Human iPSC-derived liver co-culture spheroids to model liver fibrosis

Authors:
Laura Cools , Mina Kazemzadeh Dastjerd , Ayla Smout , Vincent Merens , Yuwei Yang , Hendrik Reynaert , Nouredin Messaoudi , Vincent De Smet , Manoj Kumar , Stefaan Verhulst , Catherine Verfaillie , Leo A van Grunsven 
In:
Source: Biofabrication
Publication Date: (2024)
Issue: 16: 4
Research Area:
Gastroenterology
Toxicology
Drug Discovery
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Culture Media:
Experiment

2.5. Differentiation of iPSCs towards monocytes and macrophages
iPSC-derived monocytes (iMCs) were harvested from embryoid bodies (EBs) according to van Wilgenburg et al (2013). For this, iPSCs were made single cell using Accutase and resuspended in mTeSR-1 medium supplemented with 50 ng ml-1 BMP4, 20 ng ml-1 SCF (Peprotech, 300–07), 50 ng ml-1 VEGF and
RevitaCell (1/100) (spin EB medium) at a concentration of 150 000 cells ml-1. Cells were seeded in 10 µl
per well in a 96 ultra-low attachment U-bottom plate (Thermo Fisher Scientific, 174929) and 100 µl of spin
EB medium was added to each well. The plate was centrifuged for 3 min at 100 g. From day 1–3, medium was changed every day with spin EB medium. At day 4 all EBs were resuspended in 20 ml of differentiation medium (X-VIVO 15 medium (Lonza, BE02-060 F), 100 ng ml-1 M-CSF (PeproTech, 300-25), 25 ng ml-1 IL-3 (PeproTech, 200-03), 2 mM L-Glutamine (Lonza, BE17-605E/U1), 100 U ml-1 PenStrep (Gibco, 15140122), 0.055 mM ß-mercaptoethanol (Gibco, 31350–010)) and cultured in 5 wells of a 6-
well plate with weekly medium changes. iMCs can be harvested from the supernatant of the EBs by filtering medium containing EBs through an invertiblestrainer (40 µM, Corning, 352340). Medium containing iMCs was centrifuged at 300 g for 5 min and resuspended in Microglia Medium (ScienCell, 1901). iMCs were seeded in 12-well plates with a density of 120 000 cells/well to transdifferentiate towards iMF. Medium was changed on day 3 and 5 after seeding in 2D and iMF were harvested on day 7.

Abstract

The lack of adequate humanin vitromodels that recapitulate the cellular composition and response of the human liver to injury hampers the development of anti-fibrotic drugs. The goal of this study was to develop a human spheroid culture model to study liver fibrosis by using induced pluripotent stem cell (iPSC)-derived liver cells. iPSCs were independently differentiated towards hepatoblasts (iHepatoblasts), hepatic stellate cells (iHSCs), endothelial cells (iECs) and macrophages (iMF), before assembly into free floating spheroids by culturing cells in 96-well U-bottom plates and orbital shaking for up to 21 days to allow further maturation. Through transcriptome analysis, we show further maturation of iECs and iMF, the differentiation of the iHepatoblasts towards hepatocyte-like cells (iHeps) and the inactivation of the iHSCs by the end of the 3D culture. Moreover, these cultures display a similar expression of cell-specific marker genes (CYP3A4, PDGFRß, CD31andCD68) and sensitivity to hepatotoxicity as spheroids made using freshly isolated primary human liver cells. Furthermore, we show the functionality of the iHeps and the iHSCs by mimicking liver fibrosis through iHep-induced iHSC activation, using acetaminophen. In conclusion, we have established a reproducible human iPSC-derived liver culture model that can be used to mimic fibrosisin vitroas a replacement of primary human liver derived 3D models. The model can be used to investigate pathways involved in fibrosis development and to identify new targets for chronic liver disease therapy.