Impact of Variability in Cell Cycle Periodicity on Cell Population Dynamics

Cell cycle synchronization has been pivotal in the development of our understanding of cell population dynamics. Intriguingly, when cells are released from a synchronized state, they do not maintain synchronized cell division and rapidly become asynchronous. Here, using a combination of experiments and model simulations, we investigate this process of "cell cycle desynchronization" in cervical cancer cells (HeLa) that are arrested at the G1/S boundary. We tracked DNA content overtime at regular intervals to monitor cell cycle progression and developed a custom auto-similarity function to quantify the convergence to asynchronicity. In parallel, using experimental data, we developed a single-cell phenomenological model that returns DNA concentration across the cell cycle stages from a desynchronizing cell population. Our simulations revealed that desynchronization is primarily sensitive to cell cycle variability. We tested this prediction by introducing lipopolysaccharide to increase cellular noise, which resulted in greater cell cycle variability with an enhanced rate of desynchronization. Our results show that the desynchronization rate of cell populations can be used a proxy of the degree of variance in cell cycle periodicity.

A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery

Conditional expression of genes and observation of phenotype remain central to biological discovery. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a 'well-tempered' controller, WTC846, for precisely adjustable, graded, growth condition independent expression of genes in Saccharomyces cerevisiae. Controlled genes are expressed from a strong semisynthetic promoter repressed by the prokaryotic TetR, which also represses its own synthesis; with basal expression abolished by a second, 'zeroing' repressor. The autorepression loop lowers cell-to-cell variation while enabling precise adjustment of protein expression by a chemical inducer. WTC846 allelic strains in which the controller replaced the native promoters recapitulated known null phenotypes (CDC42, TPI1), exhibited novel overexpression phenotypes (IPL1), showed protein dosage-dependent growth rates and morphological phenotypes (CDC28, TOR2, PMA1 and the hitherto uncharacterized PBR1), and enabled cell cycle synchronization (CDC20). WTC846 defines an 'expression clamp' allowing protein dosage to be adjusted by the experimenter across the range of cellular protein abundances, with limited variation around the setpoint.

The Effects Of Cell Cycle Synchronization On The Growth Potential Of Primary Articular Chondrocytes

Rapid production of cartilaginous extracellular matrix (ECM) is required for scale up of any articular cartilage tissue engineering approach. Although several different methods have been investigated to increase the rate of cartilaginous ECM synthesis (e.g. growth factor stimulation, mechanical loading, etc.), there is evidence to suggest that cell cycle synchronization increases rate of ECM deposition. The issue with primary articular chondrocytes (PACs) is that routine methods to synchronize cells within a particular phase of the cell cycle rely on the use of monolayer culture, which is known to elicit cellular de-differentiation. This required development of a novel method of synchronizing cells within the S phase of the cell cycle during cell isolation. The objective of this study was to test whether synchronizing PACs would improve deposition of cartilaginous ECM in a three-dimensional culture model. Findings suggested that cell cycle synchronization was a viable method of improving the rate of matrix deposition in PACs.

The Effects Of Cell Cycle Synchronization On The Growth Potential Of Primary Articular Chondrocytes

Rapid production of cartilaginous extracellular matrix (ECM) is required for scale up of any articular cartilage tissue engineering approach. Although several different methods have been investigated to increase the rate of cartilaginous ECM synthesis (e.g. growth factor stimulation, mechanical loading, etc.), there is evidence to suggest that cell cycle synchronization increases rate of ECM deposition. The issue with primary articular chondrocytes (PACs) is that routine methods to synchronize cells within a particular phase of the cell cycle rely on the use of monolayer culture, which is known to elicit cellular de-differentiation. This required development of a novel method of synchronizing cells within the S phase of the cell cycle during cell isolation. The objective of this study was to test whether synchronizing PACs would improve deposition of cartilaginous ECM in a three-dimensional culture model. Findings suggested that cell cycle synchronization was a viable method of improving the rate of matrix deposition in PACs.

Effects of incubation time and method of cell cycle synchronization on collared peccary skin-derived fibroblast cell lines

The success of cloning by somatic cell nuclear transfer depends on the efficiency of nuclear reprogramming, with the cycle stage of the donor cell playing a crucial role. Therefore, the aim was to evaluate three different approaches for cell cycle synchronization: (i) serum starvation (SS) for 1 to 4 days, (ii) contact inhibition (CI) for 1 to 3 days, and (iii) using cell cycle regulatory inhibitors (dimethyl sulfoxide, cycloheximide, cytochalasin B, or 6-dimethylaminopurine) for 1 and 2 days, in terms of their effects on synchronization in G0/G1 phases and viability of collared peccary skin fibroblasts. Flow cytometry analysis revealed that SS for 4 days (79.0% ± 1.6) and CI for 3 days (78.0% ± 1.4) increased the percentage of fibroblasts in G0/G1 compared to growing cells GC, (68.1% ± 8.6). However, SS for 3 and 4 days reduced the viability evaluated by differential staining (81.4% ± 0.03 and 81.6% ± 0.06) compared to growing cells (GC, 95.9% ± 0.06). CI did not affect the viability at any of the analyzed time intervals. No cell cycle inhibitors promoted synchronization in G0/G1. These results indicate that CI for 3 days was the most efficient method for cell cycle synchronization in peccary fibroblasts.