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

Abstract

The second messenger, intracellular free calcium (Ca²⁺ ), 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 Ca²⁺ transients. Cultures that are enriched for stem/proliferative cells exhibit repetitive, high frequency (≈25 peaks h^-1 ), and short pulse width (≈25 s) Ca²⁺ 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 Ca²⁺ oscillation patterns in a consistent manner. Increasing the stiffness of the supportive matrix from 200 Pa to 3 GPa shifts Ca²⁺ transient patterns toward those resembling differentiated cells. The ability to monitor Ca²⁺ 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.