Mechanisms of Neural Stem Cell Mechanoregulation

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PROJECT SUMMARY/ABSTRACT Biophysical cues encoded in the structure, mechanics, and dimensionality of the stem cell microenvironment are now appreciated as important regulators of self-renewal and differentiation. For the past 15+ years, including two periods of R01 support, we have been exploring mechanistic and translational aspects of this regulation in hippocampal neural stem cells (NSCs), which generate new neurons into adulthood and contribute to neurological disease and repair. We have made several important contributions to the field’s understanding of stem cell mechanobiology, including the discovery that NSC lineage decisions are maximally sensitive to extracellular matrix (ECM) mechanics within a restricted temporal window, during which stiffness cues are processed by a signaling network that includes RhoA GTPase, actin/myosin, angiomotin, YAP, and β-catenin. Our discoveries raise two important questions of general interest to the stem cell field, which will serve as the foundation for our renewal application. First, what molecular mechanisms govern NSC mechanosensitive lineage commitment in three-dimensional (3D) ECMs, and how do these mechanisms differ from two-dimensional (2D) ECMs? We will build on our exciting recent discovery that the transcription factor Egr1 is a critical, 3D-specific regulator of NSC mechanosensitive lineage commitment. Second, how do stem cells dynamically integrate mechanical inputs on the time scale of minutes to hours to trigger functionally important signaling events? Here we will leverage our preliminary studies in which we have probed the timing of mechanosensitive signaling events with mismatched DNA-crosslinked viscoelastic hydrogels and optogenetic reagents that allow timed activation of RhoA and Cdc42 activation. We have two specific aims: In Aim 1, we will investigate mechanisms through which Egr1 controls mechanosensitive lineage commitment in 3D matrices using a combination of 2D and 3D engineered biomaterials, candidate-based molecular studies, and screens to identify Egr1 targets relevant to neurogenesis. In Aim 2, we will investigate how stiffness cues from 2D ECMs are triggered on the minutes-to- hours time scale are integrated over hours to days to control lineage commitment. Work in this aim builds on our observation that timed optogenetic stimulation of RhoA and incorporation of viscous (loss) properties into elastic ECMs both suppress NSC neurogenesis. We will identify critical regulatory time scales for both perturbations and determine if they influence lineage commitment through common mechanisms. To integrate aims, we will investigate the time-dependence of mechanosensitive lineage commitment in 3D ECMs and ask if RhoA stimulation and stress relaxation act through Egr1 to suppress neurogenesis. Our work will accelerate the field’s understanding of how stem cells sense and act upon mechanical signals to guide fate decisions, a problem of high fundamental and translational value. We will also marry several innovative approaches, including optogenetics, viscoelastic ECMs, genome editing, and sequencing/screening technologies. We expect that our studies will provide an intellectual roadmap that can be applied to other ECM and stem cell systems.