UC Santa Barbara

Resting membrane potential (Vmem) and calcium signaling change as a function of developmental stage and location. Electrogenic genes, encoding ion channels and other proteins regulating bioelectric state, also change spatiotemporally. Although numerous genes regulate Vmem, it remains unclear how electrogenic gene dynamics are coordinated system-wide, particularly during early differentiation from embryonic stem cells (ESCs). We measured transcriptomes during differentiation of mouse ESCs towards forebrain-like cells in vitro and used bioinformatics to predict biological processes and upstream regulators of electrogenic genes. Electrogenic genes were dynamically expressed during differentiation, and predicted shared regulatory groups included genes related to cardiovascular development and cell adhesion (and neural). Identification of predicted regulators enables studies of the role of bioelectric signaling during differentiation and may offer a more fine-tuned understanding of Vmem mechanisms in development, including channelopathies. To complement analysis of electrogenic gene dynamics, we performed physiologic measurements for Vmem and calcium signal during early ESC differentiation. Changes in Vmem and intracellular calcium concentration [Ca2+]i regulate proliferation and differentiation of embryonic cells, with prior evidence suggesting calcium signaling is downstream of Vmem. However, it remains unclear whether changes in bioelectric state and calcium signaling in precursor cells are connected with their differentiation trajectory. To address this, we differentiated mouse ESCs in monolayer towards neuroectodermal (NE) or mesendodermal (MZ) like cells, measuring Vmem and [Ca2+]i every 24 hours for 4 days. Cultures featured morphologies characteristic of NE- and MZ-like differentiation, containing regions with both flattened and dense, three-dimensional morphologies. NE-directed cells were hyperpolarized, with lower [Ca2+]i relative to MZ-directed cells prior to germ layer marker acquisition (SOX1 or Brachyury). Colonies with clustered morphologies maintained relatively depolarized Vmem and higher calcium signals, closer to those of ESCs, despite their distinct cluster appearance. Our results demonstrate that divergences in Vmem and [Ca2+]i occur prior to germ layer marker protein expression and suggest that a cell’s bioelectric state is connected with differentiation trajectory from ES cells. The regional differences identified in both Vmem and [Ca2+]i suggest that even simplified stem cell model systems can provide a useful platform in which to study how stem cells regulate membrane potentials and calcium signals during early differentiation.