(100f) Analyzing the Dynamics of Cell Cycle Transition in Differentiating Embryonic Stem Cells Through an Integrated Experimental and Modeling Approach

Embryonic stem cells (ESC) have the potential to be used in many therapeutic applications due to their ability to expand in an undifferentiated state while being able to differentiate to any cell type under specific conditions. One important characteristic vital to this behavior is the cell cycle. In somatic cells, the cell cycle is characterized by a lengthy G1 (growth) phase and a long doubling time. ESC are typically characterized by a shortened G1 phase, with an increased proportion of cells residing in the S phase (DNA replication). This leads a shorter time between mitotic events. These traits are crucial for ESC to facilitate quick self-renewal and proliferation. Although these unique cell cycle traits are central to ESC, attempts towards a thorough quantitative analysis of the cell cycle behavior in human ESC (hESC) and in reprogrammed induced pluripotent cells (iPS) is lacking thus far. Furthermore, little work has been done in the area of mechanistic information and dynamics of cell cycle transition from undifferentiated to germ layer. In this project we are using an integrated experimental and computational approach to investigate cell cycle traits with different hESC and iPS cell lines, both at the singular cell and population levels. Furthermore, we investigate the dynamics of cycle behavior transition when differentiation is induced.

Two different hESC lines (the use of which we have approval for) were used for the study, H1 and H9, as well as three iPS lines (Y1 and hFib2-iPS5, derived from dermal fibroblasts, and a type I diabetes specific line). Proportions of the cell cycle were quantified with the DNA stain propidium iodide (PI) and flow cytometry. Further characterization of the cell cycle was performed with Brdu, an S-phase proliferation maker, to extract information regarding the length of cell cycle phases. Quiescent cell state was determined by the lack of Ki67 stain. The undifferentiated cells were first analyzed to determine base level cycle phase characteristics. To study how this heterogeneous population is generated from a homogeneous starting point, the pluripotent cells were synchronized in the G2 phase by Nocodazole. Cells were released from cell synchronization, and the DNA content was analyzed every two hours to determine, temporally, how the cells revert to their asynchronous behavior. In the differentiation studies, endoderm was induced by supplementing culture media with Activin A. Four different induction pathways were compared: Activin A supplemented with either Wnt3a, Bmp4, Fgf2 or PI3K inhibition. PI, Brdu, and Ki67 stains were performed at different time points for each condition to determine the dynamics of the cell cycle phase shift. To determine the extent of differentiation, both quantitative polymerase chain reaction (qPCR) and intracellular flow cytometry were utilized. Furthermore a stochastic population based model was developed to extract mechanistic information from the experimental observations.

The simulated cell cycle data was compared to the experimental observations, and through this comparison probabilistic parameters and characteristics associated with ESC were determined. Only certain probability distributions and moments (e.g. mean and variance) were able to recapitulate experimental dynamics. It is shown that the simulated data can describe the experimental data well, being able to generate the PI flow cytometry histograms, both of the asynchronized pluripotent cells and the dynamics after release from synchronization. The doubling time predicted by the model was validated by time-lapse microscopy and Brdu analysis.  Furthermore, by performing the analysis on differentiating cells,  it is shown that different induction conditions show different behavior in terms of cell cycle, with different dynamics associated with the transition from “pluripotent cycle” (shorter doubling times and reduced G1 time) to “differentiated cycle.” Our stochastic population model is able to draw mechanistic insight regarding both the nature of the cell cycle and of differentiation from experimental observations. Although applied to undifferentiated and germ layer cells, the model could be extended to later stages of differentiation.