Novel 3D Approach to Model Non-Alcoholic Fatty Liver Disease using human Pluripotent Stem Cells

Carola Maria Morell
Samantha Grace Tilson
Rute Alexandra Tomaz
Arash Shahsavari
Andi Munteanu
Giovanni Canu
Brandon Tyler Wesley
Marion Perrin
Imbisaat Geti
Subhankar Mukhopadhyay
Francesca Mazzacuva
Paul Gissen
Jose Garcia-Bernardo
Martin Bachman
Casey Allison Rimland
Fotios Sampaziotis
Irina Mohorianu
Ludovic Vallier author has email address

Wellcome-MRC Cambridge Stem Cell Institute, JCBC Cambridge Biomedical Campus, Dept of Surgery, University of Cambridge, Cambridge, UK
Liver Disease Branch, NIDDK, NIH, Bethesda, Maryland, USA
Wellcome Trust Sanger Institute, Hinxton, UK
UCL Great Ormond Street Institute of Child Health, London, London, UK
NIHR Great Ormond Street Hospital Biomedical Research Centre, London, UK
Medicines Discovery Catapult, Alderley Park, UK
Immunopathogenesis Section, Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, MD
Berlin Institute of Health Centre for Regenerative Therapies, Berlin, Germany
Max Planck Institute for Molecular Genetics, Berlin, Germany

https://doi.org/10.7554/eLife.95042.1

Open access
Copyright information

Figures and data

3D HLCs cultures characterisation.
A) Schematic representation of HLCs embedding into RAFTs at D23. B) 2D and 3D cultures were characterised for gene expression of key hepatocyte markers (PHH and undifferentiated hiPSCs [undifferentiated control, UC] were used as positive and negative control, respectively). C) Immunofluorescence of 3D HLCs. D) ALB/AFP secretion. E) CYP3A4 expression. D) CYP3A4 basal activity. G) production of cholic acid (CA). Graphs represent average±SD of n=3-7 experiments, analysed with ANOVA.

3D HLCs cultures mimic steatosis and lipotoxicity.
A) FFAs challenge in HLCs visualised with Bodipy493/503 (green): top row, 2D HLCs treated with OA; middle row, OA treatment; bottom row, PA treatment. 3D cells were stained for albumin (yellow). B) Bodipy493/503 quantification, C) CYP3A4 activity and D) cell viability, all performed on 3D cultures. Graphs represent average±SD of n=3 experiments, analysed with ANOVA. E) RNA-Seq analyses on HLCs monocultures of disease-associated pathways. Heatmaps represent the log2 fold change of each treatment and timepoint vs control group. Red indicates an increase, whereas blue indicates a decrease in expression relative to control.

3D cultures are compatible with the growth and activation of NPCs.
A) Representative pictures of COs structures in RAFTs, positive for tdTomato reporter and biliary markers; B) biliary gene expression compared to COs grown in matrigel. C) Quiescent-like morphology of HSCs-tdTomato+ grown in collagen; TGFβ1-induced D) myofibroblast-like morphology and E) pro-fibrotic genes expression. F) M0-mStrawberry+ pancake-like shape and IBA1 expression. G) Pro-inflammatory genes expression after LPS treatment. Graphs represent average±SD of n=3-10 experiments, analysed with t-Test.

3D co-cultures promote physiologic cellular interactions.
A) Live cell imaging of HLCs-EGFP+ around COs-tdTomato+, with z-stack reconstruction showing the presence of a lumen. B) MRP2 positivity of a bile canaliculi-like network after 1-week culture (HLCs-unstained, COs-tdTomato+). C) HSCs-tdTomato+ arranged on one side of HLCs-EGFP+ clumps; D) TGFβ1 treatment induced a myofibroblast-like morphology in HSCs-tdTomato+, while surrounding HLCs-EGFP+. E) M0-mStrawberry+ diffused in the RAFT, while gradually organising on one side of the HLCs-EGFP+ clumps; after 4-weeks culture, M0-mStrawberry+ spread within HLCs-EGFP+ structures. F) ALB/AFP secretion and G) basal CYP3A4 activity in 3D mono-versus co-cultures. Graphs represent average±SD of n=3-10 experiments, analysed with t-Test.

Complex 3D co-cultures mimic liver microenvironment.
A) Cell-cell contacts were maintained throughout 4-weeks. HLCs-EGFP+ surrounded COs-tdTomato+, which in turn recruited HSCs-CFP+; the co-culture also included M0-unstained. B) CYP3A4 activity and C) ALB/AFP secretion in 3D mono-versus co-cultures. “Mix” indicates 4 cell types co-cultures. Graphs represent average±SD of n=3-4 experiments, analysed with t-Test. D) scRNA-Seq analyses of 1-week co-cultures. UMAP representation of 16 different groups that organised in 4 cellular clusters, identified by typical markers shown by violin plots.

Cell-cell communication in vitro.
CellPhone DB representation of the different cell types in 3D expressing ligands/receptors to regulate a variety of signalling. These interactions are not symmetric. The size of the dots indicates the mean-of-means (p<0.05); interactions and cell-type pairs without any dot indicate no significant interaction.

Complex 3D co-cultures promote NAFLD-like features.
A) Live cell imaging shows cellular structures after long term FFAs challenge. Top row, HSCs-CFP+ accumulated around fat laden HLCs-EGFP+, that organised around COs-tdTomato+ cells after OA treatment. 4-weeks of PA resulted in the near absence of HLCs-EGFP+ clumps and unorganised COs-tdTomato+, while HSCs-CFP+ showed an activated morphology. M0s were included in the culture, but lacked a reporter gene. Bottom row, Lipidtox Deep Red (yellow) confirmed that HLCs-EGFP+ developed a steatotic phenotype after OA treatment. B) CYP3A4 activity and C) cell viability in 1-week and 4-weeks co-cultures. D) TNFα, E) IL6, and F) TGFβ1 secretion in co-cultures. Graphs represent average±SD of n=3-10 experiments, analysed with ANOVA.

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