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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.
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This content will become publicly available on August 1, 2026
3D flexible self-folding microelectrode array for organoid electrophysiology analysis
The development of three-dimensional (3D) tissues derived from single- and multi-lineage-directed human-induced pluripotent stem cells (hiPSCs) has significantly enhanced our capacity to mimic more complex cellular and physiological environments but creates new challenges for their analysis. Electrophysiology is crucial for elucidating electrical properties within neuronal and cardiac networks; however, traditional methods are poorly adapted to capturing activity throughout the 3D tissue structure, primarily due to limited spatial resolution. To address this limitation, we have developed 3D Flexible, Self-folding Microelectrode Array (FSMEA) devices comprised of a polyimide and SU-8 photoresist bilayer or an SU-8/SU-8 bilayer, which utilizes strain differences between the layers. We demonstrate that FSMEA devices can effectively record spontaneous action potentials and local field potentials in two 3D tissues, cortical organoids ranging from 800 to 1,500 µm in diameter and human elongating multi-lineage organized cardiac (EMLOC) gastruloids. These FSMEAs represent a new class of strain-based 3D MEA devices.
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- PAR ID:
- 10652406
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Cell Biomaterials
- ISSN:
- 3050-5623
- Page Range / eLocation ID:
- 100179
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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