(232c) Linking Human Pluripotent Stem Cell Cycle Regulation and Culture Density during Expansion and Differentation

Cell cycle progression is a major regulator orchestrating human pluripotent stem cell (hPSC) self-renewal and differentiation. Self-renewing hPSCs exhibit a very short G₁ phase and proliferate faster than their differentiating counterparts. Among the stem cell environment variables, which affect the regulation of cell cycle, we focused in this study on the culture density. The association between hPSC cycle and culture density has been largely empirical.

The cycle of hPSCs was examined under various culture densities and media [1]. The fraction of cells residing in the G₁ phase increased from 25% to over 60% in high-density culture (>40 × 10⁴ cells/cm²) while hPSCs still maintained their expression of pluripotency markers such as NANOG, TRA-1-60 and TRA-1-81. Cells cultured at higher densities also upregulated their expression of the cycle inhibitor CDKN1A (p21) but not of p27 and p53. A population balance equation (PBE) model was constructed based on experimental data to rationalize in quantitative terms the link between culture density, cell cycle progression, and the states of self-renewing and differentiating hPSCs. The proliferation kinetics of each subpopulation were determined. Media commonly used for maintenance of self-renewing hPSCs exhibited the slowest kinetics of induction of differentiation (k_diff), but supplementation with BMP4 led to 14-fold higher k_diff values.

To that end, a cell-cycle fluorescence reporter system (FUCCI) [2] has been employed to further investigate the progression of the cell cycle in pluripotent and differentiating hPSCs. In conjunction with aforementioned framework, we have estimated the physiological state functions of hPSCs under different conditions. Our findings will contribute to the deepening of our understanding of cell cycle regulation during self-renewal and differentiation thereby leading to improved strategies for generating therapeutically useful progeny from hPSCs.

References:

[1] J. Wu, Y. Fan, E.S. Tzanakakis. Increased culture density is linked to decelerated proliferation, prolonged G₁ phase, and enhanced propensity for differentiation of self-renewing human pluripotent stem cells. Stem Cells Dev. 24(7):892-903, 2015.

[2] A. Sakaue-Sawano, H. Kurokawa, T. Morimura et al.  Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 132(3):487-98, 2008.