iPSC-Derived Endothelial Cells as Experimental Models for Predictive and Personalized Strategies in Cardiovascular and Cerebrovascular Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the manuscript “iPSC-derived endothelial cells as experimental models for predictive and personalized strategies in cardiovascular and cerebrovascular disease”, the authors provided a comprehensive overview of the potential of hiPSC-ECs in modeling vascular pathologies.

The manuscript is valuable, however some issue should be addressed.

"Current methods" (Line 62): The phrase "current methods allow for the analysis of pathway-level responses" is vague.

 

An important aspect that deserves further mention is the recent emergence of progesterone receptor expression in iPSCs (which persists throughout differentiation). This concept changes the understanding of how these cells respond to the environment. Progesterone is known to have effects on vasodilation, angiogenesis, vascular permeability, neuroprotective effects and blood-brain barrier (BBB) ​​stabilization. Knowing that iPSCs carry the machinery to respond to progesterone is crucial for precision medicine. This aspect should be integrated into Section 2 (Differentiation and Characterization) or the Limitations/Future Directions section (refer to DOI: 10.1007/s12015-024-10776-6).

 

Add a table for paragraph 3. Patient-specific iPSC-ECs as predictive and personalized models for cardiovascular applications.

 

For monogenic diseases, authors should discuss the importance of using isogenic controls (corrected via CRISPR/Cas9).

 

It would be usefull to add some considerations to the Organ-on-a-Chip technology.

 

Correct the paragraph numbering.

 

 

Author Response

In the manuscript “iPSC-derived endothelial cells as experimental models for predictive and personalized strategies in cardiovascular and cerebrovascular disease”, the authors provided a comprehensive overview of the potential of hiPSC-ECs in modeling vascular pathologies. The manuscript is valuable, however some issue should be addressed.

Thank you for your kind comment.

• "Current methods" (Line 62): The phrase "current methods allow for the analysis of pathway-level responses" is vague.

We have better specified the sentence as follows:

“Since iPSC-derived endothelial cells retain the donor’s genetic background and current multi-omics approaches combined with advanced computational tools enable pathway-level analyses, it is now feasible to investigate patient-specific factors underlying endothelial dysfunction and therapeutic responses in controlled experimental settings.”

• An important aspect that deserves further mention is the recent emergence of progesterone receptor expression in iPSCs (which persists throughout differentiation). This concept changes the understanding of how these cells respond to the environment. Progesterone is known to have effects on vasodilation, angiogenesis, vascular permeability, neuroprotective effects and blood-brain barrier (BBB) ​​stabilization. Knowing that iPSCs carry the machinery to respond to progesterone is crucial for precision medicine. This aspect should be integrated into Section 2 (Differentiation and Characterization) or the Limitations/Future Directions section (refer to DOI: 10.1007/s12015-024-10776-6).

Accordingly, we have highlighted this important aspect and integrated it into Section 2 (Differentiation and Characterization).

“It should be noted that Manganelli et al. demonstrated, for the first time, that reprogramming somatic cells into iPSCs leads to the constitutive expression of the progesterone receptor. Considering the crucial roles of progesterone in vasodilation, angiogenesis, vascular permeability, neuroprotection, and blood-brain barrier stabilization, it is important to understand its role during iPSC differentiation [32].”

Add a table for paragraph 3. Patient-specific iPSC-ECs as predictive and personalized models for cardiovascular applications.

Thank you for this very essential suggestion. Accordingly, we have added a new Table (Table 1) summarizing the use of iPSC-ECs as predictive and personalized models for cardiovascular and cerebrovascular applications.

• For monogenic diseases, authors should discuss the importance of using isogenic controls (corrected via CRISPR/Cas9)

In accordance with the Reviewer’s suggestion, we have discussed the relevance of using isogenic cell lines (corrected via CRISPR/Cas9) at the end of paragraph 4 as follows:

“To reduce variability arising from differences in donor genetic backgrounds, CRISPR-Cas9 technology—a highly efficient gene-editing tool—can be employed either to correct mutations in patient-derived cells or to introduce putative causative lesions into iPSCs derived from healthy individuals. This process results in the creation of isogenic cell pairs that differ by only a single genetic modification, allowing for detailed examination of the molecular and cellular phenotypes associated with specific abnormalities. These isogenic cell lines are invaluable not only for understanding the cellular impact of disease mutations, but also for supporting genetic and pharmacological screening efforts to identify underlying pathological mechanisms [64].”

• It would be useful to add some considerations to the Organ-on-a-Chip technology.

We have introduced some considerations regarding Organ-on-a-Chip technology in Section 5.

“A final element to be considered is the integration of Organ-on-Chip technology: this type of platform can replicate organ-level physiology through a cellular 3D environment, blood flow hemodynamics, mechanical forces, and organ crosstalk. In this context, integrating iPSC-ECs into Organ-on-Chips could support the generation of microphysiological systems that recapitulate specific vascular functions—such as barrier integrity, flow-mediated responses, and multicellular interactions—providing a close-to-realistic setting for studying cardiovascular and cerebrovascular diseases. For cardiovascular diseases, microfluidic platforms model their complex pathophysiology by replicating disturbed hemodynamic conditions and immune inflammation. These systems could incorporate different vessel geometry configurations, endothelial dysfunction, lipid accumulation, and inflammatory cell infiltration [79]. For cerebrovascular ones, the combination of iPSC technology with Organ-on-Chip has enabled the creation of personalized blood-brain barrier chips displaying cellular, molecular, and physiological properties typical of this structure in the in-vivo condition [80].”

• Correct the paragraph numbering.

We have corrected it.

 

Reviewer 2 Report

Comments and Suggestions for Authors

This review paper is very solid and timely. Authors not only cover iPSC-ECs updates and applications in cardiovascular disease, but also discuss in depth the limitations of current techniques, such as epigenetic issues and cell immaturity. Overall, the logic is quite clear and highly valuable for cohorts working in precision medicine and drug development.

Authors are suggested to discuss in depth the application of the clinical diagnosis. So far, this paper mainly focused on the experimental platform. However, given the huge potential of this technique in the clinic application of genotype-driven disease, a discussion on how to translate this platform technique into a clinical diagnostic tool is preferred, which will also serve as the major topic of this paper: precision medicine. For example, a standard of qualified cells for diagnosis in a clinic could be helpful, or whether there could be prepared progenitors to speed up the diagnosis turnover rate (the authors could briefly mention new techniques to accelerate cell maturation, e.g., physical stimulation or small molecules).

Authors are suggested to add a brief explanation of the electrophysiology details, such as how the ion channels of endothelial cells respond to blood flow shear stress, thereby regulating vascular tone, and how iPSC-ECs replicate these biophysical characteristics.

Another minor comment, authors are suggested to add a simple table of the gene-driven diseases mentioned in this paper, such as FH, CADASIL, and aHUS, with their core functional defects. This table will make this paper easier to read.

Overall, this is a high-quality review paper. With more discussion on the clinical translation, this paper would be more impactful.

Author Response

This review paper is very solid and timely. Authors not only cover iPSC-ECs updates and applications in cardiovascular disease, but also discuss in depth the limitations of current techniques, such as epigenetic issues and cell immaturity. Overall, the logic is quite clear and highly valuable for cohorts working in precision medicine and drug development.

Thank you very much for your kind remarks.

• Authors are suggested to discuss in depth the application of the clinical diagnosis. So far, this paper mainly focused on the experimental platform. However, given the huge potential of this technique in the clinic application of genotype-driven disease, a discussion on how to translate this platform technique into a clinical diagnostic tool is preferred, which will also serve as the major topic of this paper: precision medicine. For example, a standard of qualified cells for diagnosis in a clinic could be helpful, or whether there could be prepared progenitors to speed up the diagnosis turnover rate (the authors could briefly mention new techniques to accelerate cell maturation, e.g., physical stimulation or small molecules).

According to the reviewer’s suggestion, we have better discussed these aspects in the last two sections of our manuscript (Limitations and Future applications).

• Authors are suggested to add a brief explanation of the electrophysiology details, such as how the ion channels of endothelial cells respond to blood flow shear stress, thereby regulating vascular tone, and how iPSC-ECs replicate these biophysical characteristics.

We have added a brief explanation of the electrophysiology details in paragraph 2 as follows:

“Shear stress–sensitive ion channels play a central role in how ECs sense blood flow and regulate vascular tone. In native endothelium, mechanosensitive channels transduce fluid shear into changes in membrane potential and intracellular calcium, which activate endothelial nitric oxide synthase (eNOS), promote nitric oxide release, and thereby drive flow‑mediated vasodilation [27,28]. iPSC‑derived ECs recapitulate key aspects of this mechanotransduction cascade, as exposure to physiological shear stress induces endothelial alignment, enhances nitric oxide production, and upregulates arterial markers, resulting in the acquisition of more mature, flow‑responsive biophysical and functional properties [29].”

• Another minor comment, authors are suggested to add a simple table of the gene-driven diseases mentioned in this paper, such as FH, CADASIL, and aHUS, with their core functional defects. This table will make this paper easier to read.

In accordance with the Reviewer’s comment, we have added a new Table (Table 2) summarizing the use of iPSC-ECs as predictive and personalized models for cardiovascular and cerebrovascular applications.

Overall, this is a high-quality review paper. With more discussion on the clinical translation, this paper would be more impactful.

Thank you again for your observations and insightful suggestions, which, without any doubt, have improved the quality of our manuscript.