SRP343020 PRJNA774268 GSE186519 3D Adipose Tissue Culture Links the Organotypic Microenvironment to Improved Adipogenesis Understanding the molecular underpinnings of adipogenesis is 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 (R^2 = 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 TGFß signaling, that links the organotypic microenvironment in 3D culture to the activation and reinforcement of PPAR? and CEBP activity resulting in improved adipogenesis. Overall design: RNA sequencing by poly(A) capture was performed for adipocyte spheroid cultures developed from primary human adipocyte progenitors from the stromal vascular fraction and for corresponding freshly isolated mature adipocytes with seven donors in total. GSE186519 pubmed 34165908