Combined lineage tracing and scRNA-seq reveals unexpected first heart field predominance of human iPSC differentiation

  1. Francisco Xavier Galdos
  2. Carissa Lee
  3. Soah Lee
  4. Sharon Paige
  5. William Goodyer
  6. Sidra Xu
  7. Tahmina Samad
  8. Gabriela V Escobar
  9. Adrija Darsha
  10. Aimee Beck
  11. Rasmus O Bak
  12. Matthew H Porteus
  13. Sean Wu  Is a corresponding author
  1. Stanford University, United States
  2. Sungkyunkwan University, Republic of Korea
  3. University of California, San Diego, United States
  4. 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 ).

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Francisco Xavier Galdos

    Stanford Cardiovascular Institute, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7985-4521
  2. Carissa Lee

    Stanford Cardiovascular Institute, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Soah Lee

    Department of Pharmacy, Sungkyunkwan University, Seoul, Republic of Korea
    Competing interests
    The authors declare that no competing interests exist.
  4. Sharon Paige

    Stanford Cardiovascular Institute, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. William Goodyer

    Stanford Cardiovascular Institute, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Sidra Xu

    Stanford Cardiovascular Institute, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Tahmina Samad

    Stanford Cardiovascular Institute, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Gabriela V Escobar

    Stanford Cardiovascular Institute, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Adrija Darsha

    School of Medicine, University of California, San Diego, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Aimee Beck

    Stanford Cardiovascular Institute, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Rasmus O Bak

    Department of Biomedicine, Aarhus University, Aarhus C, Denmark
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7383-0297
  12. Matthew H Porteus

    Department of Pediatrics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3850-4648
  13. Sean Wu

    Stanford Cardiovascular Institute, Stanford University, Stanford, United States
    For correspondence
    smwu@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0000-3821

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

  1. Hina W Chaudhry, Icahn School of Medicine at Mount Sinai, United States

Version history

  1. Preprint posted: October 1, 2021 (view preprint)
  2. Received: May 6, 2022
  3. Accepted: May 27, 2023
  4. Accepted Manuscript published: June 7, 2023 (version 1)
  5. 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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  1. Francisco Xavier Galdos
  2. Carissa Lee
  3. Soah Lee
  4. Sharon Paige
  5. William Goodyer
  6. Sidra Xu
  7. Tahmina Samad
  8. Gabriela V Escobar
  9. Adrija Darsha
  10. Aimee Beck
  11. Rasmus O Bak
  12. Matthew H Porteus
  13. Sean Wu
(2023)
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

Share this article

https://doi.org/10.7554/eLife.80075

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