(W1237) CHANGES IN BEHAVIOR, ORGANIZATION, AND STATE OF HIPSC-DERIVED ENDOTHELIAL CELLS EXPOSED TO SHEAR STRESS REVEALED BY LIVE CELL MICROSCOPY AND QUANTIFICATION
Scientist Allen Institute for Cell Science, United States
Abstract: Endothelial cells (ECs) play critical roles in the vasculature and are subject to shear stress as blood flows through the vessels they line, which influences their structure, function, and morphology. Here, we use the alignment and re-alignment of human induced pluripotent stem cell-derived ECs (hiPSC-ECs) under fluid shear stress as a model system for investigating cell state transitions in a holistic manner. We differentiated endogenously tagged hiPSC lines from the Allen Cell Collection (www.allencell.org) into hiPSC-ECs and performed 3D, live cell imaging as they respond to fluid shear stress to capture changes in their morphology, behavior, and organization. We found that hiPSC-ECs exhibited distinct responses to different magnitudes of applied shear stress. Under low shear stress (0.8-6 dyn/cm2) the hiPSC-ECs elongated, collectively migrated upstream, aligned parallel relative to the direction of fluid flow, and developed VE-cadherin puncta localized to the contacts of lateral cells. When subjected to high shear stress (15-25 dyn/cm2), the cells aligned perpendicular to the direction of flow, migrated in all directions, and rarely developed VE-cadherin puncta. When the magnitude of shear stress is switched from high to low or vice versa, hiPSC-ECs changed their collective migration behavior and organization accordingly. To quantify the transition between these different cell states that occur in response to the distinct shear stress magnitudes, we developed a segmentation-free machine learning framework to extract single-cell features from 2D time-lapse image data. Using these features, we are implementing recently developed machine learning methods for stochastic dynamics to infer the underlying dynamical rules that govern the observed ensemble of single-cell behaviors. We are initially developing this workflow for images of fluorescent VE-cadherin-tagged hiPSC-ECs and plan to expand to image data of other subcellular structures and image modalities. We expect that these analyses of how the environmental cue of shear stress influences the cell state of hiPSC-ECs will improve our understanding of endothelial and cell biology and demonstrate the utility of studying cell state transitions in a holistic manner.
