Figure 2.

CD133- and CD133+ hBEC characterization in steatotic and healthy livers

(A) Total percentage of EpCAM+ cells in healthy (H), fibrotic (F), and steatotic (S) livers show no significant differences (p = 0.4336). One-way ANOVA.

(B) Total percentage of CD133− and CD133+ hBECs isolated from healthy (H) and steatotic (S) livers showing a significant increase in steatotic livers. ^∗ denotes p < 0.05 (mean ± SEM), Student’s t test.

(C) Correlation of the percentage of isolated CD133− and CD133+ hBECs according to the age of the donor (years). The graph shows a non-significant trend to decrease (R² = 0.2235 and R² = 0.1405 for CD133− and CD133+ populations, respectively). Linear regression at 95% confidence intervals (n = 9 livers of diverse etiologies, 27 to 70 years old).

(D) Correlation of the percentage of isolated CD133− and CD133+ hBECs according to the sex of the donor shows no significant differences (mean ± SEM), Student’s t test (n = 7 males, 3 females).

(E) Heatmap of normalized expression values across genes significantly differentially expressed between CD133+ and CD133− hBECs in two healthy livers (HL4 and HL5). In yellow, relative upregulation; blue, relative downregulation.

(F) Heatmap of normalized expression values across genes significantly differentially expressed between CD133+ hBECs in healthy (n = 2) and steatotic livers (n = 3). In yellow, relative upregulation; blue, relative downregulation.

(G) Bright field image showing morphological differences of CD133− and CD133+ hBEC populations in a three-dimensional Matrigel culture. Scale bars, 100 μm.

(H) CD133+ population displays significantly increased colony-forming efficiency in comparison with the CD133− population. ^∗∗∗ denotes p < 0.001 (mean ± SEM), Student’s t test (n = 4 biological replicates).

(I) Percentage of survival of CD133− and CD133+ hBECs over the course of time. ^∗∗∗∗p < 0.0001 (mean ± SEM), Mantel-Cox test.

(J) Left: chromosomes of the CD133+ hBECs cultured for over 6 months. Right: karyotype of hBECs isolated from donor livers from passage 0 to passage 10 (n = 3).

(K) CD133+ cells expanded in Matrigel culture and immunostained for cholangiocyte, hepatocyte, and progenitor cell markers. CD133+ hBECs express K19, EpCAM, and SOX9 cholangiocyte markers, as well as the progenitor markers LGR5, CD24, and CD133, while lacking hepatocyte markers (HNF4α) and mature biliary markers (AE2). Scale bars, 100 μm.

(L) Brightfield images of CD133+ hBEC organoids cultured in 3D Matrigel spheres in standard expansion media (Control) and differentiation media (Diff). Scale bars, 100 μm.

(M) Expression of genes associated with a mature cholangiocyte phenotype (cytokeratin 19 K19, EPCAM, HNF1B), hepatocytes (HNF4A, CYP3A, ALB), and progenitor cells (PROM1) normalized to GAPDH. Data include total human liver for reference (liver, gray labeled), hBECs cultured in 3D Matrigel spheres in standard expansion media (Ctrol), and differentiation media (Diff). All results displayed as relative fold increase compared to controls. ^∗ denotes p < 0.05, ^∗∗p < 0.005 (mean ± SEM), Student’s t test. (n = 3–4 per group).