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

  1. Francisco X 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 M Wu

  • Curated by eLife

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    Evaluation Summary:

    The derivation of cardiomyocytes from the first and second heart fields is a well-studied phenomenon in animal models, however, due to ethical concerns, has not been studied in human heart development. The authors utilize hiPSC technology to demonstrate that it is the FHF and SHF that give rise to cardiomyocytes which is an important step in furthering our understanding of early human heart development.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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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.

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  1. Version published to 10.7554/elife.80075 on eLife
  2. eLife

    Evaluation Summary:

    The derivation of cardiomyocytes from the first and second heart fields is a well-studied phenomenon in animal models, however, due to ethical concerns, has not been studied in human heart development. The authors utilize hiPSC technology to demonstrate that it is the FHF and SHF that give rise to cardiomyocytes which is an important step in furthering our understanding of early human heart development.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Many animal studies have shown that the first and second heart fields give rise to the heart in normal embryonic development. For obvious reasons, this has not been well-studied in humans. Thus these investigators applied hiPSC technology to recapitulate human heart development using small molecules to modulate WNT signaling and thus induce mesodermal lineage differentiation. They set up a triple reporter genetic system (TBX5-Cre/MYL2-tdTomato/CCR5-CM-Lox-STOP-Lox-TurboGFP reporter) in two hiPSC lines and demonstrated that > 90% of ventricular cardiomyocytes were derived from the TBX5/MYL2 lineage. They used RT-qPCR to verify over 12 different time points during the course of the differentiation protocol that cells begin to express markers of the FHF lineage and eventually markers of ventricular cardiomyocytes.

  4. eLife

    Reviewer #2 (Public Review):

    The theory that the heart is formed from the first and second heart field cardiac progenitor cells has been demonstrated in many vertebrate species. However, this concept was not able to be tested in humans due to ethical limitations and the inaccessibility of human embryonic tissues. In this manuscript, the authors applied hiPSCs to mimic human heart development with a widely accepted differentiation protocol using small molecules to modulate WNT signaling. They constructed a triple reporter genetic system (TBX5-Cre/MYL2-tdTomato/CCR5-CM-Lox-STOP-Lox-TurboGFP reporter) in two hiPSC lines and showed that more than 90% of ventricular cardiomyocytes were derived from the TBX5/MYL2 lineage. The authors further performed the scRNA-seq analysis at 12 different time points during differentiation and confirmed the predominance of FHF differentiation during hiPSCs differentiation. More evidence is needed to support the conclusion.

  5. eLife

    Reviewer #3 (Public Review):

    Galdos, et al., have developed a novel lineage tracing technique using genetically encoded fluorophores in human-induced pluripotent stem cells to identify first heart field cells and ventricular cardiomyocytes during differentiation. To label the FHF lineage, the authors use a CRISPR/Cas9 strategy to express a floxed TurboGfp and add a P2A-Cre recombinase sequence at the stop codon of Tbx5 in two well-characterized hiPSC lines. In these same lines, they then added a P2A-tdTomato construct at the stop codon of the ventricular cardiomyocyte-specific sarcomeric protein Myl2. They expected this strategy to allow them to identify cells as they commit to the first heart field lineage and ultimately FHF cells that differentiate into ventricular CMs, which should therefore represent LV CMs by virtue of their lineage. RT-qPCR confirms that over the course of the differentiation protocol cells begin to express well-studied markers of the FHF lineage and eventually markers of ventricular CMs. This matches the flow analysis of their lineage-tracing technique which is suggestive though not conclusive that their technique is identifying the cells it claims to identify.

    The authors found, however, that their flow data showed that the differentiation protocol they used gave rise to >90 % FHF lineage cells, most of which were also Tnnt2+ or tdTomato+ by day 30 of differentiation. None of the cells were positive for markers of the second heart field lineage. To confirm this, the authors used scRNAseq data from multiple differentiation time points to identify the paths cells follow through their Wnt-signaling-based small molecule 2D differentiation protocol. What they find suggests there are two distinct path bifurcations using this protocol. The first is between a mesodermal lineage and an endodermal lineage, and the second is, within the mesodermal cells, a bifurcation between myocardial and epicardial lineages. They compare these results to previously published datasets from murine heart field development and see that the mesodermal pathway matches murine FHF lineage development and that there is no good match for SHF lineages. They hypothesize that a 3D differentiation protocol might lead to a subset of cells developing SHF hallmarks and test this by combining the CMs from their own scRNAseq results with those from a group that developed a novel 3D differentiation protocol to form heart organoids. They identify a cluster in the 3D differentiated cells that does not appear in their own dataset and which is enriched for cells expressing SHF markers and markers of outflow tract CMs.

    Strengths:
    1. The use of a Cre/lox system to permanently label putative FHF lineage cells with TurboGFP even after reduction of Tbx5 expression will make it possible to both follow the same cells over time to better understand early human heart development and to evaluate novel differentiation protocols for which cell lineages are likely to predominate. This can then be paired with fluorophores tagged to markers of later progenitors or terminally differentiated cell types (as the authors do here with Myl2) allowing isolation of distinct cell types with known lineages at distinct stages of models of human heart development. This is a potentially quite powerful tool given the limited availability of human fetal tissue and the ethical concerns inherent to using it to study development.
    2. The authors have identified a clear weakness of using 2D differentiation protocols based on Wnt-signaling as models of human heart development. They show convincingly for two separate hiPSC lines that while the cells progress through the primitive streak and the emergence of the first heart field cells, the second heart field does not arise in this protocol. This homogeneity of the terminally differentiated cells may be beneficial in regenerative medicine contexts, but it is clear that for studying development and for pushing cells to OFT or RV CM fates, new techniques are required. They then demonstrate the promise of 3D organoid differentiation techniques in overcoming this hurdle.
    3. This manuscript also sets up a powerful workflow for evaluating cell fate decisions over pseudotime in early heart development. The authors used well-published packages to set up their datasets to meaningfully compare scRNAseq results from their own 2D differentiation experiments with those from previously published scRNAseq results of murine heart development and 3D differentiation. For the latter, they were able to combine the datasets to identify a new cluster of cells from the 3D protocol. This workflow will prove extremely beneficial in comparing cell fate outcomes arising from disparate cardiac differentiation protocols.

    Weaknesses:
    1. While demonstrating that 2D differentiation of hiPSCs is an imperfect model of development is a valuable outcome of this work, this also makes it an imperfect model in which to test the robustness of their lineage tracing technique. Nearly all of the cells are shown to progress through the FHF lineages using their fluorescent techniques. This is confirmed using scRNAseq, but this means that they are unable to give a proof of principle that their method will distinguish FHF cells from SHF cells since none of the latter arises.
    2. The authors validate their lineage tracing technique with bulk gene expression by RT-qPCR at different time points during differentiation. However, they never directly confirm that isolated TurboGFP+ cells show higher expression or protein levels of their target FHF markers nor that the TurboGFP+tdTomato+ cells are enriched for LV CMs. While their validation as it stands is highly suggestive that their lineage tracing technique works as advertised, the evidence is still only circumstantial.
    3. The section of the paper devoted to the development and validation of their lineage tracing technique is connected to the section analyzing their scRNAseq results only loosely. Having shown by their new technique and its validation that no populations positive for SHF markers are arising during their differentiation, they turn to scRNAseq to confirm this observation. The issue here is that it requires a bit of circular reasoning. Having established that better techniques are required to study human heart development to move away from relying so heavily on our understanding of murine heart development, the authors then draw their conclusion that no SHF lineages arise during the differentiation of their hiPSC lines in part by comparing them to murine heart development. This is in no way a fatal flaw to the work but it limits the ability to use the authors' techniques to draw novel distinctions between human and murine heart development.

  6. Version published to 10.1101/2021.09.30.462465v3 on bioRxiv
  7. Version published to 10.1101/2021.09.30.462465v2 on bioRxiv
  8. Version published to 10.1101/2021.09.30.462465v1 on bioRxiv