B, and -C) and class II even after differentiation of universal hiPSCs into specific lineages of the cells. These universal hiPSCs will be a promising source for stem cell therapy. At first, primary AFSCs were isolated using different media: (1) 20% fetal bovine serum (FBS) medium, (2) 20% FBS medium mixed with equal volume of amniotic fluid (FBS+AF), (3) 10% human platelet lysate (hPL) and (4) amniotic fluid (AF). The isolation of primary AFSCs using FBS+AF medium showed the highest attachment and proliferation of AFSCs. Therefore, this isolation protocol was used to establish hAFSCs in this study. It is hypothesized that hiPSCs derived from some cell lines such as fetal cells, e.g., hAFSCs, may be generated as universal hiPSCs. AFs from two or five donors were mixed and incubated in room temperature for two days and subsequently seeded on conventional tissue culture polystyrene (TCPS) plate. During mixing AFs from different donors, mononuclear cells or components of AFs in different person are supposed to generate immunogenic reaction, which targeted to different hAFSCs from each other and only “universal hAFSCs” are survived after mixing more than two donor of AFs. We succeeded to generate hiPSCs using (a) hAFSCs derived mixed AF [hiPSCs (mix)] and (b) hAFSCs from a single AF [hiPSCs (single)], where these hiPSCs were generated from transfection of pluripotent genes (Yamanaka factors) using Sendai virus vector. HLA Class Ia (HLA-A, -B, and -C) and Class II of universal hiPSCs (mix) and cardiomyocytes differentiated from universal hiPSCs (mix) were evaluated using flow cytometry. The results showed that the universal hiPSCs (mix) did not express HLA Class Ia (HLA-A, -B, and -C) and Class II even after differentiation into cardiomyocytes. Cardiomyocytes differentiated from universal hiPSCs (mix) survived and continued beating even after treatment with mononuclear cells derived from different donors. Universal hiPSCs (mix) should be suitable for stem cell therapy, because universal hiPSCs can theoretically be used to treat any patients using only one cell line.
Abstract i
摘要…………………………………………………………………………………………… ii
Index of Content iii
Index of Figure v
Index of Table xi
Chapter 1 Introduction 1
1-1 Stem Cell Application in Regenerative Medicine 1
1-2 Stem Cells 3
1-2-1 Human Pluripotent Stem Cells (hPSCs) 3
1-2-2 Human Mesenchymal Stem Cells (hMSCs) 7
1-3 Human Amniotic Fluid Stem Cells (hAFSCs) 8
1-3-1 Source of Human Amniotic Fluid Stem Cells 10
1-4 Extracellular Matrix (ECM) 13
1-5 Peripheral Blood Mononuclear Cells 14
1-6 Recent developments of universal hiPSCs 15
1-7 Goal of this research 17
Chapter 2 Materials and methods 19
2-1 Experimental materials 19
2-1-1 Cell source for cultivation 19
2-1-2 ECM-coated dish for cell culture 22
2-1-3 Differentiation medium 22
2-1-4 Characterization of stem cells 24
2-2 Experimental instruments 27
2-3 Experimental methods 27
2-3-1 Preparation of the cell culture medium and buffer solution 28
2-3-2 Generation and isolation of stem cell lines 30
2-3-3 Cultivation and passage of stem cells 34
2-3-4 Cell number and seeding density 36
2-3-5 Isolation of mononuclear cells 38
2-3-6 Live and Dead staining 39
2-3-7 Cardiomyocytes differentiation 40
2-3-8 Immunofluorescence staining 41
2-3-9 Flow cytometry measurements 43
2-3-10 Embryoid Body (EB) Formation in Vitro 43
2-3-11 Teratoma Formation in Vivo 44
Chapter 3 Results and Discussion 46
3-1 HLA class I and class II expression of several human stem cells and cancer cells 46
3-2 Cultivation of hAFSCs 47
3-2-1 The morphology of primary hAFSCs isolated using different culture medium 47
3-2-2 The morphology of single and mixing hAFSCs 50
3-2-3 Identification of the hAFSCs from surface marker analysis using flow cytometry 53
3-2-4 The morphology of hAFSCs on ECMs-coating dishes 56
3-3 Reprogramming and cultivation of hAFSCs-derived hiPSCs 58
3-3-1 Generation and isolation of hAFSCs-derived hiPSCs 59
3-3-2 Immunostaining analysis of hAFSCs-derived hiPSCs 62
3-4 Cardiomyocytes (CMs) differentiation 62
3-4-1 The morphology of hiPSCs-derived CMs 63
3-4-2 Immunostaining analysis of hiPSCs-derived CMs 65
3-5 HLA class I and class II expression and evaluation of the immunogenic reaction using mononuclear cells 65
3-5-1 HLA class I and class II expression of hESCs, hiPSCs, hAFSCs, and universal hiPSCs 66
3-5-2 Evaluation of the immunogenic reaction using mononuclear cells 69
3-6 Differentiation ability in vitro 71
3-7 Differentiation ability in vivo 72
Chapter 4 Conclusion 73
Reference 74
Supplementary Data 81
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