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

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Title:	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
Authors:	許哲瑜;Hsu, Jhe-Yu
Contributors:	化學工程與材料工程學系
Keywords:	通用多能幹細胞
Date:	2021-08-09
Issue Date:	2021-12-07 11:19:51 (UTC+8)
Publisher:	國立中央大學
Abstract:	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。;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.
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