藉由羊水幹細胞製備通用誘導多能幹細胞

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許哲瑜(Jhe-Yu Hsu) 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

藉由羊水幹細胞製備通用誘導多能幹細胞
(Universal human induced pluripotent stem cells prepared from amniotic fluid stem cells)

相關論文

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★ 可見光對羊水間葉幹細胞成骨分化之影響	★ 可見光調控神經細胞之基因表現及突觸生長
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★ 在不同培養條件下針對大腸癌細胞及組織中癌細胞進行純化、剔除及鑑定之研究	★ 羊水間葉幹細胞培養於細胞外間質改質表面其分化能力及多能性之研究
★ 人類脂肪幹細胞的膜純化法與分化能力研究	★ 具有抗藥性之大腸癌細胞株能提高癌胚抗原的表現，但並非是癌症起始細胞
★ 羊水間葉幹細胞培養於接枝細胞外間質寡肽與環狀肽具有最佳表面硬度的生醫材料,其增殖能力及多能性之研究	★ 人類體細胞從組成誘導型多能性幹細胞培養在無飼養層上

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摘要(中)

每年有數百萬人由於出生缺陷、事故和疾病遭受組織和器官的損失或損害。多能幹細胞，如人類胚胎幹細胞(hESCs)或人類誘導多能幹細胞(hiPSCs)，被認為是組織再生和醫學藥物工程的有前途的細胞來源，因為它們可以分化為任何類型的細胞。然而，移植細胞的組織相容性仍然是其臨床應用的主要挑戰，其中第一型人類白血球抗原類(HLA-A、-B、-C)分子是免疫反應的主要介質。除此之外，產生成熟的人類誘導多能幹細胞需要花費很長時間，而且需要驗證其沒有基因異常、沒有被病毒或其他病原體污染，這導致從每位患者的體細胞中生成專屬患者之人類多能幹細胞治療成本十分高昂，過程費時耗力。另一方面，在移植來自這些人類多能幹細胞的分化細胞後，通常不會產生與免疫原性相關的問題。因此，有必要建立具有各種人類白血球抗原之幹細胞庫，以避免患者產生免疫反應。此篇論文中，我報告了從多種不同捐贈者的羊水中生成通用人類誘導多能幹細胞的方法，即使在分化成特定細胞後也不表達第一型及第二類人類白血球抗原，也可以避免使用到基因編輯之技術。除了第一型及第二類人類白血球抗原，還通過流式細胞術檢測了 PD-L1、HLA-G和CD 47等免疫調節因子在通用人類誘導多能幹細胞及其原始羊水幹細胞(hAFSCs)，因為這些抗原理論上可以抑制免疫反應。即使用CD8+ T 細胞、自然殺傷細胞和巨噬細胞的同種異體單核細胞共同培養後，通用人類多能幹細胞及其分化細胞（人類間充質幹細胞）也能存活。此外，我研究了來自CD8+ T細胞和自然殺手細胞等免疫細胞對於通用人類多能幹細胞之特異免疫反應。通用人類多能幹細胞應適用於幹細胞治療，因為一支通用人類多能幹細胞理論上可治療任何患者。

摘要(英)

Millions of people suffer from loss or damage of tissue and organ every year due to birth defect, accident, and disease. Pluripotent stem cells (PSCs) such as human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs), are considered a promising cell source for tissue regeneration and medicine engineering, because they can differentiate into any type of cell lineages. However, histocompatibility of the transplanted cells remains a major challenge to their clinical application where human leukocyte antigen (HLA) class Ia (HLA-A, -B, -C) molecules are the primary mediators of immune reaction. Nevertheless, it takes long time to generate mature hiPSCs, and hiPSCs require to be verified that there is no gene abnormality and no contamination with viruses or other pathogens, which leads to a high cost of hiPSCs therapy, with laborious and time-consuming processes to generate patient-specific hiPSCs from somatic cells of each patient. On the other hand, the hiPSCs derived from each patient do not generate immunogenicity-related problems in general after the transplantation of differentiated cells from these hiPSCs. Consequently, it is necessary to establish patient-specific stem cell line or stem cells bank having specific HLA to avoid immune response of the patients. Here I report generation method of universal hiPSCs from multiple sources of human amniotic fluids without gene editing, which do not express HLA class Ia and class II even after their differentiation. Beside the HLA-Ia and II expression, immunomodulatory factors such as PD-L1, HLA-G, and CD 47 were also evaluated for universal hiPSCs and their original source cells of amniotic fluid stem cells by flow cytometry, because these antigens could suppress the immune reaction theoretically. The universal hiPSCs and their differentiated cells (human mesenchymal stem cells) survived even after treatment with allogenic mononuclear cells containing cytotoxic CD8+ T cells, natural killer cells, and macrophages. Additionally, I investigated the specific immune response of universal hiPSCs from immune cells such as CD8+ T cells and Natural Killer (NK) cells. 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.

關鍵字(中)

★ 通用多能幹細胞

關鍵字(英)

論文目次

Abstract i
摘要 iii
Index of Content iv
Index of Figure vi
Index of Table x
Chapter 1 Introduction 1
1-1 Human Pluripotent Stem Cells 1
1-1-1 Human Embryonic Stem Cells 1
1-1-2 Human Induced Pluripotent Stem Cells 2
1-1-3 hPSC for Therapeutic Application in Future 6
1-2 Human Mesenchymal Stem Cells 9
1-2-1 Human Amniotic Fluid Stem Cells (hAFSCs) 10
1-3 The Substrates for hPSCs Cultivation 14
1-4 Human Peripheral Blood Mononuclear Cells 17
1-5 Immune System 19
1-5-1 Innate Immune System 20
1-5-2 Adaptive Immune System 21
1-6 Universal hiPSCs 23
1-7 Goal of this Project 26
Chapter 2 Material and Methods 27
2-1 Experimental Materials 27
2-1-1 Cell Source for Cultivation 27
2-1-2 ECM-coated Dishes for Cell Culture 31
2-1-3 Differentiation Medium 31
2-1-4 Characterization of Stem Cells 35
2-2 Experimental Instruments 39
2-3 Experimental Methods 40
2-3-1 Preparation of the cell culture medium and buffer solution 40
2-3-2 Generation and extraction of stem cell lines 42
2-3-3 Expansion and passage of stem cells 45
2-3-4 Cell number and seeding density 47
2-3-5 Isolation of allogenic mononuclear cells 48
2-3-6 Live and Dead staining 51
2-3-7 Lactic dehydrogenase (LDH) cytotoxicity assay 51
2-3-8 Mesenchymal stem cells differentiation 52
2-3-9 Flow cytometry measurements 53
2-3-10 Immunofluorescence staining 54
2-3-11 Embryonic body formation 55
Chapter 3 Results and Discussion 57
3-1 PD-L1, HLA-G, and CD47 expression of several human stem cells and colon cancer cells 57
3-2 Cultivation of single and mixing hAFSCs 59
3-2-1 The morphology of single and mixing hAFSCs 60
3-2-2 Identification of the hAFSCs from surface marker analysis using flow cytometry 62
3-3 Reprogramming and cultivation of hAFSCs-derived hiPSCs 65
3-3-1 Generation and isolation of hAFSCs-derived hiPSCs 65
3-3-2 Pluripotency analysis of hAFSCs-derived hiPSCs 67
3-4 The tendency of immunomodulatory factors expression and evaluation of the immunogenic reaction using mononuclear cells 69
3-4-1 HLA class Ia, class II, PD-L1, HLA-G, and CD47 expression of hESCs, hiPSCs, hAFSCs, and universal hiPSCs 70
3-4-2 Evaluation of the immunogenic reaction of universal hiPSCs using allogeneic mononuclear cells 75
3-5 Universal hiPSC differentiation into mesenchymal stem cells (MSCs) 76
3-5-1 Allogenic mononuclear cells co-cultivation with hMSCs-derived from hESCs-H9 and universal hiPSCs-mix 4 78
3-5-2 HLA class Ia, class II, PD-L1, HLA-G, and CD47 expression of universal hiPSCs derived hMSCs 79
Chapter 4 Conclusion 81
Reference 82
Supplementary data 89

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指導教授

樋口亞紺(Akon Higuchi)

審核日期

2021-8-9

推文