Combined Lineage Tracing and scRNA-seq Reveals Unexpected First Heart Field Predominance of Human iPSC Differentiation

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 extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation stage human embryos. Human induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF- progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation. Overall design: scRNA-seq with oligonucleotide-based sample multiplexing for profiling cardiac differentiation across 12 timepoints and two independent hiPSC lines.

在哺乳动物发育过程中，左心室与右心室分别源自被称为第一心区（first heart field, FHF）和第二心区（second heart field, SHF）的早期心脏祖细胞群。尽管这类细胞群在非人动物模型系统中已得到广泛研究，但由于获取原肠胚阶段人类胚胎存在伦理与技术层面的限制，在活体人类组织中对其进行鉴定与研究的工作仍较为有限。人类诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）因其已被充分证实的分化为所有胚胎胚层的能力，成为模拟人类早期胚胎发生的极具前景的替代模型。本研究开发了一种TBX5/MYL2谱系追踪报告系统，可用于鉴定第一心区祖细胞及其子代细胞，包括左心室心肌细胞。此外，本研究采用基于寡核苷酸样本多重标记的单细胞RNA测序（single cell RNA sequencing, scRNA-seq）技术，对两株独立hiPSC系12个时间点的分化过程进行了全面的转录组分析。令人意外的是，通过本报告系统与scRNA-seq分析发现，采用基于小分子Wnt信号通路的二维（2D）分化方案时，细胞主要向第一心区谱系分化。我们将该数据与已发表的小鼠及三维（3D）心脏类器官scRNA-seq数据进行对比，证实了hiPSC诱导得到的子代细胞中左心室心肌细胞占比超过90%。综上，本研究为科学界提供了一种全新的高效遗传谱系追踪方法，以及一套针对hiPSC心脏分化过程的单细胞转录组图谱。实验整体设计：采用基于寡核苷酸样本多重标记的scRNA-seq技术，对两株独立hiPSC系的12个时间点的心脏分化过程进行转录组分析。