Curated Optogenetic Publication Database

Abstract: In embryonic stem cell (ESC) models for early development, spatially and temporally varying patterns of signaling and cell types emerge spontaneously. However, mechanistic insight into this dynamic self-organization is limited by a lack of methods for spatiotemporal control of signaling, and the relevance of signal dynamics and cell-to-cell variability to pattern emergence remains unknown. Here, we combine optogenetic stimulation, imaging, and transcriptomic approaches to study self-organization of human ESCs (hESC) in two-dimensional (2D) culture. Morphogen dynamics were controlled via optogenetic activation of canonical Wnt/β-catenin signaling (optoWnt), which drove broad transcriptional changes and mesendoderm differentiation at high efficiency (>99% cells). When activated within cell subpopulations, optoWnt induced cell self-organization into distinct epithelial and mesenchymal domains, mediated by changes in cell migration, an epithelial to mesenchymal-like transition, and TGF-β signaling. Furthermore, we demonstrate that such optogenetic control of cell subpopulations can be used to uncover signaling feedback mechanisms between neighboring cell types. These findings reveal that cell-to-cell variability in Wnt signaling is sufficient to generate tissue-scale patterning and establish an hESC model system for investigating feedback mechanisms relevant to early human embryogenesis.

Abstract: Precise spatial and temporal regulation of dynamic morphogen signals during human development governs the processes of cell proliferation, migration, and differentiation to form organized tissues and organs. Tissue patterns spontaneously emerge in various human pluripotent stem cell (hPSC) models. However, the lack of molecular methods for precise control over signal dynamics limits the reproducible production of tissue patterns and a mechanistic understanding of self-organization. We recently implemented an optogenetic-based OptoWnt platform for light-controllable regulation of Wnt/β-catenin signaling in hPSCs for in vitro studies. Using engineered illumination devices to generate light patterns and thus precise spatiotemporal control over Wnt activation, here we triggered spatially organized transcriptional changes and mesoderm differentiation of hPSCs. In this way, the OptoWnt system enabled robust endothelial cell differentiation and cardiac tissue patterning in vitro. Our results demonstrate that spatiotemporal regulation of signaling pathways via synthetic OptoWnt enables instructive stem cell fate engineering and tissue patterning.

Abstract: Spatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics affect cell behavior. Here, we show that optogenetic Wnt/β-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). By patterning human embryonic stem cell (hESC) cultures with varying light intensities, LAVA devices enabled dose-responsive control of optoWnt activation and Brachyury expression. Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models the embryonic presentation of Wnt signals in vitro. LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology.

Abstract: The processes of cell proliferation, differentiation, migration, and self-organization during early embryonic development are governed by dynamic, spatially and temporally varying morphogen signals. Analogous tissue patterns emerge spontaneously in embryonic stem cell (ESC) models for gastrulation, but mechanistic insight into this self-organization is limited by a lack of molecular methods to precisely control morphogen signal dynamics. Here we combine optogenetic stimulation and single-cell imaging approaches to study self-organization of human pluripotent stem cells. Precise control of morphogen signal dynamics, achieved through activation of canonical Wnt/β-catenin signaling over a broad high dynamic range (>500-fold) using an optoWnt optogenetic system, drove broad transcriptional changes and mesendoderm differentiation of human ESCs at high efficiency (>95% cells). Furthermore, activating Wnt signaling in subpopulations of ESCs in 2D and 3D cultures induced cell self-organization and morphogenesis reminiscent of human gastrulation, including changes in cell migration and epithelial to mesenchymal transition. Our findings thus reveal an instructive role for Wnt in directing cell patterning in this ESC model for gastrulation.

Abstract: Abstract not available.

Abstract: Cells are bombarded by extrinsic signals that dynamically change in time and space. Such dynamic variations can exert profound effects on behaviors, including cellular signaling, organismal development, stem cell differentiation, normal tissue function, and disease processes such as cancer. Although classical genetic tools are well suited to introduce binary perturbations, new approaches have been necessary to investigate how dynamic signal variation may regulate cell behavior. This fundamental question is increasingly being addressed with optogenetics, a field focused on engineering and harnessing light-sensitive proteins to interface with cellular signaling pathways. Channelrhodopsins initially defined optogenetics; however, through recent use of light-responsive proteins with myriad spectral and functional properties, practical applications of optogenetics currently encompass cell signaling, subcellular localization, and gene regulation. Now, important questions regarding signal integration within branch points of signaling networks, asymmetric cell responses to spatially restricted signals, and effects of signal dosage versus duration can be addressed. This review summarizes emerging technologies and applications within the expanding field of optogenetics.