Fully controlled iPSC-derived spheroids to model liver fibrosis

Introduction: Chronic liver injury leads to overproduction of scar tissue, known as fibrosis. If left untreated, long-term injury can eventually result in the development of liver cirrhosis, which leads to 1 million deaths per year worldwide. During liver injury, damaged hepatocytes activate the hepatic stellate cells (HSCs), which play a pivotal role in the wound healing response by depositing excessive amounts of extracellular matrix. If the injury persists, this ultimately results in the development of liver fibrosis. Unfortunately, there are currently no FDA-approved anti-fibrotic drugs available. This is partially due to the lack of adequate human in vitro models that recapitulate the cellular composition and function of the human liver.
Aim: The goal of this study was to develop a human spheroid model to mimic liver fibrosis by using induced pluripotent stem cell (iPSC)-derived hepatoblasts (iHepatoblasts), hepatic stellate cells (iHSCs), endothelial cells (iECs) and macrophages (iMφ).
Methods: iPSCs (Sigma IPSC0028-1VL) were independently differentiated into iHepatoblasts, iHSCs, iECs and iMφ (Kumar et al., Biomaterials, 2021), before assembly into free floating spheroids by culturing cells in 96-well U-bottom plates and orbital shaking for up to 21 days. Differential expression and Gene Ontology (GO) analysis were carried out within R on bulk RNAseq samples. Functionality of iHSCs was tested by activation with TGF-β (48h) or through iHepatocyte (iHep) damage with acetaminophen (APAP) (72h) until day 13. Primary human liver spheroids were
generated using liver resections obtained from UZ Brussel from which hepatocytes were freshly isolated with a Percoll gradient and HSCs (UV+), ECs (CD32+) and Kupffer cells (CD45+ CD163+) by FACS.
Results: Since we incorporate iHepatoblasts in spheroids that need to mature to iHeps and since HSCs play a crucial role in chronic liver diseases, we used iHep maturation and iHSC activation as readouts for the optimisation of cell ratios in the spheroids. This resulted in an optimised ratio of 2 iHSC : 1 iHepatoblast : 0.6 iEC : 0.6 iMφ. We compared differentiation towards mature iHeps in 3D co-cultures to differentiation in 2D iHep monocultures (21 days). We detected higher gene expression levels of mature hepatocyte markers at day 21 of spheroid cultures compared to 2D cultures. This suggests that differentiating iHeps in 3D in the presence of other liver cell types is beneficial for their maturation. To evaluate the liver cell types in co-culture spheroids further, we performed RNAseq at the start (day 1) and at the end (day 21) of spheroid cultures. GO analysis showed that day 1 cells were still actively in the cell cycle, while spheroids cultured for 21 days showed high expression of genes involved in metabolic processes, i.e. phase I and II metabolism, drug and bile acid metabolism and gluconeogenesis. Similar to Hep markers, EC (PECAM1) and Mφ (ITGAM) markers increased over time, suggesting further maturation of these cells in 3D as well. These findings were confirmed with immunofluorescence stainings on day 21 spheroids for CYP3A4, CD31, CD45 and PDGFRβ for Heps, ECs, iMφ and HSCs respectively. At day 21, spheroids showed decreased expression of HSC activation markers and increased expression of markers specific for quiescence, such as respectively COL1A1 and LRAT, suggesting that the iHSCs become more quiescent in these co-cultures. To mimic fibrosis, we either directly (TGF-β) or indirectly (APAP) induced iHSC activation. Both conditions led to a significant increase of HSC activation marker genes (ACTA2, COL1A1, LOXL2) indicating functionality of both iHSCs and iHeps. Lastly, our iPSC model was compared to spheroids consisting of freshly isolated primary human liver cells. Gene expression levels of cell type-specific markers such as CYP3A4 and PDGFRβ at day 7 of primary liver spheroid cultures were similar to day 13 of iPSC liver cultures, suggesting that the iPSC-derived liver spheroid culture is an adequate in vitro human liver model. Comparisons of cytotoxicity in iPSC versus primary human liver spheroids, induced by APAP and other compounds are still ongoing.
Conclusions: We have established a robust human iPSC-derived liver culture model that can be used to mimic fibrosis in vitro as a replacement of primary human 3D models. This model can be used to investigate pathways involved in fibrosis development and to identify new targets for chronic liver disease therapy.