Combined lineage tracing and scRNA-seq reveals unexpected first heart field predominance of human iPSC differentiation
- Stanford University, United States
- Sungkyunkwan University, Republic of Korea
- University of California, San Diego, United States
- Aarhus University, Denmark
Abstract
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.
Data availability
All raw data for single cell RNA-sequencing has been deposited in the GEO repository under accession number GSE202398. Accession numbers for publicly available data re-analyzed for this study can be found in Supplementary File 9. Standard code and functions used for single cell analysis are available at the following Github repositories: Seurat (https://github.com/satijalab/seurat/), ScanPy (https://github.com/scverse/scanpy), STREAM (https://github.com/pinellolab/STREAM), SoupX (https://github.com/constantAmateur/SoupX), CellRanger (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/tutorial_ov ).
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Single-cell analysis of cardiogenesis reveals basis for organ level developmental defectsNCBI Gene Expression Omnibus, GSE126128.
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A Cellular Atlas of Pitx2-Dependent Cardiac DevelopmentNCBI Gene Expression Omnibus, GSE131181.
Article and author information
Author details
Francisco Xavier Galdos
Funding
NHLBI Division of Intramural Research (F30Hl149152)
- Francisco Xavier Galdos
NHLBI Division of Intramural Research (R01HL13483004)
- Sean Wu
NIH Office of the Director (1RM1 GM131981-02)
- Sean Wu
NHLBI Division of Intramural Research (R01HL13483004)
- Sean Wu
NIH Office of the Director (NIH T32 GM007365)
- Francisco Xavier Galdos
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Hina W Chaudhry, Icahn School of Medicine at Mount Sinai, United States
Version history
- Preprint posted: October 1, 2021 (view preprint)
- Received: May 6, 2022
- Accepted: May 27, 2023
- Accepted Manuscript published: June 7, 2023 (version 1)
- Version of Record published: July 14, 2023 (version 2)
Copyright
© 2023, Galdos et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Combined lineage tracing and scRNA-seq reveals unexpected first heart field predominance of human iPSC differentiation
eLife 12:e80075.
https://doi.org/10.7554/eLife.80075
Further reading
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The brain is consisted of diverse neurons arising from a limited number of neural stem cells. Drosophila neural stem cells called neuroblasts (NBs) produces specific neural lineages of various lineage sizes depending on their location in the brain. In the Drosophila visual processing centre - the optic lobes (OLs), medulla NBs derived from the neuroepithelium (NE) give rise to neurons and glia cells of the medulla cortex. The timing and the mechanisms responsible for the cessation of medulla NBs are so far not known. In this study, we show that the termination of medulla NBs during early pupal development is determined by the exhaustion of the NE stem cell pool. Hence, altering NE-NB transition during larval neurogenesis disrupts the timely termination of medulla NBs. Medulla NBs terminate neurogenesis via a combination of apoptosis, terminal symmetric division via Prospero, and a switch to gliogenesis via Glial Cell Missing (Gcm); however, these processes occur independently of each other. We also show that temporal progression of the medulla NBs is mostly not required for their termination. As the Drosophila OL shares a similar mode of division with mammalian neurogenesis, understanding when and how these progenitors cease proliferation during development can have important implications for mammalian brain size determination and regulation of its overall function.
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