Figure 2.

Characterization of Feline Liver Organoids

(A) Representative cytological and immunofluorescent images of feline liver organoids. H&E staining showed that organoids consisted of single-layered cubical epithelium. They stained positive for epithelial marker E-cadherin (green) and were highly proliferative in culture as shown by EdU staining (green, marks S phase of the cell cycle). DAPI (blue) was used as nuclear counterstain.

(B) Gene expression analysis of feline liver organoids (n = 4 donors) in different passages (p2, p8, p14) and normal cat liver. Relative gene expression (expr.) is shown of adult stem cell, progenitor/biliary, and early and mature hepatocyte markers.

(C) Representative images of immunocyto-/histochemical stainings of feline liver organoids and normal cat liver. Organoids stained positive for progenitor/biliary markers K19, HNF1β, and BMI1. They stained negative for hepatocyte marker HepPar-1, but for albumin and ZO1 small clusters of cells within single organoids stained positive (indicated by arrowheads and arrows, respectively).

(D) Karyotyping of feline liver organoids. A representative metaphase spread is shown of a cell with a normal chromosome number (n = 38). Chromosome counts were compared between low- and high-passage number cultures (p3–p7 versus p16–p23, n = 4 donors per category) and plotted as percentage of cells with a normal chromosome number (n = 38), one gain (n = 39), one loss (n = 37), or two or more losses (n ≤ 36).