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

  1. Carola Maria Morell
  2. Samantha Grace Tilson
  3. Rute Alexandra Tomaz
  4. Arash Shahsavari
  5. Andi Munteanu
  6. Giovanni Canu
  7. Brandon Tyler Wesley
  8. Marion Perrin
  9. Imbisaat Geti
  10. Subhankar Mukhopadhyay
  11. Francesca Mazzacuva
  12. Paul Gissen
  13. Jose Garcia-Bernardo
  14. Martin Bachman
  15. Casey Allison Rimland
  16. Fotios Sampaziotis
  17. Irina Mohorianu
  18. Ludovic Vallier

  • Curated by eLife

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    eLife assessment

    The authors present an important study of a multi-cellular platform involving co-culturing of various hiPSC-derived hepatocyte like cells, cholangiocytes, stellate cells and macrophages to mimic the liver microenvironment. The aggregates are then treated with fatty acids and examined through transcriptomic and functional assays. The techniques and methodologically are sound, and the evidence supporting the conclusion is convincing, although more clinically relevant data demonstrating the effect of some potential pharmacological agents on the platform would serve to strengthen the study.

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Abstract

Non-alcoholic fatty liver disease (NAFLD) is a major health care challenge and new therapies are urgently needed. However, the mechanisms underlying disease remain to be understood. Indeed, studying NAFLD remains challenging due to the lack of model systems recapitulating the different aspects of the human pathology. Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity to address this limitation since they can be differentiated into large quantity of liver cells. Here, we took advantage of hiPSCs to develop a multi-cellular platform mimicking the complex interplays involved in NAFLD progression.hiPSCs-derived hepatocyte like cells (HLCs), cholangiocytes, stellate cells, and macrophages were co-cultured in a collagen-based 3D system to reproduce the liver microenvironment. Fatty acid treatments led to a NAFLD phenotype involving cell-cell interactions which were investigated by transcriptomic and functional analyses.Hepatic cells were grown up to 4weeks in 3D, retaining key functions and markers. Importantly, co-cultured cells spontaneously reorganised into physiologically relevant connections: HLCs arranged around biliary structures, which established contacts with stellate cells, while macrophages organised around HLCs. Fatty acid treatments induced steatosis and lipotoxicity in HLCs. Furthermore, fat-laden HLCs prompted a non-parenchymal cells response altering tissue architecture.Our multicellular platform provides a new approach to model interactions between human hepatic cells during NAFLD progression. Such approach has the potential to investigate the sequential events driving chronic liver diseases, including hepatocellular injury, inflammation and fibrosis. Furthermore, our system provides a unique and urgently needed tool to investigate the molecular mechanisms associated with NAFLD and ultimately to validate new targets for therapeutics development.

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  1. Version published to 10.7554/elife.95042.1 on eLife
  2. Version published to 10.7554/elife.95042 on eLife
  3. eLife

    eLife assessment

    The authors present an important study of a multi-cellular platform involving co-culturing of various hiPSC-derived hepatocyte like cells, cholangiocytes, stellate cells and macrophages to mimic the liver microenvironment. The aggregates are then treated with fatty acids and examined through transcriptomic and functional assays. The techniques and methodologically are sound, and the evidence supporting the conclusion is convincing, although more clinically relevant data demonstrating the effect of some potential pharmacological agents on the platform would serve to strengthen the study.

  4. eLife

    Reviewer #1 (Public Review):

    There is an undisputable need for better in vitro models recapitulating steatotic liver diseases. This article is from a group of well-known stem cell experts that use human induced pluripotent stem cells (hiPSCs) to build a multicellular steatosis model in vitro. While the model is strong for testing hepatocytes responses, it falls short on translational aspects as well as on non-parenchymal liver cells.

    (1) The authors should use the new nomenclature for the disease, MASLD / MASH, as proposed by the scientific societies (Rinella ME, et al. J Hepatol. 2023; 79(6):1542-1556. PMID: 37364790).

    (2) There has been a similar approach by the Takebe group (Ouchi R, et al., Cell Metab. 2019; 30(2):374-384, PMID: 31155493). What is different in this model?

    (3) The work is very technical and does neither provide any new mechanistic insights nor does it test any new interventions. I do see the clear technical advance in the long-term culture. However, I do not see that this system would allow modelling true "chronic" changes in MASLD, e.g. steatohepatitis and/or fibrosis.

    (4) While I am very convinced about the validity of the "hepatocyte" component in this system, the NPC compartment is insufficient. The 3D model does certainly not contain Kupffer cells (which have very distinct characteristics from "M0" macrophages) and does not contain true HSCs (LX-2 is a very insufficient model). Also, the model lacks flow conditions, which does not allow to factor in pathogenic signals from the circulation / portal vein (e.g. gut-liver axis). This will only allow very limited insights into the crosstalk between hepatocytes and NPCs.

    (5) The translational value of this model remains unclear to me. The scRNA-seq data should be meticulously compared to sc/snRNA-seq data from human MASLD livers at different stages to understand, what this system is able to model (maybe very early stages of steatosis?).

    (6) The study lacks a "use case" to study interventions, e.g. testing resmetirom or any other of the new MASLD drugs in this system.

  5. eLife

    Reviewer #2 (Public Review):

    Summary:

    The authors developed a 3D multi-cellular platform mimicking the complex interplays involved in the pathogenesis of NAFLD/NASH by employing hiPSCs-derived parenchymal and non-parenchymal cells in combination of organoids obtained from primary human cholangiocytes and the human hepatic stellate cell line LX2. They show that hiPSC-derived hepatocyte are able to accumulate intracellular lipids in fashion similar to human NAFLD and that prolonged accumulation leads to activation of inflammatory and fibrogenic pathways.

    Strengths:

    This is an original attempt to create a 3D all-human multicellular cellular platform recapitulating human NAFLD/NASH. The results are very encouraging. It is of particular note the fact that fibrogenic markers in the 3D system are not extremely (artificially) activated as in the classic 2D system. This makes the proposed platform more realistic.

    Weaknesses:

    The mixture of hiPSC-derived cells and primary or cell-line cells is understandable although potentially adding some variability to the system. The only unclear aspect is the characteristic of the collagen used to create the 3D system. Which type of collagen? Human? Which stiffness?

  6. Version published to 10.1101/2024.02.07.579290v1 on bioRxiv