(M-488) Progression of Fatty Liver Disease in a Hepatic Organoid Model

Introduction:: Organoids are tissue-like constructs that contain some of the structural, cellular, and functional abilities of in vivo tissue. As such, they are a valuable tool for in vitro development of disease models. Current in vitro organoid disease models rely on animal-derived matrices (e.g. Matrigel/Cultrex), which do not capture the high stiffness regimes present in many disease tissues (1 – 15 kPa). Additionally, the mechanical properties are inherent to the matrix and cannot be tuned. This presents a challenge for modeling the environment of tissue during disease, which can transition from soft and healthy (< 1 kPa) to stiff and diseased ( > 5 kPa).

A key issue in developing therapeutics for non-alcoholic fatty liver disease (NAFLD) is the transition in liver mechanics from a soft (healthy) to a stiff (diseased) liver. NAFLD affects ~30% of the global population and is the leading cause of end-stage liver disorders. Despite directly impacting a third of the world's population, there are currently no FDA-approved treatments for NAFLD. This is partly due to a critical gap in understanding the onset and progression of NAFLD, which presents both metabolic and mechanical changes in the liver. Identification of a mechanistic pathway has been hindered by the concurrence of multiple NAFLD symptoms: (1) lipid accumulation due to elevated levels of circulating free fatty acids (FFA), (2) metabolic dysregulation and production of oxidative species, and (3) altered mechanosignaling due to increased extracellular matrix deposition and remodeling (fibrosis).


Materials and Methods:: Using hyaluronan (HA) and an engineered elastin-like protein (ELP), we assembled HA-ELP (HELP) hydrogels, crosslinked via dynamic covalent hydrazone chemistry, to model NAFLD progression in patient-derived, induced pluripotent stem cell (iPSC) hepatic organoids (HO). By altering the concentration and crosslinking of HELP, we produced static and dynamic hydrogels that match the stiffness of healthy (~0.8 kPa), early diseased (3 kPa), and advanced diseased (6 kPa) human liver. Dynamic environments were induced by incorporation of a small molecule competitor that transiently disrupts formation of crosslinks in HELP.


Results, Conclusions, and Discussions:: HO were grown from single iPSCs into mature organoids over a 14 – 20 day culture period. Formation of HO is dependent upon both matrix stiffness and cell adhesion ligands, requiring increased RGDS concentrations in stiffer hydrogels. Formation efficiency was dramatically increased with the inclusion of 1 mg/mL laminin-111. Exposure to oleic acid, an FFA elevated in NAFLD, drove intrahepatic lipid accumulation in all conditions; however, high-stiffness hydrogels produced significantly greater volumes of lipid accumulation. Immunocytochemistry imaging of fatty liver HO reveals altered lipid droplet signatures after exposure to oleic acid, including an increased number and size. A dose-dependent response to fatty acid stimulation was observed, with increased lipid accumulation and cytotoxicity in > 500 µM oleic acid conditions. Notably, HO grown in HELP hydrogels were more sensitive to oleic acid cytotoxicity than HO grown in Matrigel, highlighting the need to capture features of the in vivo environment for disease modeling. Using Coherent Anti-Raman Spectroscopy, a non-linear microscopy technique that probes vibration frequencies, reveals environment-induced differences in lipid droplet structures, including phase and fluidity of lipids. Gene expression, in combination with lipidomic-style approaches, reveals a link between mechanosignaling and altered metabolic activity. In a series of mechanosignaling studies, we identified cyto-contractility as a putative causal link between stiff environments and exacerbated lipid accumulation.

A challenge in developing NAFLD therapeutics is the transition in liver mechanics from a soft (healthy) to a stiff (diseased) liver. To address this, we developed a small molecule approach to transiently disrupt hydrogel crosslinking to tune the mechanics of HELP on-demand during organoid culture. Importantly, we see that HO cultured in a dynamic environment that matches the transitional phase between healthy and diseased stiffness more readily adopt an exacerbated fatty liver phenotype. This highlights the growing need to study the dynamic and transitional phases between NAFLD stages.

In total, we have demonstrated a connection between altered mechanosignaling and fatty liver progression. Moving forward, we will use dynamic materials to identify signaling cascades that occur during transitional environments of healthy to disease.


Acknowledgements (Optional): :

References (Optional): :