Illuminating dynamic structural states in hiPSC through endogenous gene tagging.

Abstract

The Allen Institute for Cell Science aims to understand the principles by which human induced pluripotent stem cells (hiPSCs) establish and maintain robust dynamic localization of cellular structures, and how they transition between states during differentiation and disease. As a first step [JG1], we have created a collection of endogenously tagged hiPSC lines to illuminate cell organization. The tagged proteins include notable membrane-bound and membrane-less cellular organelles, signaling complexes, phase transition markers, transcription factors, and structural markers specific to differentiated cells. To date, the Allen Cell Collection consists of 54 single- or dual-edited lines that have undergone extensive quality control testing to ensure genomic, cell biological, and stem cell integrity. Here, we highlight our gene-editing and quality control workflow for mono- and biallelic editing of expressed or silent genes that are expressed specifically during differentiation and function as reporters of cellular state. Furthermore, we underscore our efforts to increase efficiency of the current workflow by utilizing Adeno-Associated Virus (AAV) to deliver donor DNAs and multiplexing transfection strategies for gene tagging at multiple loci. Our most recently released lines are CTCF-mEGFP and PCNA-mEGFP, for dynamic visualization of chromatin domains and replication foci, G3BP1-mEGFP to label stress granules, mono- and bi-allelic mEGFP-E-cadherin to visualize adherens junctions and DCP1a-mEGFP for P-bodies. We now have gene [RG2] [JG3]-edited cell populations for N- and VE-cadherin ready for single cell sorting. Our overarching goal is to make these tools openly available to the cell biology community to help accelerate biomedical research. In addition to our cell lines, the donor plasmids, segmented 3D images of about 200,000 cells from our lines, image analysis, visualization tools, integrated cell models and biological findings are all openly available to the research community (www.allencell.org).