A single cell transcriptional roadmap of human pacemaker cell differentiation
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Evaluation Summary:
Overall, this study explores the differentiation of human pacemaker cells from human iPSCs, demonstrating different subtypes of pacemaker cells, and highlighting the role of Wnt and TGFbeta signaling in the formation of sinoatrial note cardiomyocyte subtypes.
(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
Each heartbeat is triggered by the sinoatrial node (SAN), the primary pacemaker of the heart. Studies in animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into ‘transitional’, ‘tail’, and ‘head’ subtypes. However, the underlying molecular mechanisms, especially of human pacemaker cell development, are poorly understood. Here, we performed single cell RNA sequencing (scRNA-seq) and trajectory inference on human induced pluripotent stem cells (hiPSCs) differentiating to SAN-like cardiomyocytes (SANCMs) to construct a roadmap of transcriptional changes and lineage decisions. In differentiated SANCM, we identified distinct clusters that closely resemble different subpopulations of the in vivo SAN. Moreover, the presence of a side population of proepicardial cells suggested their shared ontogeny with SANCM, as also reported in vivo. Our results demonstrate that the divergence of SANCM and proepicardial lineages is determined by WNT signaling. Furthermore, we uncovered roles for TGFβ and WNT signaling in the branching of transitional and head SANCM subtypes, respectively. These findings provide new insights into the molecular processes involved in human pacemaker cell differentiation, opening new avenues for complex disease modeling in vitro and inform approaches for cell therapy-based regeneration of the SAN.
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Author Response:
Reviewer #1:
The manuscript by Wiesinger et al., demonstrates the differentiation of human induced pluripotent stem cells (iPSCs) into pacemaker cardiomyocytes. Authors have shown impressive analyses of sinoatrial node cardiomyocytes (SAN-CM) using scRNA-seq approach followed by a computational method namely Trajectory Inference (TI) to understand the diversification of SAN subtypes. The study further show a key role of Wnt signaling in the critical branching of pacemaker cardiomyocytes and/or pro-epicardial cells. Authors further went on to show the temporal role of Wnt and TGFbeta signaling in the formation of SAN-CM subtypes including SAN-head, SAN-tail and SAN-transitional (TZ) cells.
Strengths:
The manuscript is well written with robust and detailed experimental approach wherein authors study the SAN-CM cell …
Author Response:
Reviewer #1:
The manuscript by Wiesinger et al., demonstrates the differentiation of human induced pluripotent stem cells (iPSCs) into pacemaker cardiomyocytes. Authors have shown impressive analyses of sinoatrial node cardiomyocytes (SAN-CM) using scRNA-seq approach followed by a computational method namely Trajectory Inference (TI) to understand the diversification of SAN subtypes. The study further show a key role of Wnt signaling in the critical branching of pacemaker cardiomyocytes and/or pro-epicardial cells. Authors further went on to show the temporal role of Wnt and TGFbeta signaling in the formation of SAN-CM subtypes including SAN-head, SAN-tail and SAN-transitional (TZ) cells.
Strengths:
The manuscript is well written with robust and detailed experimental approach wherein authors study the SAN-CM cell differentiation from iPS cells and reveal the role of specific signaling pathways in directing cell fate choices. The observations may lead to potential targets for disease condition pertaining to defective pacemaker cell activity and also facilitate understanding on cardiac regeneration in general. The results do support the conclusions that the authors made. The methods described in this manuscript can be used for other similar studies and cells types to identify cell fate choices.
Weaknesses:
The study though well-executed do have a lack of conceptual novelty. The generation of SAN-CMs from iPS cells is a well-established method, so is the knowledge about SAN-head, SAN-tail and SAN-TZ subtypes and their general markers. The transcriptomic (mRNA repertoire) of SAN-CM cells of mice already shows the utility of one of the markers (VSNL1) described in the current manuscript, along with GNaO1 (Liang et al., 2021). Authors themselves discuss and agree with most of the published studies that shows the Bone Morphogenic Protein (BMP), Retinoic Acid (RA) and the implication of TGF-beta/BMP signaling in SAN development.
The contribution of this study to our understanding on SAN cells is by pinpointing the role of specific signaling pathways and validating the same in vitro. This can lead to the understanding of how subtype specific differentiation of SAN-CMs can be carried out by fine-tuning these key pathways.
We thank the reviewer for their valuable input. The first description of a method to differentiate pacemaker cells using BMP4, retinoic acid etc. was introduced in 2017 by Protze et al. In 2019, Ren et al described an alternative method utilizing activation of WNT signaling to generate pacemaker cells in vitro. To our knowledge, these protocols have not been reproduced in any other independent studies. Therefore, the generation of SANCMs from human iPS cells is not as well-established as the methods to generate other cardiomyocyte subtypes, for example, atrial cells. Furthermore, the previous studies (Protze et al, 2017 and Ren et al, 2019) did not include a detailed transcriptomic characterization of their differentiated SANCM population, and the existence of the pacemaker subpopulations in vitro remains unknown. Furthermore, we disagree that the existence of SAN-head, tail and TZ subpopulations and their markers is general knowledge. To date, there is only one study in the field (Goodyer et al, 2019) performed in mouse that characterized gene expression of these subpopulations and we believe much remains to be learnt about the molecular and functional properties of these cell types. The other studies mentioned by the reviewer (Liang et al, 2021 and others) indeed performed sequencing of the sinoatrial node but did not identify SAN subpopulations. Thus, we believe our study provides important validation for the aspects discussed above and as also mentioned by the reviewer, our study identifies signaling pathways that can be utilized for differentiation towards SANCM subpopulations in vitro.
Reviewer #2:
In the manuscript titled "A single cell transcriptional roadmap of human pacemaker cell differentiation," the authors seek to delineate the cell fate decisions that occur during the in vitro differentiation of human pacemaker cells (SANCM) from hiPSCs. The authors first compare marker expression and functional properties of differentiated SANCM and VCM cells, and establish that the SANCM cells have the expected characteristics of pacemaker cells. Single cell RNA sequencing was then used to explore the heterogeneity of the differentiated cells and illustrate the separate clustering of VCM and SANCM cells. The scRNAseq data was used to identify and characterize the different SANCM subtypes generated by the differentiation process. scRNAseq was then used to analyze samples from different stages of reprogramming and highlighted the changes in the transcriptome during the differentiation process. In addition, pseudotime analysis was performed in conjugation with pharmacological manipulation to show how WNT and TGF-beta signaling affect the stepwise progression of hiPSCs into the identified different SANCM subtypes. This study provides evidence for the presence of different SANCM subtypes generated by the SANCM differentiation process as well as illustrates the role of the WNT and TGF-beta in generating these different clusters of SANCM cells. Additional validation of the SANCM heterogeneity during the in vitro differentiation process as well as additional evidence of novel mediators of the acquisition of the unique SANCM subtype identity would strengthen the impact of this manuscript.
Specific suggestions:
- The first scRNAseq experiment highlights the transcriptional differences between VCM and SANCM clusters, however, these differences are to be expected. This data also supports the hypothesis that the SANCM differentiation leads to a heterogeneous population. Additional bioinformatic analyses into the differences between these different clusters may provide more novel insights and could provide molecular targets to explore in vivo during embryonic development. For example, the identification of Vsnl1 and Gnao1 are promising gene candidates that should be further explored during multiple timepoints of heart development and validated with quantification. This data would provide complementary evidence that this differentiation process recapitulates what happens in vivo. Immunofluorescent staining of select markers of different scRNAseq clusters should also be provided to confirm the identified cluster-specific differentially expressed genes.
- The final portion of the manuscript further establishes the specific roles of the WNT and TGF-beta components of the differentiation protocol, but requires additional experiments to show that the heterogeneity is affected at the single cell level when these pathways are altered (such as immunofluorescence staining to show that fewer cells are expressing that gene of interest rather than a systemic change seen by qPCR). Being that the significant roles of WNT and TGF-beta are to be expected due to the presence of chemical modulators of those pathways are present in the differentiation protocol, this manuscript would benefit from experiments exploring other signaling pathways that increase or decrease the efficiency in the creation of the different subtypes of SANCMs, or a more detailed evaluation of when the hiPSC based strategy begins to overlap with heart development and a characterization of the role of the newly identified genetics target(s) in SANCM subtypes differentiation in vivo.
We thank the reviewer for their suggestions. In response to comment 1, bioinformatic analysis presented in figure 3 as well as accompanying supplement files provide detailed insights into the transcriptional differences between the various SAN subpopulations and additional analysis will not add new information. We agree with the other suggestions provided by the reviewer and are currently working on obtaining additional data to support our conclusions.
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Evaluation Summary:
Overall, this study explores the differentiation of human pacemaker cells from human iPSCs, demonstrating different subtypes of pacemaker cells, and highlighting the role of Wnt and TGFbeta signaling in the formation of sinoatrial note cardiomyocyte subtypes.
(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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Reviewer #1 (Public Review):
The manuscript by Wiesinger et al., demonstrates the differentiation of human induced pluripotent stem cells (iPSCs) into pacemaker cardiomyocytes. Authors have shown impressive analyses of sinoatrial node cardiomyocytes (SAN-CM) using scRNA-seq approach followed by a computational method namely Trajectory Inference (TI) to understand the diversification of SAN subtypes. The study further show a key role of Wnt signaling in the critical branching of pacemaker cardiomyocytes and/or pro-epicardial cells. Authors further went on to show the temporal role of Wnt and TGFbeta signaling in the formation of SAN-CM subtypes including SAN-head, SAN-tail and SAN-transitional (TZ) cells.
Strengths:
The manuscript is well written with robust and detailed experimental approach wherein authors study the SAN-CM cell …
Reviewer #1 (Public Review):
The manuscript by Wiesinger et al., demonstrates the differentiation of human induced pluripotent stem cells (iPSCs) into pacemaker cardiomyocytes. Authors have shown impressive analyses of sinoatrial node cardiomyocytes (SAN-CM) using scRNA-seq approach followed by a computational method namely Trajectory Inference (TI) to understand the diversification of SAN subtypes. The study further show a key role of Wnt signaling in the critical branching of pacemaker cardiomyocytes and/or pro-epicardial cells. Authors further went on to show the temporal role of Wnt and TGFbeta signaling in the formation of SAN-CM subtypes including SAN-head, SAN-tail and SAN-transitional (TZ) cells.
Strengths:
The manuscript is well written with robust and detailed experimental approach wherein authors study the SAN-CM cell differentiation from iPS cells and reveal the role of specific signaling pathways in directing cell fate choices. The observations may lead to potential targets for disease condition pertaining to defective pacemaker cell activity and also facilitate understanding on cardiac regeneration in general. The results do support the conclusions that the authors made. The methods described in this manuscript can be used for other similar studies and cells types to identify cell fate choices.
Weaknesses:
The study though well-executed do have a lack of conceptual novelty. The generation of SAN-CMs from iPS cells is a well-established method, so is the knowledge about SAN-head, SAN-tail and SAN-TZ subtypes and their general markers. The transcriptomic (mRNA repertoire) of SAN-CM cells of mice already shows the utility of one of the markers (VSNL1) described in the current manuscript, along with GNaO1 (Liang et al., 2021). Authors themselves discuss and agree with most of the published studies that shows the Bone Morphogenic Protein (BMP), Retinoic Acid (RA) and the implication of TGF-beta/BMP signaling in SAN development.
The contribution of this study to our understanding on SAN cells is by pinpointing the role of specific signaling pathways and validating the same in vitro. This can lead to the understanding of how subtype specific differentiation of SAN-CMs can be carried out by fine-tuning these key pathways.
-
Reviewer #2 (Public Review):
In the manuscript titled "A single cell transcriptional roadmap of human pacemaker cell differentiation," the authors seek to delineate the cell fate decisions that occur during the in vitro differentiation of human pacemaker cells (SANCM) from hiPSCs. The authors first compare marker expression and functional properties of differentiated SANCM and VCM cells, and establish that the SANCM cells have the expected characteristics of pacemaker cells. Single cell RNA sequencing was then used to explore the heterogeneity of the differentiated cells and illustrate the separate clustering of VCM and SANCM cells. The scRNAseq data was used to identify and characterize the different SANCM subtypes generated by the differentiation process. scRNAseq was then used to analyze samples from different stages of reprogramming …
Reviewer #2 (Public Review):
In the manuscript titled "A single cell transcriptional roadmap of human pacemaker cell differentiation," the authors seek to delineate the cell fate decisions that occur during the in vitro differentiation of human pacemaker cells (SANCM) from hiPSCs. The authors first compare marker expression and functional properties of differentiated SANCM and VCM cells, and establish that the SANCM cells have the expected characteristics of pacemaker cells. Single cell RNA sequencing was then used to explore the heterogeneity of the differentiated cells and illustrate the separate clustering of VCM and SANCM cells. The scRNAseq data was used to identify and characterize the different SANCM subtypes generated by the differentiation process. scRNAseq was then used to analyze samples from different stages of reprogramming and highlighted the changes in the transcriptome during the differentiation process. In addition, pseudotime analysis was performed in conjugation with pharmacological manipulation to show how WNT and TGF-beta signaling affect the stepwise progression of hiPSCs into the identified different SANCM subtypes. This study provides evidence for the presence of different SANCM subtypes generated by the SANCM differentiation process as well as illustrates the role of the WNT and TGF-beta in generating these different clusters of SANCM cells. Additional validation of the SANCM heterogeneity during the in vitro differentiation process as well as additional evidence of novel mediators of the acquisition of the unique SANCM subtype identity would strengthen the impact of this manuscript.
Specific suggestions:
1. The first scRNAseq experiment highlights the transcriptional differences between VCM and SANCM clusters, however, these differences are to be expected. This data also supports the hypothesis that the SANCM differentiation leads to a heterogeneous population. Additional bioinformatic analyses into the differences between these different clusters may provide more novel insights and could provide molecular targets to explore in vivo during embryonic development. For example, the identification of Vsnl1 and Gnao1 are promising gene candidates that should be further explored during multiple timepoints of heart development and validated with quantification. This data would provide complementary evidence that this differentiation process recapitulates what happens in vivo. Immunofluorescent staining of select markers of different scRNAseq clusters should also be provided to confirm the identified cluster-specific differentially expressed genes.
2. The final portion of the manuscript further establishes the specific roles of the WNT and TGF-beta components of the differentiation protocol, but requires additional experiments to show that the heterogeneity is affected at the single cell level when these pathways are altered (such as immunofluorescence staining to show that fewer cells are expressing that gene of interest rather than a systemic change seen by qPCR). Being that the significant roles of WNT and TGF-beta are to be expected due to the presence of chemical modulators of those pathways are present in the differentiation protocol, this manuscript would benefit from experiments exploring other signaling pathways that increase or decrease the efficiency in the creation of the different subtypes of SANCMs, or a more detailed evaluation of when the hiPSC based strategy begins to overlap with heart development and a characterization of the role of the newly identified genetics target(s) in SANCM subtypes differentiation in vivo.
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