Impedance flow cytometry gauges proliferative capacity by detecting TRPC1 expression

Abstract

When examined, the expansion of many stem cell classes has been shown to be facilitated by mechanically-regulated calcium entry from the extracellular space that also helps direct their developmental programs towards mechanosensitive tissues such as muscle, bone, and connective tissues. Cation channels of the transient receptor potential C class (TRPC) are the predominant conduit for calcium entry into proliferating myoblasts. Nonetheless, methods to non-invasively study this calcium-entry pathway are still in their infancy. Here we show that a microfluidic configuration of impedancebased flow cytometry (IFC) provides a method to detect TRP channel expression in cells at high throughput. Using this technology we discern changes in the IFC signal that correlates with the functional expression of TRPC1 channels and coincides with cell proliferation. Pharmacological agents, mechanical conditions or malignant states that alter the expression of TRPC1 channels are reflected in the IFC signal accordingly, whereas pharmacological agents that alter cation-permeation through TRPC1 channels, or ionophores that independently increase calcium entry across the membrane, have little effect. Our results suggest that IFC detects changes in whole-cell membrane organization associated with TRPC1 activation and surface expression, rather than cation permeation through the channel per se. IFC-based technologies thus have the potential to identify living stem cells in their earliest stages of expansion without staining or chemical fixation. V C 2014 International Society for Advancement of Cytometry

TRPC6 in simulated microgravity of intervertebral disc cells

“…In agreement with our results obtained with human IVD cells, murine myoblasts exposed to simulated microgravity or SKF-96365 exhibited reduced proliferation and cell cycle arrest within the G2/M phase [28,31]. This reflects the fact that proliferative capacity may concurrently correlate with mechanosensitivity and TRPC function [31].…”

“…This reflects the fact that proliferative capacity may concurrently correlate with mechanosensitivity and TRPC function [31]. However, myoblasts seem to rely on modulation of TRPC1 rather than TRPC6, indicating that the exact role of these TRP channels appears to be tissue and/or species dependent.…”

“…TRP channels are therefore interesting candidates when aiming to link IVD mechanobiology with IVD ageing and degeneration. Interestingly, simulated microgravity was shown to depress TRPC1 expression associated with an accumulation of cells into G2/M, effectively stalling proliferation of mouse myoblasts [28,31].…”

Functional KCa1.1 channels are crucial for regulating the proliferation, migration and differentiation of human primary skeletal myoblasts

“…To record the basal intracellular calcium levels and the effects of paxilline in large cell population for a prolonged duration, we recorded continuously by FCM. 21, 22 DM1 myoblasts displayed a significantly higher Indo-1 ratio than normal myoblasts (Figure 2a). Blocking KCa1.1 with paxilline induced an increase in intracellular calcium levels in normal myoblasts and to a lower extent in DM1 myoblasts (Figure 2b).…”

Transient receptor potential vanilloid 2‐mediated shear‐stress responses in C2C12 myoblasts are regulated by serum and extracellular matrix

“…The recruitment of distinct calcium entry and release channels endows specificity to the selection of diverse calcium‐activated enzymes and genes at each stage of myogenesis. Notably, mechanically gated transient receptor potential (TRP) channels of the canonical and vanilloid classes invoke CICR at the early stages of myogenesis (14, 19) and concomitantly function as store‐operated channels (12, 18, 20), and alterations in their normal functioning profoundly alter skeletal muscle development (20–23).…”