CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes

Sci Rep. 2021 Feb 4;11(1):3026. doi: 10.1038/s41598-021-81860-x.

Abstract

Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics
  • Cardiac Myosins / genetics
  • Cell Differentiation / genetics*
  • Green Fluorescent Proteins
  • Heart Atria / cytology
  • Heart Atria / growth & development*
  • Heart Atria / metabolism
  • Heart Ventricles / cytology
  • Heart Ventricles / growth & development
  • Heart Ventricles / metabolism
  • Humans
  • Induced Pluripotent Stem Cells / metabolism*
  • Induced Pluripotent Stem Cells / pathology
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / metabolism*
  • Myosin Light Chains / genetics

Substances

  • Cyan Fluorescent Protein
  • Myosin Light Chains
  • myosin light chain 2
  • Green Fluorescent Proteins
  • Cardiac Myosins