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Dynamic cell contacts between periportal mesenchyme and ductal epithelium act as a rheostat for liver cell proliferation

1st December 2022

Organoid cultures have emerged as an alternative in vitro system to recapitulate tissue in culture to study tissue biology and their use for biomedical applications. Liver organoids have now been established for multiple species from induced pluripotent stem cells, embryonic stem cells and adult tissue derived cells, however, there is limited understanding about how those 3D cultures compare to the original tissue structure and function at different stages of the organ development and repair.
The adult liver epithelium comprises of hepatocytes and biliary ducts lined by ductal cells. The epithelium and duct cells are mostly in homeostasis, but they proliferate swiftly upon damage enabling rapid healing and regeneration.

In this study, the authors established ductal-mesenchymal organoids to investigate how liver self-organisation and determine how internal stability is maintained and the switch to promote injury and repair.
Mesenchymal cells (Msc cells) — hepatic stellate cells and portal fibroblasts — express various mitogens and growth factors essential for regeneration and organoid formation. The authors observed that Msc cells regulate ductal cells in two paradoxical ways: secreting pro-proliferative signals yet inducing growth arrest via cell contact. This opens a new concept on how culturing methods could recapitulate physiological liver architecture.

This study changes the paradigm of the role of Msc cells within the liver and highlights the importance of the relative abundance of cell-to-cell contact not just the absolute number of cells within the cellular niche. Cell proliferation within the niche structure (whether in vivo or in vitro) will be dictated by both secreted mitogen factors and also direct cell-cell inhibitory contact during different phases of the damage-regeneration responses. This is particularly important when developing in vitro cellular models that mimic precisely stages of damage and regeneration of native tissues for biomedical and pharmaceutical testing purposes.

  • Results from mice fed with a 0.1% DDC diet for 5 days and allowed to recover for 7 and 38 days (early and end phase, respectively), demonstrated that Msc and ductal cells proliferate at different rates during homeostasis, damage, and regeneration:
  • Liver damage led to increased proliferation of ductal cells, which resulted in a decreased ratio of Msc:ductal cells. This caused a significant decrease in the number of cell contacts between both populations. By the early phase of recovery, Msc cells increased in number and re-established cell contacts with ductal cells. By end phase of the recovery both Msc and ductal cells returned to their steady-state numbers, ratios, special disposition, and cell contacts.
  • Immunostaining and RNA sequencing analysis demonstrated a high degree of Msc cells’ heterogeneity. Three distinct clusters were identified: Acta2-Lrat/Reln-, and Cd34-expressing cells.
  • Platelet derived growth factor receptor alpha (PDGFRα+) and stem cell antigen 1 (SCA1) (PDGFRα+ SCA1+) Msc cells appeared to be exclusively localized at the portal tract near to liver ductal cells, physically wrapping the ductal epithelium.
  • Co-staining of PDGFRα+SCA1+ Msc cells with ductal cells in matrigel co-cultures indicated that Msc cells sustain organoid formation.
  • To imitate adult portal tract’s architecture the researchers resuspended Msc and ductal cells in agarose microgel droplets and loaded them onto custom-designed microfluidic flow devices to encapsulate both cell types. Co-encapsulated organoids exhibited complex structures at great efficacy, similar to in vivo structures.
  • To further investigate the molecular basis of these effects, the authors analysed RNA profiles of both cell types. Ductal cells expressed Notch1, and Notch2Tgfbr1Tgfbr2 and Hippo pathway downstream effectors Yap1 and Wwtr1(TAZ), while Msc expressed Notch and Tgfb ligands. Efficacy of organoid formation in medium containing the signalling pathway inhibitors was disrupted, with Notch signalling inhibitors having the most significant effects.
  • Using matrigel co-cultures of freshly sorted cells from genetically modified mice the authors confirmed that Notch signalling inhibits ductal cell proliferation.