It is important to note that even after the first 5 minutes of mESC culture in 2.2% O2, increases are observed in HIF-1a expression and reactive O2 species. At 12–24 hours of hypoxia, cell-cycle regulatory proteins such as cyclins D1 and E, CDK2 and CDK4 along with retinoblastoma phosphorylation increase yielding a larger fraction of mESCs in the S phase and higher overall cell numbers. Accelerated cell proliferation in aggregate regions of hypoxia will lead to increased O2 consumption further depressing pO2 levels. As our model indicates, an increasing fraction of cells will reside in the hypoxic region of the aggregates which grow continuously. The ‘cut-off’ time of cell exposure to low pO2 before hypoxia-induced changes become irreversible is unknown. Such information will be essential in formulating strategies ) to achieve ESC aggregate sizes so that a high cell fraction remains pluripotent for downstream differentiation to a desired phenotype. The effects of O2 on gene expression are mediated largely by HIF transcription factors and as noted, mESCs upregulate HIF-1a within 5 minutes of exposure to 2.2% O2. Cells in our cultures did not exhibit CYT 11387 significant differences in HIF-1a expression under different agitation rates. This may be attributed to the average cluster diameter, which generally ranged between 200-300 mm, leading to rather limited fractions of cells exposed to 2.2% O2 or less. Yet, we acknowledge that HIF-1a transcripts may not be representative of the corresponding protein amount and activity levels. Moreover, the qPCR results are population ‘averages’ and HIF expression variation at locales with different pO2 cannot be discerned. Lastly, changes due to fluctuating O2 can be brought about by HIF-independent mechanisms such as the environmental sensing mammalian target of rapamycin . Although this study focused on the transport of O2 in ESC aggregates in dish and stirred-suspension cultures, the concurrent availability and consumption of other components such as nutrients and factors for self-renewal or differentiation should be considered. A steady state analysis of the diffusion of glucose and cytokines in human EBs was recently presented. The diffusivities of glucose and cytokine were lower than that of O2 suggesting that their transport places further limits on ESC aggregates to avoid cell starvation and death. Such limitations on transport may actually hinder the growth of aggregates and cause their size to stabilize. Interestingly enough, Cameron et al. reported that over 21 days hESC aggregates displayed a maximum size at day 10 ranging between 400 and 500 mm without further increase. These observations support the choice of the Gompertz equation for modeling aggregate growth due to cell proliferation. This discrepancy may be explained by the fact that experimental data were obtained from static cultures with relatively constant bulk concentration of substrates due to daily medium changes whereas realistically these concentrations should decrease markedly over time. The maximum radius attained by ESC aggregates