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標題: | 心臟前驅細胞:追蹤,放大以及分化的命運 Human Cardiac Precursor Cells: Tracing, Proliferation and Commitment |
作者: | Shu-Chuan Tsao 曹書銓 |
指導教授: | 陳文彬(Wen-Pin Chen) |
關鍵字: | 心臟前驅細胞,心肌細胞,譜系追蹤,跳躍子,S-腺苷甲硫胺酸,心肌分化, cardiac precursor cells,cardiomyocytes,lineage tracing,transposon,S-adenosyl methionine,myogenic differentiation, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 細胞治療中利用人類誘導多功能性幹細胞/胚胎幹細胞分化而生的心肌細胞來補償心肌梗塞後心臟中所流失的心肌細胞是當今可行的方法之一。然而,如何有效地擴增特定譜系細胞以用於心肌新生,血管新生或血管形成的方法仍需要進一步的研究才能說明。心臟前驅細胞(CPC)的代謝體學表徵可能有助於研究心肌新生的機制。 我們的研究中發現CPC培養下的培養液可以誘發中內胚層細胞的心肌分化。進一步從CE-TOFMS的分析中發現,CPC中的S-腺苷甲硫胺酸(SAM)含量較CM來的高。接著利用跳躍子基因編輯MYH6表達報告基因hiPSC,在分化過程中給予SAM的抑制劑-環亮胺酸(CLC) 來揭示甲基化調節因子在肌源性分化以及細胞自我擴增中所扮演的關鍵作用,又以分化第六天影響最為關鍵,同時也是CPC相關轉錄因子之基因表現開始上升的時間。 接著,我們建立跳躍子標記的譜系追蹤系統,用來驗證調節細胞擴增及分化命運的表觀調節機制。MYH6-Ugm標記的hiPSC藉由剪切和黏貼可被跳躍的基因進入基因組中,進而使MYH6啟動子驅動的H2GFP-mCherryGPI標記心肌細胞。在在分化的第10天,跳動的CMs同時也表達報告基因,表示不會影響到成肌的分化潛能,並且隨著CMs成熟度的增加,其增殖能力也會降低。為了在hiPSC中建立CPC追踪系統,我們使用跳躍子的方式分別以NKX2.5和ISL1的增強子和啟動子標記CPC,並揭示hiPSC心肌分化過程中報告基因的表達量。在對這些帶有報告基因的CPC進行篩選並培養至d20進行分化命運的驗證,從ICC染色顯示NKX2.5 CPC會成為表達SA(α輔機動蛋白2)和smMHC(平滑肌肌球蛋白重鏈)的細胞,是為CM和平滑肌細胞的特徵。 總結來說,這項研究顯示出作為CPC培養液中的代謝物,甲基化調節劑SAM在心肌分化扮演著至關重要的角色。我們建立了跳躍子譜系追踪系統來標記心臟分化過程中的CM和CPC。而在不影響跳躍子標記後的幹細胞分化出之心肌功能的前提之下,NKX2.5 + CPCs具有成為CM和平滑肌細胞的潛力。CPC跳躍子譜系追踪系統可用於分離我們感興趣的CPC細胞,其強大的標記功能也能用來揭示CPC5中不同譜系的詳細代謝組情況,進而了解CPC分化及放大的表觀調節機轉。 除此之外,我們的CPC跳躍子標記系統具有GPI-mCherry報告基因,可以標記到細胞質中的膜狀胞器,包括胞吐小體,因而在未來,我們可以收集有表達報告基因胞吐小體並進一步檢測其對心肌分化的影響。 Cell therapy with human iPSC/ESC-derived cardiomyocytes is a feasible approach for the compensation of the lost myocardium in post-MI hearts. However, it needs further investigation to elucidate the method that can efficiently expand the specific lineage cells for the purpose of cardiomyogenesis, angiogenesis or vascularization. Metabolomic characterization of cardiac precursor cells (CPC) may be helpful to investigate the machinery of cardiomyogenesis. Our study found that metabolites in CPC condition medium can induce myogenic differentiation from mesendodermal cells. Furthermore, from CE-TOFMS analysis we found high level of S-adenosyl methionine (SAM) in CPC in comparison with CMs. We use the transposon edited MYH6-reporter hiPSC under the treatment of SAM inhibitor, cycloleucine (CLC), to reveal the crucial role of methylation regulators in myogenic differentiation and cell self-renewal at cardiac differentiation d6, the time with several CPC transcription factors (TFs) transcription level start to increase. Next, we establish transposon-lineage tracing system for the validation of epigenetic mechanism in regulating cell proliferation and commitment. In MYH6-Ugm labeled hiPSC, cardiomyocytes were labeled with H2GFP-mCherryGPI driving by MYH6 promoter through the cassette of cut-and-paste transposable element to jump into genome. The myogenic differentiation potential will not be affected by beating CMs express reporter at d10 of differentiation, and its proliferation increases as it became much more mature. To build up the CPC tracing system in hiPSC, we use transposon to label CPC by NKX2.5 and ISL1 enhancer and promoter respectively and reveal the reporter expression level during hiPSC cardiac differentiation. After sorting these reporter positive CPCs and culture until d20 for commitment validation, ICC stain showed that NKX2.5+ CPC would commit into cells that express both SA and smMHC, the characterizations of CMs and smooth muscle cells. In conclusion, this study revealed that methylation regulator SAM, as the metabolite on CPC condition medium, is important for cardiac differentiation. We established transposon-lineage tracing system to label CMs and CPCs during cardiac differentiation. Under the premises of without affecting cardiac function after transposon labeling, NKX2.5+ CPCs had the commitment of becoming CM and smooth muscle cells. CPC transposon-lineage tracing system can be used to isolate cells that we are interested in, with its powerful ability to reveal more detail metabolome profile of different lineages of CPC. Besides, our CPC transposon-labeling system have GPI-mCherry reporter which can label membrane vesicles, including exosomes. In the future, we can collect reporter positive exosome and examine its cardiac differentiation potency. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19942 |
DOI: | 10.6342/NTU202003398 |
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顯示於系所單位: | 藥理學科所 |
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