A Platform for Investigating Nuclear Organization and its Changes During Human iPSC Differentiation

Abstract

The Allen Institute for Cell Science is developing a state space of stem cell structural signatures to understand the principles by which cells reorganize during the cell cycle and differentiation. We have developed a pipeline to generate high-replicate, dynamic image data on cell organization and activities using endogenous fluorescently tagged human induced pluripotent stem cells (hiPSCs) (the Allen Cell Collection at www.allencell.org). Each of the ∼25 lines express a monoallelic EGFP-tagged protein representing a cellular structure. For each structure, we take advantage of thousands of replicate high-resolution 3D images to develop quantitative image-based assays, segmentations, analyses, and computational models. We are initiating an analogous study of the nucleus, with a goal of creating a “state space of nuclear signatures.” We are endogenously tagging an initial set of eight key nuclear landmark proteins with EGFP and analyzing their sub-nuclear localization as the cells progress through the cell cycle and differentiate into cardiomyocytes. These landmarks include the nucleolus, nuclear lamina, nuclear pores, cohesin, CTCF, histones, and heterochromatin. We are also developing methods to quantify the change in localization patterns for these nuclear landmark proteins in the undifferentiated hiPSCs vs. cardiomyocytes derived from them. Preliminary image analyses, for example, reveal that H2B in cardiomyocytes displays less variation in both signal intensity and local spatial structure, generating a “smoother” protein distribution pattern within the nucleus and suggesting that our analysis approaches can detect changes in localization patterns of nuclear landmark proteins. We are also using single-cell RNAseq, FISH, and single-cell activity assays to conjoin gene expression with organization and phenotype. These cell lines and our analyses will further be integrated with genome-wide assays of chromatin architecture together with the 4D Nucleome Project and once fully characterized will be distributed publicly.