Multiplex live single-cell transcriptional analysis demarcates cellular functional heterogeneity

Area of Science:

• Cell Biology
• Molecular Biology
• Stem Cell Research

Background:

• Understanding cellular heterogeneity is crucial for development, physiology, and disease.
• Existing methods often lack real-time, single-cell resolution for gene expression and physiology.

Purpose of the Study:

• To develop and validate a novel live-cell imaging strategy for simultaneous gene expression and physiological assessment.
• To investigate the functional evolution of early myocardial subpopulations during human pluripotent stem cell differentiation.

Main Methods:

• Utilized fluorescently labeled mRNA-specific anti-sense RNA probes and dsRNA-binding protein.
• Employed Förster Resonance Energy Transfer (FRET) for real-time, single-cell gene expression detection.
• Combined live-cell mRNA imaging with detailed physiological phenotyping.

Main Results:

• Successfully identified distinct myocardial subpopulations expressing specific structural proteins (myosin heavy chain α, myosin light chain 2a) in real-time.
• Captured the dynamic functional changes of these subpopulations during lineage specification.
• Demonstrated the capability to link gene expression profiles with physiological characteristics at the single-cell level.

Conclusions:

• The developed live-cell mRNA imaging approach offers unprecedented resolution for studying cellular heterogeneity.
• This technology has broad applications in biological research where cellular diversity is significant.
• Provides a powerful tool for understanding cell differentiation and disease mechanisms.