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Abstract Metabolic dysfunction‐associated steatohepatitis (MASH) is a leading cause of chronic liver disease with few therapeutic options. To narrow the translational gap in the development of pharmacological MASH treatments, a 3D liver model from primary human hepatocytes and non‐parenchymal cells derived from patients with histologically confirmed MASH was established. The model closely mirrors disease‐relevant endpoints, such as steatosis, inflammation and fibrosis, and multi‐omics analyses show excellent alignment with biopsy data from 306 MASH patients and 77 controls. By combining high‐content imaging with scalable biochemical assays and chemogenomic screening, multiple novel targets with anti‐steatotic, anti‐inflammatory, and anti‐fibrotic effects are identified. Among these, activation of the muscarinic M1 receptor (CHRM1) and inhibition of the TRPM8 cation channel result in strong anti‐fibrotic effects, which are confirmed using orthogonal genetic assays. Strikingly, using biosensors based on bioluminescence resonance energy transfer, a functional interaction along a novel MASH signaling axis in which CHRM1 inhibits TRPM8 via Gq/11 and phospholipase C‐mediated depletion of phosphatidylinositol 4,5‐bisphosphate can be demonstrated. Combined, this study presents the first patient‐derived 3D MASH model, identifies a novel signaling module with anti‐fibrotic effects, and highlights the potential of organotypic culture systems for phenotype‐based chemogenomic drug target identification at scale.

Abstract Obesity and type 2 diabetes are strongly associated with adipose tissue dysfunction and impaired adipogenesis. Understanding the molecular underpinnings that control adipogenesis is thus of fundamental importance for the development of novel therapeutics against metabolic disorders. However, translational approaches are hampered as current models do not accurately recapitulate adipogenesis. Here, a scaffold‐free versatile 3D adipocyte culture platform with chemically defined conditions is presented in which primary human preadipocytes accurately recapitulate adipogenesis. Following differentiation, multi‐omics profiling and functional tests demonstrate that 3D adipocyte cultures feature mature molecular and cellular phenotypes similar to freshly isolated mature adipocytes. Spheroids exhibit physiologically relevant gene expression signatures with 4704 differentially expressed genes compared to conventional 2D cultures (false discovery rate < 0.05), including the concerted expression of factors shaping the adipogenic niche. Furthermore, lipid profiles of >1000 lipid species closely resemble patterns of the corresponding isogenic mature adipocytes in vivo (R2 = 0.97). Integration of multi‐omics signatures with analyses of the activity profiles of 503 transcription factors using global promoter motif inference reveals a complex signaling network, involving YAP, Hedgehog, and TGFβ signaling, that links the organotypic microenvironment in 3D culture to the activation and reinforcement of PPARγ and CEBP activity resulting in improved adipogenesis.