單細胞定序分析肺臟幹/先驅細胞分化成第I型肺泡上皮細胞過程中的基因及子群變化

Abstract

肺臟表皮由多種不同功能的表皮細胞所組成，由於直接與吸入的氣體接觸，肺臟表皮會持續地受到來自外界環境因子的刺激與傷害，有鑑於此，負責進行損傷修復與維持正常生理狀況下細胞汰換的肺臟幹/先驅細胞的存在是非常重要的。在先前的研究中，我們從新生小鼠的肺部中發現了一群稀少的肺臟幹/先驅細胞族群，這群細胞會表現具有特異性的細胞標誌，柯薩奇病毒/腺病毒受體(CAR)，利用此細胞標誌可以分離出此細胞族群，並將其命名為mPSCsCAR+。這群細胞在培養七到十天後會分化成近似第I型肺泡上皮細胞。為了釐清mPSCsCAR+作為肺臟幹細胞的角色以及與其他已被研究過的肺臟幹/先驅細胞的相似程度，我們蒐集了其他研究提出的肺臟幹細胞所表現的細胞標誌，並利用單細胞定序分析這些細胞標誌在mPSCsCAR+的表現量，結果顯示，某些細胞標誌在所有的mPSCsCAR+都會表現，表示mPSCsCAR+可能與表現那些細胞標誌的細胞族群有很高的相關程度。此外，我們發現乙型轉化生長因子(TGF-β)的訊息傳遞路徑在mPSCsCAR+分化成第I型肺泡上皮細胞的過程中是不可或缺的，但同時此路徑的活化也會導致α-平滑肌動蛋白(α-SMA)以及其他與肺纖維化相關的基因表現量上升。在加入地塞米松(Dexamethasone)及乙型轉化生長因子受體抑制劑之後，α-平滑肌動蛋白與其他肺纖維化相關基因的表現量有顯著下降，此外，與細胞連接相關的蛋白，如上皮細胞黏著分子(EpCAM)及緊密連接蛋白(caludin18)的表現量也有所提升，顯示地塞米松與乙型轉化生長因子受體抑制劑對於肺纖維化的過程有潛在的抑制效果。然而，我們觀察到加入藥物後並不是所有細胞的α-平滑肌動蛋白表現量都會下降，不同細胞對藥物的反應不一致的現象使我們認為此細胞可能是一個異質化的族群。藉由單細胞定序的技術，我們可以從不同處理的細胞組別中分別分離出具有不同特徵的亞族群，並嘗試釐清不同組別中亞族群之間的關聯。

The lung epithelium composed of various types of epithelial cells with their distinct functions is directly exposed to the external environment and continually undergoes injury caused by environmental factors from inhaled air. Given the reason, the existence of stem/progenitor cells responsible for injury repair and normal turnover at steady state is crucial. Recently, we have identified a rare population of mouse pulmonary stem/progenitor cells (mPSCs) from neonatal mice, which express a specific marker, coxsackievirus/adenovirus receptor (CAR) and were named mPSCsCAR+. They have the ability to differentiate into alveolar type I-like (ATI-like) cells in 7-10 days. To clarify the role and position of mPSCsCAR+ and the similarity to other proposed progenitor populations, we evaluated the expression of proposed stem/progenitor cell markers in mPSCsCAR+ by single-cell RNA sequencing. According to the gene expression, all of our cells would express some markers proposed by the previous studies, indicating our cells might be similar to the populations mentioned in those studies. Besides, we found the expression of α-SMA and other fibrosis-related genes were upregulated during the differentiation process by TGF-β signaling pathway which is necessary for differentiation of mPSCsCAR+ into ATI-like cells. After treating with dexamethasone and TGF-β receptor inhibitor, the expression of α-SMA and other fibrosis-related genes were downregulated. In addition, the cell junction protein, EpCAM and claudin18 were upregulated, indicating the potential therapeutic effect of TGF-β receptor inhibitor on fibrotic process. At the same time, we observed differentiated cells displayed different responses to TGF-β receptor inhibitor by expressing different levels of α-SMA, indicating our cells might be a heterogeneous population. By single-cell RNA sequencing, we identified the subpopulations with different characteristics in mPSCsCAR+, untreated and drug-treated differentiated cells, and attempted to clarify the relationships of subpopulations between each group.