Figure 3.

Both hepCLiPs and bilCLiPs exhibit BEC-like phenotypes under 2D culture, while they become more hepatocyte-like under the 3D culture

(A) Schematic of strategy to establish hepCLiPs and bilCLiPs.

(B) Heatmap of hepatic and BEC markers as assessed by qRT-PCR with clonal hepCLiPs (n = 23), bilCLiPs (n = 11), fresh hepatocytes (n = 6), and fresh BECs (n = 5).

(C) Schematic of 3D culture-based hepatic induction of hepCLiPs and bilCLiPs. Images obtained for one of the hepCLiP clones are shown as a representative example. Scale bars: 100 μm.

(D) Gene set enrichment analysis (GSEA) comparing hepCLiPs cultured under 2D and 3D conditions using sets of hepatocyte-enriched genes (n = 3,432) compared with BECs and BEC-enriched genes (n = 2,048) compared with hepatocytes (Merrell et al., 2021) (n = 3 hepCLiP clones established from two donors; n = 3 bilCLiP clones established from one donor).

(E) PCA mapping of hepCLiPs and bilCLiPs cultured in 2D and 3D (n = 3) along with in vivo reprogrammed cells (n = 3). In vivo samples were harvested from normal mouse livers and those under hepatobiliary reprogramming induced by challenging the mice with 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). For in vivo reprogramming experiments, the cells were harvested by FACS.

(F) Heatmap of hepatic (n = 3,432) and BEC (n = 2,048) marker genes as assessed by RNA-seq with clonal hepCLiPs (n = 3 clones from two donors) and bilCLiPs (n = 3 clones from one donor) along with DDC-induced in vivo reprogrammed cells (n = 3).