Engineering Chimeric Cardio-Vascular Assembloids Using Human iPSC-Derived Cardiomyocytes and Vascular Rings

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

“Engineering Chimeric Cardio-Vascular Assembloids Using Human iPSC-derived Cardiomyocytes and Vascular Rings”
Lead Author: Hannah Klör

This manuscript describes a co-culture system in which human iPSC-derived cardiomyocytes are combined with vascular ring segments, with the aim of incorporating adventitial cell populations such as smooth muscle cells and macrophages into engineered cardiac tissue. The idea of using larger vessel structures to increase tissue complexity and better approximate the myocardial environment is interesting and relevant.

The experimental approach is generally straightforward and uses standard, well-established methods. There are no major concerns regarding the technical execution of the experiments.

That said, the overall level of novelty is somewhat limited. The study builds on existing co-culture and assembloid approaches rather than introducing a clearly distinct conceptual or technical advance. The inclusion of adventitial components is a useful extension, but the manuscript would benefit from a clearer demonstration of what this adds beyond current models.

The results are presented in a logical sequence. Figure 1 appropriately shows the generation and characterisation of cardiomyocytes from both mouse and human iPSCs. Figure 2 demonstrates vascular sprouting from aortic rings embedded in collagen, and the presence of macrophages from both adventitial and bone marrow sources. These observations are valid, but largely descriptive and not unexpected.

Figure 3 is intended to be the central figure of the manuscript, showing the spatial relationship between cardiomyocytes and vascular structures. However, the data here are not fully convincing. In particular, panels G, H, and I do not clearly demonstrate integration between the vascular and cardiac components due to lack of co-staining between vascular/macrophages and cardiomyocytes. It is difficult to identify endothelial structures or confirm their morphology, and nuclear staining is not sufficiently clear to support the interpretation. Higher-resolution imaging with co-staining would be needed to substantiate claims of vascular sprouting into cardiomyocyte clusters or meaningful cellular interaction.

Similarly, while the supplementary videos show contractile activity, Figure 4 does not clearly demonstrate cardiomyocyte viability or functional status in the co-culture system.

The manuscript also highlights the presence of macrophages of different origins as a key feature. However, this aspect is not explored in any depth. There is no investigation of their functional roles, nor any comparison between macrophages derived from adventitial versus bone marrow sources albeit went through cre-lox processes to discriminate the origins. As a result, this point remains descriptive and does not substantially strengthen the study.

Overall, while the model is a reasonable step toward more complex cardiac tissue systems, the current work lacks depth in terms of mechanistic insight and functional validation. The conclusions tend to go beyond what is directly supported by the data, particularly with respect to cellular integration and the role of adventitial components.

In summary, this is an initial effort with a reasonable experimental foundation, but it would benefit from more precise experimentation, rigorous analysis, improved imaging, and clearer evidence to support the main claims. Further work exploring the functional contribution of the added cell types, and more precise documentation of their interactions, would significantly strengthen the manuscript.

Author Response

Dear Reviewer,

Thank you for your valuable time and constructive feedback, which has helped us improve the manuscript. Please find below our detailed point-by-point response to your comments.

On behalf of all co-authors,
Philipp Wörsdörfer

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This manuscript describes a co-culture system in which human iPSC-derived cardiomyocytes are combined with vascular ring segments, with the aim of incorporating adventitial cell populations such as smooth muscle cells and macrophages into engineered cardiac tissue. The idea of using larger vessel structures to increase tissue complexity and better approximate the myocardial environment is interesting and relevant.
The experimental approach is generally straightforward and uses standard, well-established methods. There are no major concerns regarding the technical execution of the experiments.

We thank the reviewer for their valuable time and constructive feedback, which helped us improve the manuscript. We appreciate the positive assessment of our work as "interesting and relevant," as well as the acknowledgment that there are "no major concerns regarding the technical execution of the experiments."

That said, the overall level of novelty is somewhat limited. The study builds on existing co-culture and assembloid approaches rather than introducing a clearly distinct conceptual or technical advance. The inclusion of adventitial components is a useful extension, but the manuscript would benefit from a clearer demonstration of what this adds beyond current models.

We explore an assembloid approach that combines mature vascular tissue with induced pluripotent stem cell (iPSC)-derived cell types. To our knowledge, this approach has not yet been reported. We believe that the described method is novel and interesting because it offers two significant advantages:

First, it has the potential to enhance the maturation of iPSC-derived tissue.

Certain cell types, such as cardiomyocytes, are difficult to obtain from donors and expand as organoids, while others, e.g., intestinal epithelial cells, are widely used for in vitro modeling. This limitation arises for two main reasons: (i) cardiac tissue biopsies are considerably more difficult to obtain than intestinal biopsies, and (ii) primary cardiomyocytes are more difficult to isolate and maintain in culture than epithelial cells. Therefore, using induced pluripotent stem cells (iPSCs) is the only viable alternative.

However, iPSC-derived cardiomyocytes resemble fetal tissue more than adult tissue.
This necessitates the development of strategies to enhance their maturation for meaningful modeling applications. Previous studies have demonstrated that coculturing with fibroblasts, endothelial cells, and macrophages promotes cardiomyocyte maturation. Notably, these supporting cell types are present in and can be derived from adventitial tissue, which contains a versatile stem and progenitor cell niche. Thus, using mature vascular tissue as a cellular niche provides a biologically relevant microenvironment that potentially supports improved maturation.

Second, enabling connection to a perfusion system is important because even when organoids develop intrinsic capillary-like networks, they remain limited in size and functionality due to a lack of perfusion, which often results in necrotic cores. Incorporating large-diameter vessels into the assembloid model allows for direct integration with a perfusion system. These perfused vessels can supply nutrients and oxygen to the attached cardiac tissue via capillary connections. Therefore, integrating macroscopic vessels is a significant technical advancement.

We revised the manuscript to more clearly highlight these aspects and more effectively emphasize the novelty and significance of the approach.

The results are presented in a logical sequence. Figure 1 appropriately shows the generation and characterisation of cardiomyocytes from both mouse and human iPSCs. Figure 2 demonstrates vascular sprouting from aortic rings embedded in collagen, and the presence of macrophages from both adventitial and bone marrow sources. These observations are valid, but largely descriptive and not unexpected.

We agree that Figure 2 is largely descriptive and that the findings are not unexpected. However, it is important to note that these data demonstrate that the coculture system produces all of the major cell types that have been reported to enhance cardiomyocyte maturation, including fibroblasts, endothelial cells, and tissue-resident macrophages. This is a key advantage of co-culturing induced pluripotent stem cell (iPSC)-derived cardiomyocytes with vascular tissue because it creates a cellular environment that is physiologically relevant and has the potential to support cardiomyocyte maturation.

Figure 3 is intended to be the central figure of the manuscript, showing the spatial relationship between cardiomyocytes and vascular structures. However, the data here are not fully convincing. In particular, panels G, H, and I do not clearly demonstrate integration between the vascular and cardiac components due to lack of co-staining between vascular/macrophages and cardiomyocytes. It is difficult to identify endothelial structures or confirm their morphology, and nuclear staining is not sufficiently clear to support the interpretation. Higher-resolution imaging with costaining would be needed to substantiate claims of vascular sprouting into cardiomyocyte clusters or meaningful cellular interaction.

We agree with the reviewer and have revised Figure 3 accordingly. Specifically, we restructured Figure 3D by removing redundant images and adding new stainings that more clearly demonstrate the interaction between endothelial cells and cardiomyocytes. The original figure contained overlapping information in panels 3D, D′, E, 3F, and 3G. Therefore, panels D, D′, and G were removed.
Additionally, panels H and I in the original Figure 3 did not effectively demonstrate the integration of vascular and cardiac components, as the reviewer correctly noted.
These panels have also been removed. We have added new stainings in revised panels G–K, along with two corresponding supplemental videos. The new stainings include both whole-mount and paraffin sections and clearly illustrate endothelial sprouts extending from the vascular adventitia into the surrounding cardiac tissue.

Similarly, while the supplementary videos show contractile activity, Figure 4 does not clearly demonstrate cardiomyocyte viability or functional status in the co-culture system.

We agree with the reviewer. The original Figure 4 only included H&E staining of the human cardiovascular assembloid, which did not sufficiently demonstrate the viability or functional status of the cardiomyocytes.

During the revision process, we conducted additional immunofluorescence analyses on paraffin sections. The new data clearly show the presence of the cardiomyocyte markers TNNT2 and ACTN2 within the cardiac compartment and the endothelial marker CD31 and the macrophage marker IBA1 within the vascular wall and cardiac tissue of the assembloid.

Importantly, revised Figure 4G shows the characteristic cross-striated, sarcomere-like staining pattern of ACTN2, which indicates the structural maturation of the cardiomyocytes. Together with the clearly observable spontaneous contractile activity shown in Supplementary Video 4, we believe that these new data provide strong evidence for the viability and functional status of the cardiac tissue within our coculture system.

The manuscript also highlights the presence of macrophages of different origins as a key feature. However, this aspect is not explored in any depth. There is no investigation of their functional roles, nor any comparison between macrophages derived from adventitial versus bone marrow sources albeit went through cre-lox processes to discriminate the origins. As a result, this point remains descriptive and does not substantially strengthen the study.

The question of macrophage origin is particularly relevant because tissue-resident macrophages play critical roles in cardiac development, functional maintenance, electrical conduction, repair, and regeneration. These macrophages arise during early hematopoiesis or from adventitial stem cells, in contrast to monocyte-derived macrophages. Thus, demonstrating the presence of tissue-resident macrophages in our cultures and their ability to originate from the vascular adventitia in the described co-culture model was important to us. We agree with the reviewer that these descriptive findings are an initial step and that further studies are required to determine if these macrophages contribute to enhanced tissue maturation under our culture conditions. We have added a corresponding paragraph to the revised manuscript to discuss this point.

Overall, while the model is a reasonable step toward more complex cardiac tissue systems, the current work lacks depth in terms of mechanistic insight and functional validation. The conclusions tend to go beyond what is directly supported by the data, particularly with respect to cellular integration and the role of adventitial components.

We carefully reviewed the manuscript to identify and revise or remove statements and conclusions that extend beyond what is directly supported by the data. This was done to avoid overinterpretation. Additionally, we expanded the discussion to more clearly address the study's limitations and outline important directions for future experiments.

In summary, this is an initial effort with a reasonable experimental foundation, but it would benefit from more precise experimentation, rigorous analysis, improved imaging, and clearer evidence to support the main claims. Further work exploring the functional contribution of the added cell types, and more precise documentation of their interactions, would significantly strengthen the manuscript.

Thanks to the reviewer’s constructive feedback, we were able to strengthen the manuscript by incorporating improved imaging data and additional experimental evidence, as outlined above.

We agree that functional testing and integrating the system into a perfusion setup are highly relevant and will be a focus of our future work. However, such experiments are technically demanding and time-intensive and cannot be completed within the timeframe of the current revision.

Going forward, we aim to develop the necessary methodologies and establish appropriate collaborations to demonstrate enhanced cardiac maturation. This includes measurements of sarcomere length by transmission electron microscopy, electrophysiological characterization, and metabolic profiling at different time points in culture, with and without vessel segments present.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript by Hannah Klör et al. describes vessel-cardiomyocyte assembloids created by combining human iPSC-derived cardiomyocytes with mouse or human vascular rings.
The concept of incorporating large-caliber vessel segments while preserving the adventitial compartment is interesting and holds potential value for studying cardiomyocyte-stromal interactions. The authors demonstrate that the myocardial component maintains contractility, and that endothelial sprouting

Major comments:

1. All results are presented as representative images without quantification or statistical analysis. Key parameters (sprout number/length, CD34 and F4/80 cell counts, TNNT2 area) must be quantified with clearly defined n numbers and appropriate statistical tests.

2. The claim that adventitial co-culture supports hiPSC-CM maturation is central to the study but unsupported by data. Comparative functional analysis (e.g., electrophysiology, calcium kinetics, or maturation marker expression) between assembloids and cardiomyocyte-only controls is required, or the claim should be substantially moderated.

3. The entirely human model is supported only by a brightfield image and an H&E section. Immunofluorescence analysis equivalent to that performed for the mouse aorta model is needed to validate this translationally important finding.

Minor comments:

1. The contractility videos (S1–S4) are referenced but cannot be assessed in this review format. The authors should indicate the frame rate, duration, and magnification for each video. Quantification of beating rate (beats per minute) from these recordings would strengthen the functional characterization.

2. "All animal experiments were conducted in accordance with local animal protection laws and institutional guidelines" is insufficient. Also, the ethics committee name and approval number for human saphenous vein use should be explicitly stated in the methods.

3. In-text citations are formatted as superscript numbers (e.g., hiPSC¹) throughout the manuscript. The journal's default citation style requires bracketed numbers (e.g., [1]). Please reformat all in-text citation accordingly.

Author Response

Dear Reviewer,

Thank you for your valuable time and constructive feedback, which has helped us improve the manuscript. Please find below our detailed point-by-point response to your comments.

On behalf of all co-authors,
Philipp Wörsdörfer

-----

This manuscript by Hannah Klör et al. describes vessel-cardiomyocyte assembloids created by combining human iPSC-derived cardiomyocytes with mouse or human vascular rings.
The concept of incorporating large-caliber vessel segments while preserving the adventitial compartment is interesting and holds potential value for studying cardiomyocyte-stromal interactions. The authors demonstrate that the myocardial component maintains contractility, and that endothelial sprouting

We thank the reviewer for their valuable time and thoughtful feedback on our manuscript. We addressed several key points raised in the review. A detailed overview of all changes is provided in the point-by-point response below. We believe the revisions substantially improve the manuscript and hope the reviewer agrees.

Major comments:
1. All results are presented as representative images without quantification or statistical analysis. Key parameters (sprout number/length, CD34 and F4/80 cell counts, TNNT2 area) must be quantified with clearly defined n numbers and appropriate statistical tests.

We agree with the reviewer that the manuscript lacks quantification and statistical analyses. This is primarily because the present study is intended as a proof of concept for a novel methodology, cardio-vascular assembloids, rather than a comparative analysis of different conditions or time points. We did not consider a comparative analysis essential to the main conclusions of the study. The primary objective at this stage is to demonstrate the feasibility of the system and its correct assembly, which can be appropriately addressed by qualitative and descriptive analyses.

Furthermore, early-stage systems, such as newly established assembloid models, often exhibit variability between constructs and rely on evolving protocols. Therefore, premature quantification may be difficult to interpret and potentially misleading.

Nevertheless, we considered including quantitative analyses during the revision phase. However, meaningful and robust quantification could not be reliably derived from the available sectioned material. In our view, such analyses would require whole-mount staining of multiple assembloids collected at defined time points. For example, this would be necessary to properly assess vascular network formation. Unfortunately, performing these additional experiments was not feasible within the given timeframe for revisions. We hope the reviewer understands this limitation.

2. The claim that adventitial co-culture supports hiPSC-CM maturation is central to the study but unsupported by data. Comparative functional analysis (e.g., electrophysiology, calcium kinetics, or maturation marker expression) between assembloids and cardiomyocyte-only controls is required, or the claim should be substantially moderated.

At this stage, we can conclude that we have successfully established an assembloid model containing all the key cell types that have been reported to enhance cardiomyocyte maturation. These cell types arise from the vascular adventitia and establish close interactions with the cardiomyocytes. Although we hypothesize that this environment may promote maturation, providing direct functional proof remains challenging.

Such analyses would require advanced methodologies, including electron microscopy to assess sarcomere length at defined culture time points and under controlled conditions (e.g., with or without vascular segments), as well as electrophysiological and metabolic measurements compatible with the 3D culture setup.

Developing these techniques, identifying suitable collaborators, and generating a sufficient number of biological replicates to enable statistically meaningful analyses are time-intensive processes. Therefore, these experiments cannot be performed within the timeframe of the current revision.

In line with the reviewer’s suggestion, we have carefully revised the manuscript to avoid overestimating our findings. We have also explicitly discussed the study's limitations and the key experiments that will be required in future work.

3. The entirely human model is supported only by a brightfield image and an H&E section. Immunofluorescence analysis equivalent to that performed for the mouse aorta model is needed to validate this translationally important finding.

We agree with the reviewer. The original Figure 4 only included H&E staining of the human cardiovascular assembloid, which was insufficient to support the model's translational relevance. During the revision process, we performed additional immunofluorescence analyses on paraffin sections. The results clearly demonstrate the presence of the cardiomyocyte markers TNNT2 and ACTN2 within the cardiac compartment, as well as CD31⁺ endothelial cells and IBA1⁺ macrophages in the vascular wall and cardiac tissue of the assembloid. Importantly, revised Figure 4G shows the characteristic cross-striated, sarcomere-like staining pattern of ACTN2, which is indicative of the structural maturation of the cardiomyocytes. Together with the observable spontaneous contractile activity shown in Supplementary Video 4, these findings demonstrate that the approach initially established with murine vessel segments can be successfully translated into a fully human model system.

Minor comments:
1. The contractility videos (S1–S4) are referenced but cannot be assessed in this review format. The authors should indicate the frame rate, duration, and magnification for each video. Quantification of beating rate (beats per minute) from these recordings would strengthen the functional characterization.

We apologize that the video files could not be accessed during the review process. The videos are important because they represent an integral part of the study and provide strong support for the functionality of our culture system.
We will inform the editors of the issue and hope it can be resolved promptly.

2. "All animal experiments were conducted in accordance with local animal protection laws and institutional guidelines" is insufficient. Also, the ethics committee name and approval number for human saphenous vein use should be explicitly stated in the methods.

Mice were bred in the specific pathogen-free animal facility of the Center for Experimental Molecular Medicine at the University of Würzburg under a 12:12 h light–dark cycle, with ad libitum access to standard chow and autoclaved drinking water. All procedures were performed in accordance with German federal guidelines and local regulations (Regierung von Unterfranken, Würzburg, Germany). Under these regulations, decapitation of mice followed by tissue collection (e.g., the aorta) from sacrificed animals for in vitro experiments does not require an animal experiment permit or specific ethical approval.

The use of human saphenous veins obtained from cadavers donated to our anatomical institute does not require additional ethical approval, as the the legally reviewed institutional body donation agreement includes also the explicit consent for the use of tissues for scientific research purposes (the official version of this agreement in German language is publicly available at the webpage of the Institute of Anatomy and Cell Biology, Würzburg). Furthermore, no identifiable personal data were involved in this study.

3. In-text citations are formatted as superscript numbers (e.g., hiPSC1) throughout the manuscript. The journal's default citation style requires bracketed numbers (e.g., [1]). Please reformat all in-text citation accordingly.

We apologize for the formatting error and have updated all in-text citations to align with MDPI's citation style.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The revised manuscript has addressed several key concerns, particularly by adding immunofluorescence characterization of the fully human vessel-cardiomyocyte assembloid and by moderating claims regarding cardiomyocyte maturation. The study should now be evaluated primarily as a proof-of-concept methodological report rather than a mechanistic or quantitative functional study.
Although the lack of quantification, biological replicate numbers, and statistical analysis remains a limitation, the authors now acknowledge that further histological, ultrastructural, electrophysiological, and functional studies will be required. I therefore consider the manuscript acceptable after minor revisions.

The authors should clearly state the exploratory nature of the study and add available sample numbers for representative images and videos wherever possible.
For all representative images and videos, the authors should provide, at minimum, the number of assembloids, vessel rings, donors, and independent differentiations in which similar observations were made. This information is important even if the data are presented as representative observations, because it would help readers assess the reproducibility and generalizability of the findings.
The language in the Abstract and Conclusions should also be tempered. Terms including "drug testing", "disease modeling", and "functional remodeling", go beyond what has been directly demonstrated in the present study.
Since these remain future applications rather than validated outcomes, expressions such as "potential platform" or "may be useful for" would be more appropriate.
Similarly, the statement in the Abstract that the model is "particularly suited for drug testing by intravascular delivery" should be substantially softened, because the perfusion system appears to be presented only as a future concept in Figure 5 and has not been functionally validated in the current study.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf