摘要：Gap junctions (GJs), which are proteinaceous channels, couple adjacent cells by permitting direct exchange of intracellular molecules with low molecular weights. GJ intercellular communication (GJIC) plays a critical role in regulating behaviors of human embryonic stem cells (hESCs), affecting their proliferation and differentiation. Here we report a novel use of sonoporation that enables single cell intracellular dye loading and dynamic visualization/quantification of GJIC in hESC colonies. By applying a short ultrasound pulse to excite single microbubbles tethered to cell membranes, a transient pore on the cell membrane (sonoporation) is generated which allows intracellular loading of dye molecules and influx of Ca2 into single hESCs. We employ live imaging for continuous visualization of intercellular dye transfer and Ca2 diffusion in hESC colonies. We quantify cell–cell permeability based on dye diffusion using mass transport models. Our results reveal heterogeneous intercellular connectivity and a variety of spatiotemporal characteristics of intercellular Ca2 waves in hESC colonies induced by sonoporation of single cells.
其他摘要：Abstract Gap junctions (GJs), which are proteinaceous channels, couple adjacent cells by permitting direct exchange of intracellular molecules with low molecular weights. GJ intercellular communication (GJIC) plays a critical role in regulating behaviors of human embryonic stem cells (hESCs), affecting their proliferation and differentiation. Here we report a novel use of sonoporation that enables single cell intracellular dye loading and dynamic visualization/quantification of GJIC in hESC colonies. By applying a short ultrasound pulse to excite single microbubbles tethered to cell membranes, a transient pore on the cell membrane (sonoporation) is generated which allows intracellular loading of dye molecules and influx of Ca 2 into single hESCs. We employ live imaging for continuous visualization of intercellular dye transfer and Ca 2 diffusion in hESC colonies. We quantify cell–cell permeability based on dye diffusion using mass transport models. Our results reveal heterogeneous intercellular connectivity and a variety of spatiotemporal characteristics of intercellular Ca 2 waves in hESC colonies induced by sonoporation of single cells.
