Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis

Area of Science:

• Cardiovascular Biology
• Stem Cell Biology
• Genomics

Background:

• Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are valuable for disease modeling and drug screening.
• Their clinical utility is hampered by immaturity and cellular heterogeneity.
• Understanding the origins of this heterogeneity is crucial for advancing hiPSC-CM applications.

Purpose of the Study:

• To investigate the sources of cellular heterogeneity during hiPSC-CM differentiation.
• To identify key transcriptional regulators driving distinct cardiomyocyte populations.
• To establish a platform for analyzing in vitro cellular heterogeneity.

Main Methods:

• Employed single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing (bulk RNA-seq) over time.
• Integrated transcriptomic and splicing analyses.
• Utilized CRISPR genome editing, ChIP-seq, patch clamp, calcium imaging, and CyTOF to assess transcription factor roles (NR2F2, TBX5, HEY2).

Main Results:

• Identified over six distinct single-cell populations during hiPSC-CM differentiation, with several co-existing at day 30.
• Dissected the functional impact of NR2F2, TBX5, and HEY2 on cardiomyocyte subpopulations.
• Demonstrated the utility of integrative genomics for characterizing cellular heterogeneity.

Conclusions:

• Cellular heterogeneity in hiPSC-CMs arises from distinct transcriptional programs.
• Specific transcription factors play critical roles in shaping cardiomyocyte populations.
• This study provides a robust framework for understanding and potentially controlling hiPSC-CM heterogeneity.