A Rainbow Reporter Tracks Single Cells and Reveals Heterogeneous Cellular Dynamics among Pluripotent Stem Cells and their Differentiated Derivatives

Abstract

Recent single cell analyses have found molecular heterogeneities within populations of pluripotent stem cells (PSCs). A tool that tracks single cell lineages and their phenotypes longitudinally would reveal whether heterogeneity extends beyond molecular identity. Hence, we generated a stable Cre-inducible rainbow reporter human PSC line that provides up to 18 unique membrane-targeted fluorescent barcodes. These barcodes enable repeated assessments of single cells as they clonally expand, change morphology, and migrate. Owing to the cellular resolution of this reporter, we identified subsets of PSCs with enhanced clonal expansion, synchronized cell divisions, and persistent localization to colony edges. Reporter expression was stably maintained throughout directed differentiation into cardiac myocytes, cortical neurons, and hepatoblasts. Repeated examination of neural differentiation revealed self-assembled cortical tissues derive from clonally dominant progenitors. Collectively, these findings demonstrate the broad utility and easy implementation of this reporter line for tracking single cell behavior.

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16high Cells In Vivo

“…To determine the long-term proliferative capacity of Tom + p16 high cells, we first labeled p16 high cells in 10-week-old mice by TAM treatment for 5 days and analyzed them 2 and 14 weeks after treatment. Given that prior cell tracing experiments clearly demonstrated that proliferating cells formed a cluster in tissues (Boone et al, 2019;El-Nachef et al, 2020;Ueno and Weissman, 2006), we examined whether Tom + p16 high cells formed clusters in the kidneys, lungs, and livers. We first partitioned all fields into squares with 25-fold larger average area of a targeted single Tom + p16 high cell and then calculated the number of Tom + p16 high cells in each square (Figure S2B).…”

Dissecting nephron morphogenesis using kidney organoids from human pluripotent stem cells

“…In this regard, hPSC-derived kidney organoids may represent an affordable model system for studying such dynamic processes in the human context. To this end, the combination of advanced imaging techniques together with hPSC reporter lines for the expression of key marker genes [61][62][63] and/or reporter systems for tracking of single cells and their progeny in an unbiased manner [64] will be crucial to monitor cellular behaviors at high spatiotemporal resolution. Additionally, techniques for spatiotemporal perturbation of relevant genetic, biochemical and mechanical effectors will be key to systematically test the interaction between these parameters [41,68].…”

Fluorescent PSC-Derived Cardiomyocyte Reporter Lines: Generation Approaches and Their Applications in Cardiovascular Medicine

“…Uniquely, hiPSC-CM reporter cells provide insights into human cardiogenesis without embryo donor requirements and without the concerns over species differences encountered with mouse models. Future multi-coloured reporter lines [ 46 ] may enable the discovery of novel cardiac progenitors, and the improvement of CM derivation and differentiation protocols.…”