171 6 iPSC-derived liver organoids as a tool to study Medium Chain Acyl-CoA Dehydrogenase deficienc RESULTS Generating induced pluripotent stem cells from control and MCADD patient fibroblasts Control and MCADD fibroblasts were reprogrammed into iPSCs following a previously published protocol33. Immunofluorescence confirmed the expression of NANOG, OCT4, SOX2, SSEA-4, Tra-1-60 and Tra-1-81 indicating the pluripotency of the iPSCs (Supplementary figure 1). Generating expandable hepatic organoids (EHOs) from control and MCADD iPSCs In order to differentiate iPSCs into hepatic organoids, a previously published protocol 34 was slightly adapted (Figure 1a): to supplement Wnt3a and hRspon1, conditioned medium was used instead of recombinant proteins. At day 8 the cells were transferred from 2D culture and embedded into BME domes and cultured in expansion medium (EM) for at least 10 days. Morphological changes in the cells were observed at the different stages of the differentiation (Figure 1b). While most of the organoids were cystic and contained clear lumina, more complex structures could also be observed. Some organoids formed “lobule-like” structures emerging from a central structure (Figure 1b). At this stage, expandable hepatic organoids (EHO) could be cryopreserved and - after thawing - expanded in culture for several passages without any clear morphological changes. In order to assess cell identity at the different stages of differentiation, the gene expression profile of the organoids was characterized (Figure 1c-e). We defined different developmental stages including iPSCs, definite endoderm (DE), hepatoblasts (HB), and EHOs (Figure 1a and b), and compared them to human liver biopsies. Endoderm-specific markers SOX17, FOXA2, GATA4, and GATA6 were all induced at DE stage. FOXA2, GATA4, and GATA6 stayed higher than in the iPSCs at all stages, while SOX17 declined in EHOs, resembling the liver reference (Figure 1c). Early hepatic specification markers TBX3 and HNF4-α were already observed in the DE stage but peaked in the EHOs. For alpha-fetoprotein (AFP), a fetal hepatocyte marker, we observed a peak in expression at the stage of EHOs and barely any presence in the earlier stages, nor in liver tissue. The hepatic marker albumin was not observed until later stages of the differentiation (HB) and showed its maximal expression in the EHOs. Interestingly, the albumin expression was very high in the EHOs and almost comparable to that in human liver (Figure 1d). Finally, the expression of early biliary markers CK19 and SOX19 (Figure 1e) illustrates the presence of more than one cell type in the organoids. Together, these results
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