Stem/Proliferative and Differentiated Cells within Primary Murine Colonic Epithelium Display Distinct Intracellular Free Ca²⁺ Signal Codes

Abstract

The second messenger, intracellular free calcium (Ca 2+ ), acts to transduce mitogenic and differentiation signals incoming to the colonic epithelium. A self-renewing monolayer of primary murine colonic epithelial cells is formed over a soft, transparent hydrogel matrix for the scalable analysis of intracellular Ca 2+ transients. Cultures that are enriched for stem/proliferative cells exhibit repetitive, high frequency (≈25 peaks h −1 ), and short pulse width (≈25 s) Ca 2+ transients. Upon cell differentiation the transient frequency declines by 50% and pulse width widens by 200%. Metabolites and growth factors that are known to modulate stem cell proliferation and differentiation through Wnt and Notch signaling pathways, including CHIR-99021, N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), bone morphogenetic proteins (BMPs), and butyrate, also modulate Ca 2+ oscillation patterns in a consistent manner. Increasing the stiffness of the supportive matrix from 200 Pa to 3 GPa shifts Ca 2+ transient patterns toward those resembling differentiated cells. The ability to monitor Ca 2+ oscillations with the spatial and temporal resolution offered by this platform, combined with its amenability to high-content screens, provides a powerful tool for investigating real-time communication within a wide range of primary tissues in addition to the colonic epithelium.

Store-Operated Ca2+ Entry as a Putative Target of Flecainide for the Treatment of Arrhythmogenic Cardiomyopathy

“…Spontaneous intracellular Ca 2+ oscillations have been described not only in BM-hMSCs [ 54 , 83 , 84 ] but also also in human adipose tissue-derived MSCs (AD-hMSCs) [ 51 , 85 ]. Intracellular Ca 2+ oscillations regulate stem cell differentiation by stimulating the expression of genes that control cellular fate and silencing those that are required for self-renewal [ 52 , 86 , 87 , 88 , 89 , 90 ]. The spatio-temporal profile of spontaneous Ca 2+ activity can selectively change during hMSC differentiation in tissue-specific lineages by showing either an increase or a decrease in the spike frequency [ 52 ].…”