Single-Cell RNA Sequencing Analysis of Proliferative and Differentiated Hepatic Organoids Generated from hPSC-Derived Hepatocyte-Like Cells, using STEMdiff™ Hepatic Organoid Growth Medium and STEMdiff™ Hepatic Organoid Differentiation Medium

Functionally relevant in vitro human hepatocyte models are critical for drug safety and efficacy screening, and liver research. However, currently available models, such as primary human hepatocytes and immortalized cell lines, often either rapidly de-differentiate or lack metabolic maturity. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) and hepatic organoids represent scalable and patient-representative alternatives to conventional models, particularly for long-term cell-based assays. STEMdiff™ Hepatic Organoid media are designed to support the establishment, expansion, and differentiation of proliferative and differentiated hepatic organoids, derived from HLCs generated using the STEMdiff™ Hepatocyte Kit. Here, we compare the composition of proliferative and differentiated HLC-derived hepatic organoids, cultured in STEMdiff™ Hepatic Organoid Growth Medium and STEMdiff™ Hepatic Organoid Differentiation Medium, respectively. Overall design: Hepatocyte-like cells were generated from induced pluripotent stem cell line WLS-1C using STEMdiff™ Hepatocyte Kit, and then embedded into Matrigel domes to generate proliferative hepatic organoids using STEMdiff™ Hepatic Organoid Growth Medium (OGM). Once established, organoids were fragmented and cryopreserved using CryoStor10. To generate samples for sequencing, cryopreserved organoid fragments were recovered and cultured in OGM and passaged twice using a 7-day passaging schedule, seeding ~1000 fragments per 30uL Matrigel dome per passage. Organoids were passaged a third time in OGM and on day 7, they were either harvested for characterization of proliferative hepatic organoids, or switched to STEMdiff™ Hepatic Organoid Differentiation Medium (ODM) for a further 10 days for maturation in 3D before harvest for characterization. To harvest both proliferative and differentiated organoid conditions, Gentle Cell Dissociation Reagent was added to Matrigel domes for organoid recovery, followed by dissociation to single cells using the Animal Component-Free Cell Dissociation Kit. Cell concentration was determined using a Nucleocounter NC-250. Single cell capture and library preparation was performed using the 10X Genomics Chromium platform.