176 Chapter 6 medium-chain acyl-CoA. Given the indications of higher number of peroxisomes in hepatic cells36, we decided to first further differentiate the EHOs into mature EHOs (Mat-EHOs) in order to stimulate peroxisome proliferation. EHOs were kept in expansion medium (EM) for 4-5 days after passage and then transferred to maturation medium (MM) (Figure 3a). Maturation medium contains different cytokines that allow the EHOs to mature into a more hepatocyte-like state. EHOs in MM underwent morphological changes, shrinking in size and developing thicker outer edges. (Figure 3b). Maturation was confirmed by upregulation of mature hepatocyte markers HNF4-α, AFP, Albumin and CYP2C9. Matured organoids also showed lower expression of cholangiocyte markers TBX3 and CK19. Moreover, genes coding for enzymes involved in peroxisomal fatty-acid oxidation (ABCD1, ACOX1, and CROT) were significantly upregulated (Figure 3c). Finally, Mat-EHOs secreted higher levels of albumin into the supernatant than EHOs, in line with a more hepatocyte-like phenotype (Figure 3d).
RkJQdWJsaXNoZXIy MTk4NDMw