About the non-cell autonomous inflammation and fibrogenic effects
Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive severe disease characterized by hepatic lipid accumulation, inflammation, and fibrosis, which lacks any approved drug therapy. Novel approaches to identify therapeutic candidates that predict clinical responses are needed. Human pre-clinical models, including organoids/spheroids, are powerful models for drug testing and discovery. The selection of donors to model drug responses is, however, a critical step to providing translational readouts and is currently a challenge.
We have previously developed a human-derived 3D spheroid model containing primary human hepatocytes, Kupffer cells, liver endothelial cells, and stellate cells from healthy donors. By inducing a MASH-inducing cocktail, we were able to recapitulate the main hallmarks of MASH, including steatosis, inflammation, and fibrosis.
Here, we aim to generate patient-derived liver spheroids to model MASH hallmarks. We selected primary human hepatocytes from 2 donors with a NAFLD Activity Score (NAS) of 4. We then generated liver spheroids with these steatotic primary hepatocytes in combination with primary Kupffer, endothelial, and stellate cells obtained from healthy donors. To understand the non-cell autonomous effects of hepatocytes in non-parenchymal cells, we investigated the progression of MASH hallmarks in control versus MASH-prone culture conditions.
We first monitored the cell viability and spheroid aggregation markers, including size, ATP levels, and lactate dehydrogenase (LDH) leakage, and showed comparable levels between healthy and steatotic donors. Then, we performed an extensive histological and immunohistochemical characterization with hepatic (Albumin), liver endothelial cells (CD31), Kupffer cells (CD68), and stellate cells (Vimentin) markers revealing the presence of all these cell types.
We showed that triglyceride levels were elevated in the 2 steatotic donors under MASH culture stimuli compared to healthy hepatocyte donors. Remarkably, only 1 donor showed initial increased triglycerides under control culture conditions. In this case, we showed an association between primed steatosis and increased basal fibrogenesis, which was induced even in the absence of MASH culture stimuli.
In conclusion, this study reveals the importance of testing several donors to recapitulate the diversity of MASH pathophysiology in the phenotype of multicellular spheroids. We suggest that slow versus fast progression into fibrosis of MASH patients is captured by these patient-derived multicellular spheroids.
The donor-dependent phenotypic differences in this study offer a unique opportunity to test drug responses and understand the underlying genetic diversity of MASH etiology. Future biobanks with larger pools of patient-derived liver spheroids will be instrumental in identifying efficacious therapeutic approaches.
Download our poster about non-cell autonomous inflammation and fibrogenic effects in multicellular liver spheroids
