Modeling Metabolic Dysfunction-Associated Steatotic Liver Disease and Subsequent Progression with Stem Cell-Derived Hepatocytes

Introduction: : As one of the most common liver disorders, metabolic-dysfunction associated steatotic liver disease (MASLD) affects nearly 40% of adults worldwide. MASLD is known to be highly correlated with other health conditions, such as obesity and type 2 diabetes, suggesting the potential cause of its original development. Nevertheless, the pathogenesis of MASLD has not been comprehensively revealed. Metabolic-dysfunction associated steatohepatitis (MASH), a more severe stage of MASLD with increased inflammation, is susceptible to fibrosis, thereby leaving permanent damage of liver scarring. Weight loss at the MASLD stage has been the consensus on circumventing the progression to cirrhosis, yet there is currently no FDA-approved medication targeting MASLD. Therefore, models that can recapitulate critical features of MASLD are in dire need to be developed for better elucidation of disease pathogenesis and evaluation of drug screening.
The established models of MASLD mainly utilize immortalized hepatocytes, primary human hepatocytes, or animal models. Still, drawbacks include partial functional loss, limited in vitro lifetime, or species variation, which fail to vividly display human liver characteristics accurately. Recently generated MASLD models derived from human induced pluripotent stem cells (hiPSCs) depict a novel approach with promising progress. However, these modeling strategies have several limitations, including the inability to investigate the crosstalk among various cell lineages.

Materials and

Methods: : Firstly, hiPSCs were differentiated into definitive endoderm, which was then differentiated toward hepatocyte-like cells (HLCs) with a cocktail of growth factors. During the maturation phase between days 16 and 21, cells underwent either 2D or 3D culture to generate cell sheets or hepatic aggregates (HA). Characterization of both HLCs and HAs was carried out with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunofluorescence (IF), and multiple functional assays, including Periodic Acid-Schiff (PAS) staining, indocyanine green (ICG) uptake, urea detection, cytochrome P450 3A4 (CYP3A4) activity, and albumin enzyme-linked immunosorbent assays (ELISA). Both HLC and HA can be induced with free fatty acids to elicit steatosis at optimized dosage. Further assays, including qRT-PCR and albumin ELISA, were performed to assess MASLD-like hepatic functional decrease upon induction. Raman spectroscopy and imaging confirmed the uptake and localization of saturated and unsaturated lipid species in the HAs induced by palmitic and oleic acid. During the induction, the conditioned media were collected daily from both 2D and 3D MASLD models, and were applied to human dermal fibroblasts (HDFs) every day for the same duration. Activation of HDF was evaluated by qRT-PCR, IF, and ELISA. Multiplex assay for cytokine secretion was performed to explore the hepatokines secreted by our 2D MASLD model, suggesting potential cues that mediate the onset of MASH and progression to fibrosis. All experiments were conducted for 3 biological replicates except for the multiplex assay.

Results, Conclusions, and Discussions:: We first showed the successful differentiation of HLCs with upregulated gene expression of ALB, SERPINA, and SREBF1. IF images of HNF4α, ALB, and A1AT also indicated that the cells we derived via 2D or 3D culture were of hepatic lineage. The HLCs were proven functional, given the positive staining of PAS and ICG, as well as albumin secretion and CYP3A4 activity upon induction. After the induction of free fatty acids, both HLC and HA revealed an increase in lipid accumulation compared to the control. Large vacuoles resembling hepatocellular ballooning phenotype, which is a diagnostic feature of MASH, were observed in OA-induced HA. Lower albumin secretion and SREBF1 expression were found to demonstrate reduced hepatic functionality of our 2D MASLD model. In order to study the paracrine effect of hepatocytes on hepatic stellate cells, which is a main contributor to liver fibrosis, human dermal fibroblasts were cultured with conditioned media from the MASLD model. The HDFs receiving signals from the MALSD model were activated, resulting in increased extracellular matrix deposition, which was corroborated by the IF intensity of collagen I and fibronectin. Moreover, gene upregulation of ACTA2, COL1A1, and LAMC1 was detected in HDF stimulated by the 2D MASLD model. Amplified cytokine secretion, such as IL-6 and IL-8, was detected in the activated HDF, but not in the conditioned media from the MASLD model. This suggests the MASLD model secreted hepatokines that lead to the onset of inflammation. From the multiplex assay, we identified several potential hepatokines, such as RANTES and MCP4.
Here, we demonstrate the ability to generate functional HLCs in 2D and 3D differentiation conditions, which can induce a fibrotic phenotype in stromal cells upon supplementation of free fatty acids. Importantly, by measuring the secretome of HLCs exposed to OA and PA conditions, we were able to capture a unique signature of hepatokines mirrored in clinical observations of MASH. Collectively, our tractable system demonstrates the utility of hiPSC-derived HLCs for modeling aspects of MASLD in addition to identifying novel factors implicated in liver disease.

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