Multiplex live single-cell transcriptional analysis demarcates cellular functional heterogeneity

Abstract

A fundamental goal in the biological sciences is to determine how individual cells with varied gene expression profiles and diverse functional characteristics contribute to development, physiology, and disease. Here, we report a novel strategy to assess gene expression and cell physiology in single living cells. Our approach utilizes fluorescently labeled mRNA-specific anti-sense RNA probes and dsRNA-binding protein to identify the expression of specific genes in real-time at single-cell resolution via FRET. We use this technology to identify distinct myocardial subpopulations expressing the structural proteins myosin heavy chain α and myosin light chain 2a in real-time during early differentiation of human pluripotent stem cells. We combine this live-cell gene expression analysis with detailed physiologic phenotyping to capture the functional evolution of these early myocardial subpopulations during lineage specification and diversification. This live-cell mRNA imaging approach will have wide ranging application wherever heterogeneity plays an important biological role.

Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing

“…S8, D and E). Our observation that a fraction of ∆Ex51 cardiomyocytes did not exhibit an arrhythmic phenotype could stem from the transcriptional, structural, and functional heterogeneity of iPSC-derived cardiomyocytes ( 41 , 42 ), whereby cardiomyocytes from different lineages (i.e., nodal, atrial, and ventricular) could display variable susceptibilities to arrhythmias. A similar observation was reported in other studies investigating the electrophysiological properties of iPSC-derived cardiomyocytes in the setting of diseases that affect cardiac electrophysiology ( 40 , 43 , 44 ).…”

“…Calcium imaging of human iPSC-derived cardiomyocytes was performed as previously described ( 41 ). Beating cardiomyocytes were dissociated into a single-cell suspension and seeded on a glass-bottom dish at single-cell density.…”

Nrf1 promotes heart regeneration and repair by regulating proteostasis and redox balance

“…hPSC derived from a healthy male donor was directly obtained from the UT Southwestern Wellstone Myoediting Core. hPSCs culture and differentiation were performed as previously described 46 . Briefly, hPSCs were cultured on Matrigel-coated tissue culture polystyrene plates and maintained in Essential 8 media (Thermo Fisher).…”

Engineering of the Lrp/AsnC‐type transcriptional regulator DecR as a genetically encoded biosensor for multilevel optimization of L‐cysteine biosynthesis pathway in Escherichia coli

“…The percentage of bound substrate was calculated based on its contribution to the total fluorescence of the respective channel. The apparent binding affinity K d was calculated according to previous studies (Atmanli et al, 2019;Sobhy et al, 2019) using the flowing equation:…”