Abstract 14922: Combined Lineage Tracing and Scrna-Seq Reveals Unexpected First Heart Field Predominance of Human Ipsc Differentiation

Abstract

Introduction: During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been studied in non-human model systems, human induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis. A major question is whether hiPSCs can model cardiac progenitor development as seen in animal systems. Hypothesis: Dual TBX5/MYL2 lineage tracing and time course single cell RNA sequencing can uncover the identity of human cardiac progenitor lineage in vitro. Methods: Using CRISPR/Cas9 genome editing we introduced a TBX5/MYL2 fluorescent reporter lineage tracing system into two independent hiPSC lines to identify FHF-progenitors and their descendants. Using a standard biphasic WNT modulation protocol, we differentiated hiPSCs into cardiomyocytes and characterized the expression of TBX5-lineage positive and MYL2 cardiomyocytes between day 3 and day 30 of differentiation. To further characterize the cell populations, present during differentiation, we conducted single cell RNA sequencing on differentiating hiPSCs across 12 timepoints with oligonucleotide-based sample multiplexing. Results: Our reporter system revealed the surprising finding that using a standard WNT modulation small molecule protocol across two cell lines yielded 93.2%±0.80% and 92.1%± 1.60% TBX5-lineage positive cardiomyocytes, respectively. Among MYL2 expressing ventricular cardiomyocytes, we observed 99.0%±0.21% and 93.2±0.84% as TBX5-lineage positive, respectively. ScRNA-seq analysis and trajectory inference revealed high expression of known FHF gene markers relative to murine developmental data and identified a clear bifurcation of cardiac progenitor populations into myocardial and epicardial lineage consistent with a FHF differentiation trajectory. Lastly, a comparison of our data with scRNA-seq data from a recently published 3D protocol reveals the identity of a SHF progenitor population within a 3D system. Conclusions: Our work provides a powerful new genetic lineage tracing approach, as well as a single cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.