A single-cell transcriptomic analysis reveals precise pathways and regulatory mechanisms underlying hepatoblast differentiation

How the bi-potential hepatoblasts differentiate into hepatocytes and cholangiocytes remains unclear. Here, using single-cell transcriptomic analysis of hepatoblasts, hepatocytes, and cholangiocytes sorted from E10.5 to E17.5 mouse embryos, we found that hepatoblast-to-hepatocyte differentiation occurred gradually followed a linear default pathway. As more cells became fully differentiated hepatocytes, the number of proliferating cells decreased. Surprisingly, the proliferating and quiescent hepatoblasts exhibited homogeneous differentiation states at a given developmental stage. This unique feature enabled us to combine the single-cell and bulk-cell analyses to define the precise timing of the hepatoblast-to-hepatocyte transition, which occurs between E13.5 and E15.5. In contrast to hepatocyte development at almost all levels, hepatoblast-to-cholangiocyte differentiation underwent a sharp detour from the default pathway. New cholangiocyte generation occurred continuously between E11.5 and E14.5, but their maturation states at a given developmental stage were heterogeneous. Even more surprising, the number of proliferating cells increased as more progenitor cells differentiated into mature cholangiocytes. Based on an observation from the single-cell analysis, we also discovered that the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) signaling pathway promoted cholangiocyte maturation. CONCLUSIONS: Our studies have defined distinct pathways for hepatocyte and cholangiocyte development in vivo, which are critically important for understanding basic liver biology and developing effective strategies to induce stem cells to differentiate towards specific hepatic cell fates in vitro. The overall goal of this study was to define the precise road map and understand the underlying mechanisms for hepatobiliary lineages development. Specifically, we performed single-cell RNA-seq in sorted hepatoblasts, hepatocytes and cholangiocytes from E10.5-E17.5 mouse fetal livers. To precisely identify the time point of hepatoblast to hepatocyte transition, we performed bulk-cell RNA-seq using 2 x 10^5 sorted hepatoblasts/hepatocytes from E10.5 to E18.5. To study whether MAPK pathway promotes cholangiocyte maturation, we performed single-cell RNA-seq in sorted cells from liver explants treated with TPPB ((2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino) benzolactam, a PKC agonist) or U0126 (a MEK1/2 inhibitor).双潜能肝母细胞（bi-potential hepatoblasts）向肝细胞（hepatocytes）与胆管上皮细胞（cholangiocytes）的分化机制至今尚未阐明。本研究针对从E10.5至E17.5小鼠胚胎中分选获得的肝母细胞、肝细胞及胆管上皮细胞开展单细胞转录组分析（single-cell transcriptomic analysis），结果显示肝母细胞向肝细胞的分化遵循线性默认通路，且进程逐步推进。随着完全分化的肝细胞比例提升，增殖细胞的数量逐渐减少。值得注意的是，在同一发育阶段，增殖态与静息态的肝母细胞呈现出均一的分化状态。这一独特特征使得我们能够结合单细胞与批量细胞分析（bulk-cell analysis），精准定位肝母细胞向肝细胞的转换节点，该过程发生于E13.5至E15.5之间。与肝细胞发育的多数层面不同，肝母细胞向胆管上皮细胞的分化脱离了上述默认通路，呈现出显著的迂回分支。新生胆管上皮细胞的生成持续于E11.5至E14.5阶段，但同一发育时点下，其成熟状态存在明显异质性。更出乎意料的是，随着更多祖细胞分化为成熟胆管上皮细胞，增殖细胞的数量反而有所增加。基于单细胞分析的观测结果，本研究还发现蛋白激酶C（protein kinase C，PKC）/丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信号通路可促进胆管上皮细胞的成熟。结论：本研究明确了体内肝细胞与胆管上皮细胞发育的两条差异化通路，这一发现对于理解肝脏基础生物学，以及开发体外诱导干细胞定向分化为特定肝系细胞的有效策略具有关键意义。本研究的整体目标是明确肝胆系发育的精准路线图，并解析其背后的调控机制。具体而言，我们对从E10.5至E17.5小鼠胎肝中分选得到的肝母细胞、肝细胞及胆管上皮细胞进行了单细胞RNA测序（single-cell RNA-seq）。为精准鉴定肝母细胞向肝细胞转换的时间节点，我们使用从E10.5至E18.5分选获得的2×10^5个肝母细胞/肝细胞开展了批量细胞RNA测序（bulk-cell RNA-seq）。为探究MAPK通路是否促进胆管上皮细胞成熟，我们对经TPPB[((2S,5S)-(E,E)-8-(5-(4-(三氟甲基)苯基)-2,4-戊二烯酰氨基)苯并内酰胺，一种PKC激动剂]或U0126（一种MEK1/2抑制剂）处理的肝外植体中的分选细胞进行了单细胞RNA测序（single-cell RNA-seq）。