Table 1.

Single Cell RNA sequencing experiments of liver, or cells differentiated into liver cells.

Species, stage	Number of cells sequenced	Method used and Read depth	Main findings related to cholangiocytes	Additional notes	Reference
Mouse Adult female liver 6–10 weeks and fetal liver E14.5	>50 mouse tissues 60,000 cells total; 3730 cells from fetal liver; 6426 cells from adult liver	Microwell-Seq Proof of principle in cell lines shows saturated sequencing yields 6,500 genes from 55,000 transcripts per cell. Sequencing depth used for tissues not stated.	Adult liver scRNA seq identified (in addition to several other cell types) 4 types of hepatocytes: pericentral, periportal, Fabp1-high, and mt-Nd4 high; and identified two types of epithelial (biliary) cells: undefined, and Spp1-high.	Including cell lines/cultures, >400,000 cells were sequenced in this paper. Liver not explicitly discussed in main text, some data in supplementary figures and data available and explorable at http://bis.zju.edu.cn/MCA. Fetal liver is mainly immune cells, as well as AFP-high hepatocytes and stem/progenitor cells.	[138]
Mouse E11.5, 12.5, 13.5, 14.5, 16.5, 18.5, P2.5 whole liver and P3.25 Epcam-sorted cells	E11.5-P2.5 dissociated and randomly picked on a C1 RNA-Seq IFC (Fluidigm).P3.25 FACS sorted for Epcam 557 cells from dissociated liver, 52 from Epcam-sorted P3.25	C1 Fluidigm chip For dissociated liver: unique mapped reads 1.1 -3.8million per cell. 3000-6000 genes per cell with FPKM>1. For Epcam -sorted cells, 2000 genes per cell at same sequencing depth and mapping rate.	Cholangiocytes isolated as Epcam positive cells showed high Spp1 expression, and higher expression of Jag1/Notch2 and Hes1 than hepatoblasts. Comparison of embryonic hepatoblasts with Epcam+ cholangiocytes at P3.25 showed that the two E11.5 hepatoblasts (but not later embryonic hepatoblasts) clustered with the cholangiocytes, suggesting hepatoblasts may commit to this fate earlier than previously thought.	Hepatoblast/mesenchymal hybrid cells co-express Dlk1 and Vimentin. Cdh1 is proposed as a highly specific and sensitive marker for isolation of embryonic hepatoblasts.	[16]
Mouse E9.5, E10.5 & E11.5 liver.	Organs dissected and trypsinized, individual cells mouth pipetted to lysis buffer. 332 sequenced cells from liver, 320 used after QC for further analyses	Modified STRT protocol An average of 6361 genes per cell from 0.43 million UMI transcripts.	E9.5-E11.5 liver possibly contains multiple clusters of mesoderm-derived cells, one clear cluster of epithelial cells and possibly several clusters of hematopoietic cells. Epithelial cells with mesenchymal features: some Epcam/Cdh1 positive cells in liver also express Vimentin. Dlk1 expression not described.	1916 cells in total sequenced. Cells with fewer than 2000 genes/cell removed –> 1819 were used in analyses, from embryonic mouse including forebrain, hindbrain, skin, heart, somite, lung, liver, and intestine.	[15]
Human In vitro: 2D culture of iPSCs (TkDA3–4, University of Tokyo) undergoing hepatic differentiation and 3D culture of liver bud organoids derived from hepatic cells differentiated from the iPS cell line, cocultured with HUVECS (Lonza) and MSCs (Lonza) In vivo: Adult (three donors: donor 1, female, 55; donor 2, male, 65; donor 3, male, 21) and fetal (two donors, gestation weeks 10.5 and 17.5) Mouse E14.5, E15.5, and E16.5	Liver bud organoid cells: Liver bud organoids, different constellations of cells: 177 cells dissociated, no selection. Isolation of adult human liver cells: 256 cells from human adult liver. Protocol of hepatocyte or other cell isolation from adult liver published in [82]; liver is dissociated and cell types separated using centrifugation steps. Isolation of fetal human cells: 238 cells from fetal stages, dissociated and briefly cultured (12h) on laminin-coated plates to remove red blood cells, followed re-dissociation of cells. Isolation of mouse hepatoblasts: 92 cells from mouse liver, dissociated, erythrocytes were lysed, and magnetic bead sorted for Dlk1.	C1 Fluidigm chip 1–5 million reads per cell. Cells were excluded from further analyses if they had < 100,000 reads, < 1,000 expressed genes or failed to express housekeeping genes ACTB or GAPDH	This manuscript does not explicitly identify cholangiocytes, but provides valuable insight into which culture systems better support in vitro differentiation faithful to in vivo hepatoblast growth.	iPSC-derived hepatoblasts undergoing culture in liver bud organoids more closely resemble fetal liver hepatic cells than do 2D cultured iPSC-derived hepatoblasts. Ligand-receptor pair analyses of co-cultured cells in organoids showed a KDR/VEGFA signaling pair in which VEGFA secreted by immature hepatocytes stimulates KDR on endothelial cells, which in turn support hepatoblast growth.	[8]
Human Naïve-like H9 iPSCs, primed iPSCs, and embryoid bodies.	Cells allowed to differentiate into embryoid bodies vitro and dissociated for analysis. 482 cells were identified as liver cells. 498 cells identified as epithelial.	C1 Fluidigm chip 175,000 transcripts per cell, ca 5000 genes per cell	Epithelial cell cluster is SOX9 and FOXP1 positive, and differentiation is regulated by Hippo and AMPK pathways. This could be a liver epithelial (biliary) population, or other epithelial cells.	4822 cells sequenced in total that passed quality control, of which 2636 were embryoid body cells. Day 8 embryoid bodies included liver-like cells characterized by APOA1, TTR, FGB and AFP.	[81]
Human Reanalysis of cells in [8]	See [8]	See [8]	Hypoxia induces hepatic differentiation accompanied by TGFB1 and TGFB3 suppression. However, extensive hypoxia increases TGFBs and cholangiocyte marker expression. Single cell RNA seq suggests the source of TGFB, from previously published non-hypoxia experiments. No focus on biliary cells.	TGFB2 is expressed in mesenchymal cells (MCs) while both TGFB1 and TGFB3 are expressed in ECs and MCs. TGFB receptor 1 (TGFBR1) is expressed in fetal hepatocytes and MCs.	[18]