Abstract
The balance between cell quiescence and proliferation is fundamental to tissue physiology and homeostasis. Recent studies have shown that quiescence is not a passive and homogeneous state but actively maintained and heterogeneous. These cellular characteristics associated with quiescence were observed primarily in cultured cells under a static medium. However, cells in vivo face different microenvironmental conditions, particularly, under interstitial fluid flows distributed through extracellular matrices. Interstitial fluid flow exerts shear stress on cells and matrix strain, and results in continuous replacement of extracellular factors. In this study, we analyzed individual cells under varying fluid flow rates in microfluidic devices. We found quiescence characteristics previously identified under conventional static medium, including serum signal-dependant quiescence entry and exit and time-dependant quiescence deepening, are also present under continuous fluid flow. Furthermore, increasing the flow rate drives cells to shallower quiescence and become more likely to reenter the cell cycle upon growth stimulation. This effect is due to flow-induced physical and biochemical cues. Specifically, increasing shear stress or extracellular factor replacement individually, without altering other parameters, results in shallow quiescence. We show our experimental results can be quantitatively explained by a mathematical model connecting extracellular fluid flow to an Rb-E2f bistable switch that regulates the quiescence-to-proliferation transition. Our findings uncover a previously unappreciated mechanism that likely underlies the heterogeneous responses of quiescent cells for tissue repair and regeneration in different physiological tissue microenvironments.
Highlights
Quiescence is a dormant, non-proliferative cellular state
We examined the effects of extracellular fluid flow on cellular quiescence depth using a microfluidic system with a controllable medium flow rate
Cells in microfluidic devices were cultured in serum-starvation medium (0.02% serum) for 4 days under a given extracellular fluid flow (5 or 20 μl/h)
Summary
Quiescence is a dormant, non-proliferative cellular state. Quiescent cells, still maintain the potential to proliferate upon physiological signals, making them distinct from other dormant cells that are irreversibly arrested, such as those in senescence or terminal differentiation. Activating quiescent cells (e.g., adult stem and progenitor cells) to proliferate is fundamental to tissue homeostasis and repair (Coller et al, 2006; Wilson et al, 2008; Li and Clevers, 2010; Cheung and Rando, 2013). Recent studies have revealed that quiescence is rather actively maintained and highly heterogeneous (Coller et al, 2006; Sang et al, 2008; Cheung and Rando, 2013; Spencer et al, 2013; Wang et al, 2017). The dysregulation of cellular quiescence depth can lead to disrupted tissue homeostasis, exhibiting either an insufficient number of growing cells due to an abnormally deep quiescence, or a depleted pool of quiescent stem and progenitor cells due to an abnormally shallow quiescence (Orford and Scadden, 2008; Cheung and Rando, 2013; Fujimaki and Yao, 2020)
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