Dual TBX5-Lineage and MYL2 Reporter System For Identification of Left Ventricular Cardiomyocytes During Human Induced Pluripotent Stem Cell Differentiation

Abstract

Rationale Patient-derived induced pluripotent stem cells (iPSCs) present an exciting avenue for the modeling congenital heart disease. While hiPSC cardiac differentiations generate various cell types, the presence of mixed cell populations can confound interpretation of study results, particularly in the case of modeling structural congenital heart defects where lesions affect specific chambers of the heart. During cardiac development, the left and the right ventricles arise from distinct cardiac progenitor populations known as the first and second heart fields, respectively. Currently, availability of a lineage tracing tool to identify the descendants of these progenitors in the human iPSC system is lacking and such a tool would allow for the identification of left and right ventricular cardiomyocytes for modeling of chamber specific congenital heart defects in vitro. Objectives Genetically engineer a heart field-specific lineage tracing and a ventricular specific genetic reporter system in human iPSCs to identify left and right ventricular cardiomyocytes in vitro. Methods and Results We gene targeted a TBX5-based Cre-LoxP lineage tracing system via CRISPR/Cas9 genome editing into an hiPSC line from a healthy male patient. We also replaced the stop codon of the ventricular-specific Myosin Light Chain-2 (MYL2) gene with a P2A-TdTomato construct to allow for the identification of ventricular cardiomyocytes through the course of differentiation. Using a standard small molecular biphasic WNT modulation protocol, we conducted multiple independent differentiations and analyzed by FACS the percentage of lineage positive and troponin-T (TNNT2+) positive cardiomyocytes at multiple timepoints during differentiation. Analysis of GFP+ (TBX5-lineage+) cells out of TNNT2+ cells identified a gradual increase in GFP expression beginning at day 11 of differentiation and results in nearly 100% of TNNT2+ cardiomyocytes exhibiting GFP expression. GFP+ expression among MYL2-Tomato+ cells confirmed the predominance of TBX5-lineage+ ventricular cardiomyocytes. Analysis of gene expression across differentiation confirmed the predominance of LV marker genes and the absence or downregulation of SHF and RV markers. Conclusions Here, we genetically engineered a triple-targeted dual-fluorescent hiPSC reporter line that allows for the identification of TBX5-lineage positive ventricular cardiomyocytes. Gene expression analysis confirms the predominance of a left ventricular phenotype consistent with the fluorescence reporter expression. In summary, we provide a powerful tool for identifying and isolating chamber-specific left ventricular cardiomyocytes.