(637b) Engineered Matrices Reveal Stiffness-Mediated Progression of Fatty Liver Disease

The progression of non-alcoholic fatty liver disease (NAFLD) is characterized by concurrent lipid accumulation and hardening of the liver. Despite an increasing prevalence, reduction in quality of life, and economic burden, there are no FDA-approved treatments. This is partly due to a critical gap in mechanistic insight into the interplay between excess lipid accumulation and stiffness. Current models of NAFLD rely on animal models, which do not accurately demonstrate disease progression, or rely on animal-derived matrices (e.g. Matrigel/Cultrex), which do not capture the high stiffness regimes present in diseased liver (1 – 15 kPa). Additionally, such matrices do not allow for independent presentation of biochemical cues associated with progression of liver disease such as hyaluronan (HA), elastin, and fibronectin (FN).

Engineered matrices comprised of benzaldehyde-modified HA and a hydrazine-modified elastin-like protein (ELP) with FN-derived cell adhesion motif (RGDS) allow for independent presentation of biochemical cues inherent to the diseased liver and tuning of biophysical properties. However, to achieve the elevated stiffness in liver disease (> 5 kPa) within an HA-ELP (HELP) hydrogel requires increased crosslinking density, resulting in rapid gelation times that do not allow for adequate mixing of cells and materials. To address this, we have identified a library of small molecules with either hydrazine- or aldehyde-functionality, which reversibly compete for bond formation with benzaldehyde-HA or hydrazine-ELP, increasing the gelation time. After gelation, the small molecule competitors freely diffuse out of the system, allowing for complete crosslinking of the hydrogel. Models of reaction kinetics and diffusion were built to identify relevant timescales of complete crosslinking of the hydrogel and were empirically validated using modulus recovery studies (small-angle oscillatory shear). Using HELP and small molecule competitors, we have produced hydrogels that match the stiffness of healthy (~ 0.8 kPa), early NAFLD (3 kPa), and advanced NAFLD (6 kPa) human liver.

To investigate NAFLD progression, we have developed a 3-dimensional (3D) HELP culture platform that supports formation and growth of human induced pluripotent stem cell (hiPSC) derived hepatic organoids (HO). hiPSC-derived HO have a diverse cellular composition (hepatocytes, cholangiocytes), and retain metabolic and structural functions of the liver, enabling development of an in vitro model that captures essential features of NAFLD. Formation of HO was found to be dependent upon both stiffness and cell adhesion, requiring increased concentrations (RGDS) in stiff hydrogels. Exposure to oleic acid, a fatty acid associated with NAFLD, enhanced lipid accumulation in all conditions; however high stiffness hydrogels produced significantly greater volumes of lipid accumulation. Oleic acid exposure also drove metabolic dysregulation, increasing metabolic activity. A key protein involved in fatty acid intake, CD36, was elevated in stiff environments, highlighting a potential link between ECM environment and the progression of NAFLD. Taken together, these results highlight the unresolved role of the surrounding environment in driving disease progression and the potential of an organoid in vitro model to provide mechanistic insight into NAFLD.