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. Overall design: 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 × 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).