Multi omics reveals mesodermal fate bias and enables predictive cell state control in human pluripotent stem cell biomanufacturing

Despite accelerating interest in using human induced pluripotent stem cell (hiPSC)–derived products for disease modeling and therapeutic development, there is substantial evidence that conventional culture approaches do not fully recapitulate natural embryonic nor lineage committed states. It remains poorly understood how in vitro environmental conditions cause divergence from natural developmental trajectories, and current strategies emphasize restricted characterization of phenotype without appreciating the complexity of biology in maintaining pluripotency and driving differentiation. To address this knowledge gap, we examined hiPSC cell state during short term culture in stirred suspension bioprocesses under varying oxygen and agitation conditions. We profiled intracellular metabolic, transcriptional, and proteomic changes to characterize cellular responses to engineered environments and implications for cell phenotype. Using a random forest framework, we modeled population dynamics over time across metabolic and transcriptional programs and mapped those predictions onto hallmark biological signatures. This integrative approach captures and identifies environmentally reinforced programs, offering a framework to guide optimization of pluripotent cell state maintenance and differentiation.