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.
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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Deficient Anterior pituitary with common Variable Immune Deficiency (DAVID) syndrome results from NFKB2 heterozygous mutations, causing adrenocorticotropic hormone deficiency (ACTHD) and primary hypogammaglobulinemia. While NFKB signaling plays a crucial role in the immune system, its connection to endocrine symptoms is unclear. We established a human disease model to investigate the role of NFKB2 in pituitary development by creating pituitary organoids from CRISPR/Cas9-edited human induced pluripotent stem cells (hiPSCs). Introducing homozygous TBX19K146R/K146R missense pathogenic variant in hiPSC, an allele found in congenital isolated ACTHD, led to a strong reduction of corticotrophs number in pituitary organoids. Then, we characterized the development of organoids harboring NFKB2D865G/D865G mutations found in DAVID patients. NFKB2D865G/D865G mutation acted at different levels of development with mutant organoids displaying changes in the expression of genes involved on pituitary progenitor generation (HESX1, PITX1, LHX3), hypothalamic secreted factors (BMP4, FGF8, FGF10), epithelial-to-mesenchymal transition, lineage precursors development (TBX19, POU1F1) and corticotrophs terminal differentiation (PCSK1, POMC), and showed drastic reduction in the number of corticotrophs. Our results provide strong evidence for the direct role of NFKB2 mutations in the endocrine phenotype observed in patients leading to a new classification of a NFKB2 variant of previously unknown clinical significance as pathogenic in pituitary development.
