Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here researchers from the Max Planck Institute for Evolutionary Anthropology use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. They then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. They find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. The researchers benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. They use a receptor–ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. This molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.
Exploring human hepatic differentiation from pluripotency in 2D culture and 3D LB organoids
a, scRNA-seq was performed on cells during hepatocyte-like differentiation from pluripotency in 2D culture and in 3D LBs. Time points: day 0, iPS cell, 80 cells; day 6, DE, 70 cells; day 8, HE, 113 cells; day 14, IH, 81 cells; day 21, MH, 81 cells; day 0 input EC, 74 cells; day 0 input MC, 104 cells. Day 3 LBs: HE-LB, 54 cells; EC-LB, 53 cells; MC-LB, 67 cells. The 2D and 3D data are from the same two differentiation batches. Six LB replicates were analysed. b, Pairwise correlation lineage network reveals a differentiation topology from iPS cells through DE–HE–IH–MH. Correlation threshold, Pearson’s r > 0.4. c, Pseudotime ordering of 2D cells shows changes in gene expression (log2(fragments per kilobase of mapped reads, FPKM)) from pluripotency to hepatocyte-like fate. Time point shown above the heatmap. Column, single cell; row, gene. d, Cell network with cells coloured on the basis of gene expression that distinguishes the lineage. e, Brightfield and confocal image of a 3-day LB with HE (tagged with enhanced green fluorescent protein, eGFP) and EC (Kusabira Orange, KO-Red) labelled. MCs are unlabelled. Scale bar, 100 μm. f, PCA separates cells on the basis of SNPs. g, PCA separates input and LB cells, suggesting distinct transcriptional changes upon LB formation. h, SOM (50 × 50 grid) constructed from input and LB single-cell transcriptomes showing scaled metagene expression. Black lines demarcate overexpressed metagene signatures. Top left heatmap shows metagene signature score for each input and LB cell population. Black represents the strongest score. ER, endoplasmic reticulum; MSC, mesenchymal stem cell. i, Mean profiles for each input and LB cell type are shown. Arrows mark overexpressed signatures; lineage-defining signatures (E, O, and C) are starred