Product Manager, MEA Systems Axion BioSystems Atlanta, Georgia, United States
Abstract: The liver is essential for critical physiological processes, such as detoxification, protein synthesis, metabolism, and hormone regulation. Despite its remarkable in vivo regenerative capacity, in vitro expansion of hepatocytes remains challenging. Induced pluripotent stem cells (iPSCs) offer a versatile source for generating hepatic cells and can be used to create hepatic organoids, which mimic liver structure and function. Effective differentiation of iPSCs into hepatic organoids requires monitoring both differentiation and organoid development. This study outlines a novel non-invasive workflow for analyzing iPSC-derived hepatic organoid development. iPSCs (StemCell Technologies) were differentiated into hepatic progenitor cells (HPCs) and hepatocyte-like cells (HLCs) using supplier protocols. HLCs represent a more advanced stage of hepatocyte lineage differentiation. Morphological changes during differentiation were monitored using the Omni live-cell imager. Subsequently, HPCs and HLCs were embedded in individual Matrigel domes to form organoids. Organoids were cultured for one week, passaged, and then imaged every 4 hours for 72 hours using the Omni to measure organoid area, diameter, and roundness. Clear morphological differences were observed across various differentiation stages. iPSCs had a highly compact, small-cell morphology, while definitive endoderm cells were even smaller and more compact. In contrast, HPCs were larger with a tightly packed cobblestone morphology. HLCs were the largest cells, maintaining the tightly packed cobblestone arrangement. Both HPC- and HLC-derived organoids were spherical with a prominent lumen. The average roundness across all time points was similar for both organoid types (0.85). Initially, HPC-derived organoids were smaller (diameter: 53 µm; area: 3027 µm²) than to HLC-derived organoids (diameter: 67 µm; area: 6885 µm²) but grew 3.6-fold over 72 hours as compared to 3.0-fold for HLC-derived organoids, indicating growth differences that may be related to cell maturity. This non-invasive workflow highlights the utility of live-cell imaging for real-time monitoring of iPSC differentiation and hepatic organoid formation, advancing liver research and applications in disease modelling and therapy.
