单细胞测序分析小鼠肝卵圆细胞的分化

Single-Cell Sequencing Analysis of the Differentiation of Mouse Hepatic Oval Cells

摘要: 目的：探讨肝卵圆细胞HOCs分化为胆管上皮细胞的分化过程和分子机制，肝卵圆细胞(Hepatic oval cells, HOCs)是肝脏中具有双向分化潜能的干细胞样细胞，能够在肝脏严重损伤时分化为肝细胞和胆管上皮细胞以恢复肝脏。为系统性地明确HOCs在肝脏再生中的分化作用机制，本研究由小鼠肝卵圆细胞培养诱导分化为胆管上皮细胞的高通量单细胞转录组测序揭示HOCs分化为胆管上皮细胞中特定状态的细胞亚群和调控分化的关键作用机制。方法：HOCs在体外条件下联合表皮生长因子、干细胞生长因子、白血病抑制因子的诱导方案培养细胞，定向诱导分化为胆管上皮细胞。进行单细胞测序检测，整合测序数据进行质量控制、PCA降维分析、细胞聚类分群和t-SNE。结果：呈卵圆形、折光性强的HOCs诱导分化后细胞形态变为长梭形、铺路石样生长。单细胞测序数据的PCA降维分析将HOCs分化的细胞分为17个cluster，由HOCs的特异性marker基因Twf1和胆管细胞特异性标记物Krt19在各个亚群中的表达量异同和细胞分化的clustertree重新将HOCs分为6个细胞亚群。结论：由HOCs分化为胆管上皮细胞的单细胞测序数据鉴定出5种具有异质性的细胞类型，分别为肝卵圆细胞1 (Hepatic Oval Cell 1)、前胆管细胞1 (Pre-Cholangiocytes 1)、前胆管细胞2 (Pre-Cholangiocytes 2)、前胆管细胞3 (Pre-Cholangiocytes 3)、肝卵圆细胞2 (Hepatic Oval Cell 2)，还有一群未能鉴定明确的细胞类型未知细胞(Unknown)。

Abstract: Objective: This study aims to investigate the differentiation process and molecular mechanisms of hepatic oval cells (HOCs) into bile duct epithelial cells. HOCs, known as hepatic oval cells, are stem cell-like cells in the liver with bidirectional differentiation potential, capable of differentiating into hepatocytes and bile duct epithelial cells to restore liver function under severe liver injury conditions. To systematically elucidate the differentiation mechanisms of HOCs in liver regeneration, this study utilizes high-throughput single-cell transcriptome sequencing of cultured mouse hepatic oval cells induced to differentiate into bile duct epithelial cells. The findings reveal specific cellular subpopulations and key regulatory mechanisms involved in the differentiation of HOCs into bile duct epithelial cells. Methods: Hepatic oval cells (HOCs) were cultured under in vitro conditions with a combination of epidermal growth factor, stem cell growth factor, and leukemia inhibitory factor to induce cell differentiation into bile duct epithelial cells. Single-cell sequencing was performed to detect cellular transcription profiles. Integrated sequencing data underwent quality control, principal component analysis (PCA) for dimensionality reduction, cell clustering, and t-distributed stochastic neighbor embedding (t-SNE) analysis. Results: Following induction, the morphology of HOCs changed from oval-shaped with strong birefringence to elongated spindle-shaped cells with cobblestone-like growth pattern. Principal component analysis (PCA) of single-cell sequencing data separated the differentiated cells into 17 clusters. Re-evaluation of HOCs differentiation using cluster tree analysis based on the expression levels of specific marker genes, such as Twf1 for HOCs and Krt19 for bile duct cells, resulted in the classification of HOCs into 6 distinct cellular subpopulations. Conclusion: Single-cell sequencing data identified five distinct heterogeneous cell types derived from the differentiation of HOCs into bile duct epithelial cells. These cell types were designated as Hepatic Oval Cell 1, Pre-Cholangiocytes 1, Pre-Cholangiocytes 2, Pre-Cholangiocytes 3, and Hepatic Oval Cell 2. Additionally, there was a group of cells that could not be definitively identified, labeled as “Unknown” cells.