Abstract: Over the past decade, the Allen Institute for Cell Science has developed and distributed tools to study human induced pluripotent stem cells (hiPSCs), including fluorescently tagged lines, images of 2D hiPSC colonies, and image analysis workflows. However, 2D cultures cannot replicate the complex multicellular interactions seen in 3D environments. To address this, we present the adherent hiPSC lumenoid model, a simple and tractable 3D system for investigating cellular dynamics in a physiologically relevant context. Lumenoids are hollow, acinar structures with a single central lumen surrounded by a coherent, edgeless, apical-in/basal-out, pluripotent epithelium. Lumenoid formation is initiated by adding dilute extracellular matrix (Matrigel) to 2D colonies. This triggers a cascade of cellular events, including apical contraction, colony edge lifting, and epithelial closure via a purse-string-like mechanism, creating a topologically closed lumen within ~6-24 hours. Once formed, lumenoids grow into large, often spherical 3D structures as the lumen inflates. Lumenoid formation is accompanied by de novo basal secretion of basement membrane (BM) material, and fully formed lumenoids are encased in a BM shell. During an epithelial-to-mesenchymal transition (EMT) induced by WNT activation, lumenoid cells delaminate and migrate through newly formed holes in the BM, revealing key cell-cell and cell-matrix interactions involved in this critical process. The simple geometry of lumenoids, their genetic tractability, and their reproducibility offer significant advantages over other 3D culture models. Importantly, lumenoids remain stably anchored to the coverslip, allowing for long-term live imaging in high throughput. They are also retained through media changes, fixation, and immunolabeling, enabling seamless downstream multimodal data integration. Using this system and fluorescently tagged lines from the Allen Cell Collection, we have generated hundreds of 60-hour 3D timelapse movies capturing lumenoid formation, growth, and EMT, all publicly available alongside open-source analysis tools. Moving forward, this assay will enable holistic integration of cell dynamics, molecular features, and local context in a reproducible, experimentally tractable, physiologically relevant 3D system.
