The selectivity provides a robust system for studying the emergence of spatial organization during the transition of a cell population from a pluripotent to a largely homogenous neural progenitor state. Open in a separate window Fig. that occurs Trifloxystrobin in response to intercellular perturbation. These findings suggest an integral role of gap junction communication in the temporal coordination of emergent patterning during early differentiation and neural commitment of pluripotent stem cells. Introduction The spatial organization of heterogeneous cells within multicellular systems, such as tissues and organs, is a primary determinant in deriving their respective functionality1. During embryogenesis, pluripotent cells migrate and differentiate to form complex multicellular structures in a reliable and reproducible manner. An incomplete understanding of the dynamic signaling mechanisms that affect differentiation and morphogenic patterning limits faithful and accurate replication of emergent behavior in vitro. To create more sophisticated engineered living systems (ELS), it is necessary to elucidate the collective impact of the numerous processes that shape multicellular constructs during normal development. Embryonic stem cells (ESCs) are an excellent model system for mimicking aspects of embryonic morphogenesis and investigating the various modes of communication amongst pluripotent populations2. The process of secretion, diffusion, and uptake of molecules is a well-established mechanism of biochemical communication across tissues, with the formation of extracellular morphogen gradients providing positional information that instructs cell Cav2 fate decisions during differentiation, both in vitro and in vivo3C5. However, emerging evidence in recent years suggests that direct cell-cell communication plays an equally significant role in pattern formation during morphogenesis6C10. Ascertaining the role of intercellular communication as a regulator of differentiation is crucial for deciphering the diversity of spatial cues present during developmental processes and for the future derivation of more complex ELS. Gap junction communication (GJC) provides direct channels that facilitate intercellular diffusion of small molecules (<1?kDa) between the cytosol of adjacent cells. Gap junctions assemble from hemichannels of connexin proteins present in the plasma membrane of adjacent cells and the connexin composition of Trifloxystrobin each channel dictates the permeability of specific metabolites11. Furthermore, the transcription and translation of connexin isotypes is regulated by cellular phenotype, allowing cells to exercise considerable dynamic control over intercellular connectivity during differentiation and tissue development12. The collective GJC across a population of cells produces an intercellular network of cells with fluid connectivity. The versatility of GJ-connectivity creates vast potential for the development of intracellular gradients of small molecules - such as cAMP, ATP, and serotonin - that influence many downstream metabolic and transcriptional processes governing cell-fate decisions13C17. Unfortunately, accurately interpreting molecular gradients within a network of differentiating ESCs is challenging due to the close-packed density of epithelial cells Trifloxystrobin and development of gradients across various length scales. While some sensors Trifloxystrobin are capable of discerning concentration gradients of small molecules, many rely on FRET-based detections and have noted limitations18. Specifically, bleed through of the FRET-donor can skew measurements and an inherently low signal-to-noise ratio severely limits the sensitivity of these sensors. Furthermore, while several techniques exist for characterizing GJ transport19,20, they typically offer limited capability to quantify Trifloxystrobin fluctuations in connectivity at a single-cell resolution simultaneously with the transport behavior at the population level. The difficulty of quantifying the influence of individual cells on the intercellular network is compounded when considering connectivity that can both modulate and be modulated by dynamical differentiation processes occurring throughout the population. For such instances, computational modeling offers an attractive approach, in combination with single-cell transport data, to investigate the dynamics of multicellular GJ communication and its relationship with differentiation. In this work, we quantified intercellular transport rates from single cells within ESC colonies, identified cell cycle state as a modulator of these rates, and used this knowledge to construct a computational model of intercellular transport in a multicellular system. This agent-based model, regulated by cell cycle and considering growth,.