Background <p>The manufacturing of stem cell-based therapeutics hinges on models linking product characteristics to bioprocess conditions and cellular physiology. Typical quantitative models of cellular systems rely on population-average properties but fail to inform about the state of the cells based on critical quality attributes. Frameworks capturing the inherent heterogeneous nature of isogenic stem cell populations depend on physiological state functions (PSFs), which represent distributions of rates of cellular content change, division and differentiation. Such modalities can offer invaluable biological insights and facilitate the robust biomanufacturing of stem cell products, but obtaining the PSFs presents significant challenges.</p> Methods <p>Population balance equation (PBE) modeling was implemented to derive stem cell PSFs. Their extraction from experimental data requires the acquisition of relevant distributions for newborn and dividing cells. These subpopulations for human embryonic and induced pluripotent stem cells were identified and analyzed with multiplex flow cytometry. Solution of the PBE model featuring appropriate PSFs was achieved via interval-of-quiescence techniques.</p> Results <p>Rate distributions of synthesis, division and differentiation of human stem cells are presented. These PSFs were calculated for the first time capitalizing on experimental analysis of stem cell ensembles, including mitotic and newborn cells. Here, the PSFs were linked to the pluripotency marker POU5F1 (OCT4) as a descriptor of hPSC state and they followed a similar unimodal distribution over the OCT4 cargo for the stem cell lines examined. Exogenous lactate, which decelerates growth without affecting pluripotency marker expression, suppressed the PSF range revealing notable differences across the stem cell lines. The findings also pointed to line-specific effects induced by stressors such as high extracellular lactate. Lastly, the derivation was demonstrated of PSFs based on intracellular NANOG utilizing the PSF distributions extracted for OCT4.</p> Conclusions <p>The work provides a first account of the derivation of rate distributions – rather than population averages – of stem cell physiological properties including division and change in OCT4 content. This enables the implementation of modeling frameworks for the rigorous quantitative description of hPSC populations that is important for addressing fundamental biological questions about pluripotency and differentiation, and critical in the biomanufacturing of hPSC-based therapeutics.</p>