The Synthetic Extracellular Matrix as a Maestro of the In Vitro Stem Cell Niche: Orchestrating Fate and Function

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In recent years, the concept of the stem cell niche has expanded beyond soluble signaling factors to include the extracellular matrix (ECM) as a dynamic and instructive microenvironment. Increasing evidence demonstrates that ECM composition, mechanical properties, and spatiotemporal dynamics actively regulate self-renewal, fate decisions, and lineage specification of stem cells. In parallel, the limitations of animal-derived, poorly defined matrices such as Matrigel have become increasingly evident, particularly with respect to reproducibility, mechanistic interpretation, and clinical translation. Consequently, synthetic and fully defined ECM systems have emerged as powerful tools for engineering stem cell niches with precise biochemical and biophysical control.

This review comprehensively discusses the role of the ECM as a key regulator of stem cell behavior, with a particular focus on synthetic ECMs as alternatives to Matrigel. The authors systematically describe the structural and functional limitations of Matrigel, followed by an extensive overview of synthetic ECM materials, fabrication strategies, and their applications in human pluripotent stem cell (hPSC) culture, lineage-specific differentiation, and organoid development. By covering ectodermal, endodermal, and mesodermal lineages, the manuscript provides a broad survey of the literature and highlights the potential of synthetic ECMs as tunable, reproducible, and clinically relevant niche platforms. Overall, the review aims to position synthetic ECMs as “maestros” orchestrating stem cell fate and function.

Major concerns:
1. Context-dependent comparison of Matrigel, natural ECMs, and synthetic ECMs
While the manuscript clearly describes the limitations of Matrigel and comprehensively highlights the advantages of synthetic ECMs, the comparison among Matrigel, natural ECMs, and synthetic ECMs remains largely one-directional. As currently written, the narrative may convey the impression that synthetic ECMs are universally superior, without sufficiently emphasizing that the optimal choice of ECM is highly context-dependent.

In practice, ECM selection depends strongly on the experimental purpose, including exploratory basic research, differentiation optimization, mechanistic studies requiring quantitative control, or clinical-grade and GMP-compliant applications. Therefore, the manuscript would benefit significantly from a more balanced and decision-oriented comparison.

The authors are strongly encouraged to either:
(i) include a comparative table summarizing the respective strengths and limitations of Matrigel, natural ECMs, and synthetic ECMs across key criteria (e.g., reproducibility, tunability, biological complexity, clinical translatability, scalability, and cost), or
(ii) more clearly state in the main text under which experimental conditions and research objectives synthetic ECMs represent the most appropriate or optimal choice.

Such a revision would enhance the conceptual clarity of the review and increase its practical value for a broad readership.

2. Insufficient conceptual integration between stem cell niche biology and ECM design
Although the manuscript frames synthetic ECMs as “maestros” of the stem cell niche, this concept remains largely descriptive rather than being translated into a unifying design framework. Specifically, the review does not sufficiently generalize how individual ECM properties—such as stiffness, viscoelasticity, ligand presentation, degradability, and temporal dynamics—systematically regulate stem cell fate decisions across different biological contexts.

To strengthen the conceptual impact of the review, the authors are encouraged to more specifically integrate stem cell niche biology with materials design principles. For example, summarizing how specific ECM parameters map onto distinct cellular responses or developmental outcomes would help distill generalizable rules from the extensive body of literature reviewed. The inclusion of a schematic model or a conceptual summary figure illustrating these relationships could further enhance clarity.

Addressing this point would elevate the manuscript from a comprehensive literature survey to a more theory-guided and design-oriented review, thereby substantially strengthening its overall impact.

3. Temporal balance and positioning of recent advances
Although the manuscript cites a broad range of literature spanning foundational to recent studies, advances from the last two to three years are not always clearly distinguished or contextualized as emerging trends. As a result, readers may find it difficult to identify the current frontiers of the field.

To improve this aspect, the authors could add short subsections or concluding paragraphs highlighting “recent advances” within major sections, and explicitly contrast newer approaches (e.g., dynamic or viscoelastic ECMs, stress-relaxation control, time-dependent ligand presentation, or advanced synthetic designs) with earlier static ECM systems.

This would help readers better appreciate how the field is evolving and where future opportunities lie.

Minor points:
a. Redundancy and suggestions for streamlining
Redundancy is noticeable in Section 5 (particularly Sections 5.1–5.3), where similar introductory statements regarding ECM importance, Matrigel limitations, and advantages of synthetic ECMs are repeatedly used for different lineages. As a concrete improvement, the authors could consolidate general introductory statements into the beginning of Section 5, and focus subsequent subsections on lineage- or organ-specific insights, relying more heavily on Table 2 for detailed examples. This would reduce repetition while preserving informational content.

b. Improving alignment between Figures 1–2 and the main text
Figures 1 and 2 are conceptually informative but are not always clearly integrated into the narrative. To enhance their effectiveness: For Figure 1, explicit references such as “as summarized in Figure 1A/B” might be better to be added in Sections 2 and 3 when discussing structural and functional differences between Matrigel and synthetic ECMs. For Figure 2, individual fabrication methods (e.g., electrospinning, bioprinting, cryogelation) are to be directly referenced in the text when they are introduced. These changes would improve clarity and strengthen the connection between visual and textual content.

c. Minor grammatical and formatting issues
A few representative examples include:
Lines 79–80:
  “It has been shown that insulin-like growth factor 1 and epidermal growth factor were detected … [17] a later study,”
  The sentence is incomplete.
Also, some inconsistency is noticeable in the use of the singular and plural forms of “ECM” and “ECMs,” which could be corrected during revision.

Author Response

In recent years, the concept of the stem cell niche has expanded beyond soluble signaling factors to include the extracellular matrix (ECM) as a dynamic and instructive microenvironment. Increasing evidence demonstrates that ECM composition, mechanical properties, and spatiotemporal dynamics actively regulate self-renewal, fate decisions, and lineage specification of stem cells. In parallel, the limitations of animal-derived, poorly defined matrices such as Matrigel have become increasingly evident, particularly with respect to reproducibility, mechanistic interpretation, and clinical translation. Consequently, synthetic and fully defined ECM systems have emerged as powerful tools for engineering stem cell niches with precise biochemical and biophysical control.

This review comprehensively discusses the role of the ECM as a key regulator of stem cell behavior, with a particular focus on synthetic ECMs as alternatives to Matrigel. The authors systematically describe the structural and functional limitations of Matrigel, followed by an extensive overview of synthetic ECM materials, fabrication strategies, and their applications in human pluripotent stem cell (hPSC) culture, lineage-specific differentiation, and organoid development. By covering ectodermal, endodermal, and mesodermal lineages, the manuscript provides a broad survey of the literature and highlights the potential of synthetic ECMs as tunable, reproducible, and clinically relevant niche platforms. Overall, the review aims to position synthetic ECMs as “maestros” orchestrating stem cell fate and function.

Major concerns:
1. Context-dependent comparison of Matrigel, natural ECMs, and synthetic ECMs
While the manuscript clearly describes the limitations of Matrigel and comprehensively highlights the advantages of synthetic ECMs, the comparison among Matrigel, natural ECMs, and synthetic ECMs remains largely one-directional. As currently written, the narrative may convey the impression that synthetic ECMs are universally superior, without sufficiently emphasizing that the optimal choice of ECM is highly context-dependent.

In practice, ECM selection depends strongly on the experimental purpose, including exploratory basic research, differentiation optimization, mechanistic studies requiring quantitative control, or clinical-grade and GMP-compliant applications. Therefore, the manuscript would benefit significantly from a more balanced and decision-oriented comparison.

The authors are strongly encouraged to either:
(i) include a comparative table summarizing the respective strengths and limitations of Matrigel, natural ECMs, and synthetic ECMs across key criteria (e.g., reproducibility, tunability, biological complexity, clinical translatability, scalability, and cost), or
(ii) more clearly state in the main text under which experimental conditions and research objectives synthetic ECMs represent the most appropriate or optimal choice.

Such a revision would enhance the conceptual clarity of the review and increase its practical value for a broad readership.

Thanks to the reviewer for this constructive comment. We have now included a separate ‘Section 3: Comparison of Matrigel, Natural ECMs, and Synthetic ECMs’, where we addressed all the above-mentioned points. We also included a comparison table name ‘Table 1: Strengths and limitations of Matrigel, Natural ECMs, and Synthetic ECMs’. We hope this will be helpful.

2. Insufficient conceptual integration between stem cell niche biology and ECM design
   Although the manuscript frames synthetic ECMs as “maestros” of the stem cell niche, this concept remains largely descriptive rather than being translated into a unifying design framework. Specifically, the review does not sufficiently generalize how individual ECM properties—such as stiffness, viscoelasticity, ligand presentation, degradability, and temporal dynamics—systematically regulate stem cell fate decisions across different biological contexts.

To strengthen the conceptual impact of the review, the authors are encouraged to more specifically integrate stem cell niche biology with materials design principles. For example, summarizing how specific ECM parameters map onto distinct cellular responses or developmental outcomes would help distill generalizable rules from the extensive body of literature reviewed. The inclusion of a schematic model or a conceptual summary figure illustrating these relationships could further enhance clarity.

Addressing this point would elevate the manuscript from a comprehensive literature survey to a more theory-guided and design-oriented review, thereby substantially strengthening its overall impact.

Thanks to the reviewer for this constructive comment. We have now included ‘Section 6: Synthetic ECM properties regulating stem cell fate specification’, where we addressed every points raised by the reviewer and discussed the individual properties of synthetic ECM and their impact on stem cell niche biology. We hope the reviewer will find it useful. We also included another figure (Figure 3: Mechanical properties of extracellular matrix and stem cell differentiation) to enhance the clarity.

3. Temporal balance and positioning of recent advances
   Although the manuscript cites a broad range of literature spanning foundational to recent studies, advances from the last two to three years are not always clearly distinguished or contextualized as emerging trends. As a result, readers may find it difficult to identify the current frontiers of the field.

To improve this aspect, the authors could add short subsections or concluding paragraphs highlighting “recent advances” within major sections, and explicitly contrast newer approaches (e.g., dynamic or viscoelastic ECMs, stress-relaxation control, time-dependent ligand presentation, or advanced synthetic designs) with earlier static ECM systems.

This would help readers better appreciate how the field is evolving and where future opportunities lie.

Thanks to the reviewer for this constructive comment. We have now included a separate ‘Section 7: Recent advances and emerging trends in synthetic ECM development’. Here we discussed the recent advances in ECM synthesis chemistries, ligand choices, bioprinting strategies, and AI/ML based synthetic ECM design. We hope the reviewer will find it impactful.

Minor points:
a. Redundancy and suggestions for streamlining
Redundancy is noticeable in Section 5 (particularly Sections 5.1–5.3), where similar introductory statements regarding ECM importance, Matrigel limitations, and advantages of synthetic ECMs are repeatedly used for different lineages. As a concrete improvement, the authors could consolidate general introductory statements into the beginning of Section 5, and focus subsequent subsections on lineage- or organ-specific insights, relying more heavily on Table 2 for detailed examples. This would reduce repetition while preserving informational content.

Thanks to the reviewer for the comment. We have significantly added more information in the sections 5.1 to 5.3 (Now it is 6.1 to 6.3). We didn’t find any significant redundancy in information in these sections. We hope the reviewer will agree on this.

b. Improving alignment between Figures 1–2 and the main text
Figures 1 and 2 are conceptually informative but are not always clearly integrated into the narrative. To enhance their effectiveness: For Figure 1, explicit references such as “as summarized in Figure 1A/B” might be better to be added in Sections 2 and 3 when discussing structural and functional differences between Matrigel and synthetic ECMs. For Figure 2, individual fabrication methods (e.g., electrospinning, bioprinting, cryogelation) are to be directly referenced in the text when they are introduced. These changes would improve clarity and strengthen the connection between visual and textual content.

Thanks to the reviewer for the comment. We have put our best effort to align the Figures and Tables citations in the main text. We hope the reviewer will find it satisfactory.

c. Minor grammatical and formatting issues
A few representative examples include:
Lines 79–80:
“It has been shown that insulin-like growth factor 1 and epidermal growth factor were detected … [17] a later study,”
The sentence is incomplete.

Thanks to the author for pointing out the error. We have now fixed it with complete sentence.

Also, some inconsistency is noticeable in the use of the singular and plural forms of “ECM” and “ECMs,” which could be corrected during revision.

Thanks to the reviewer for the comment. The singular and plural forms of ECM were written in context. The plural form, ECMs were written to mean more than one ECM in the text.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Subhajit Giri et al. is very well written and presents an interesting study. Many thanks to the authors for this valuable contribution. However, a few areas, as mentioned below, should be addressed to further enhance the manuscript.

Comments to the authors:

Major comments

1. In Introduction, authors please define the “synthetic ECM “shortly.
2. For the better flow and clarity, authors are kindly requested to organize the Introduction part into separate paragraphs.
3. Please provide the specific knowledge gap that this review aims to address in the introduction.
4. In section 3, please explain in detail how different types of the scaffold architecture influence the stem cell organization and differentiation.
5. Why synthetic ECMs are better option than Matrigel for hPSC culture in terms of reproducibility, scale-up and future clinical applications. Please mention them briefly in section 4.
6. Explain briefly in section 5.1.2., how the synthetic ECMs could be useful for AMD, especially for RPE transplantation and function?
7. In the section discussing Hepatic Lineages, please elaborate how the synthetic ECM systems perform compared to Matrigel, focusing insulin secretion.
8. In the section 5.3, based on the current provided evidence, please explain which of the synthetic ECM systems appear most promising for the future cardiac diseases therapy?
9. How the synthetic ECM based kidney organoids could be used in regenerative therapy? Please elaborate it in terms of functional capabilities.
10. Please expand on the functional maturity of the osteoprogenitor or bone organoids prepared using synthetic ECM scaffold in terms of mineralization and regenerative capacity, compared to conventional scaffold system.
11. The authors mentioned about the novel hydrogel “Amikagel” in section 5.2.3., could the authors explain shortly how it might influence functional maturation of β-cells?
12. Could the authors please describe how the features of the scaffold like stiffness or structure affect vSMC behaviour, what cellular mechanism or signalling pathways drive these changes? Please mention them.
13. Most of the sections contain long paragraphs, authors are requested to divide them into separate paragraphs to improve the flow.

Minor comments

1. Please abbreviate (ECM; Line number 35, FGF2; Line number 82,) at their first occurrence in the manuscript. Please check for others as well throughout the manuscript.
2. Please avoid re-abbreviating (MSCs; line number 364, EBs; line number 635).
3. Please abbreviate “Retinal pigment epithelial cells” as RPE in 327 and use the abbreviation consistently and check in the table 2 as well.
4. In lines 582 and 627, authors mentioned “numerous studies”, but no references are provided.
5. A minor typographical correction- a full stop is missing at the end of line 458.
6. The reference number 64 seems to be somewhat outdated. Please replace it with more recent reference.

Thank you and good luck!

Author Response

The manuscript by Subhajit Giri et al. is very well written and presents an interesting study. Many thanks to the authors for this valuable contribution. However, a few areas, as mentioned below, should be addressed to further enhance the manuscript.

Comments to the authors:

Major comments

1. In Introduction, authors please define the “synthetic ECM “shortly.

Thanks to the reviewer for the suggestions. A brief introduction of ‘synthetic ECM’ is now added to the ‘Introduction’ section.

2. For the better flow and clarity, authors are kindly requested to organize the Introduction part into separate paragraphs.

Thanks to the reviewer for the suggestions. The ‘Introduction’ section has now three paragraphs.

3. Please provide the specific knowledge gap that this review aims to address in the introduction.

Thanks to the reviewer for the suggestions. The ‘Introduction’ section includes the final paragraph to discuss the suggested topic.

4. In section 3, please explain in detail how different types of the scaffold architecture influence the stem cell organization and differentiation.

Thanks to the reviewer for the suggestions. A paragraph has been added to the ‘Section 4: Synthetic ECM scaffolds’, on the suggested topic.

5. Why synthetic ECMs are better option than Matrigel for hPSC culture in terms of reproducibility, scale-up and future clinical applications. Please mention them briefly in section 4.

Thanks to the reviewer for the suggestions. The limitations of Matrigel has been discussed in length section 2, and the benefits, reproducibility, clinical application of synthetic ECM was discussed in a context dependent manner in several occasion in section 3 and 4. To avoid the redundancy, we did not include it the section 4 as a separate paragraph. We hope reviewer will agree with our justification.

6. Explain briefly in section 5.1.2., how the synthetic ECMs could be useful for AMD, especially for RPE transplantation and function?

Thanks to the reviewer for the suggestions. Though in original draft, we discussed the synthetic ECM usage for clinical grade RPE production, we still included a brief explanation in the context of AMD in the revised version.

7. In the section discussing Hepatic Lineages, please elaborate how the synthetic ECM systems perform compared to Matrigel, focusing insulin secretion.

Thanks to the reviewer for the suggestions. We have discussed the synthetic ECM performance in insulin secretion in the section ‘6.2.3. Pancreatic lineage’, as insulin secretion not necessarily depends on hepatic lineages. I hope reviewer will find it useful.

8. In the section 5.3, based on the current provided evidence, please explain which of the synthetic ECM systems appear most promising for the future cardiac diseases therapy?

Thanks to the reviewer for the suggestion. We have now included a short paragraph discussing the above matter in the subsection ‘6.3.1. Cardiac lineages’.

9. How the synthetic ECM based kidney organoids could be used in regenerative therapy? Please elaborate it in terms of functional capabilities.

Thanks to the reviewer for the suggestions. We discussed the suggested topic as a separate paragraph under subsection ‘6.3.2. Renal lineages’.

10. Please expand on the functional maturity of the osteoprogenitor or bone organoids prepared using synthetic ECM scaffold in terms of mineralization and regenerative capacity, compared to conventional scaffold system.

Thanks to the reviewer for the suggestions. We briefly discussed the suggested topic in the first paragraph under subsection 6.3.4.

11. The authors mentioned about the novel hydrogel “Amikagel” in section 5.2.3., could the authors explain shortly how it might influence functional maturation of β-cells?

Thanks to the reviewer for the suggestions. We discussed the suggested topic in the first paragraph under section 6.2.3.

12. Could the authors please describe how the features of the scaffold like stiffness or structure affect vSMC behaviour, what cellular mechanism or signalling pathways drive these changes? Please mention them.

Thanks to the reviewer for the suggestions. We discussed the suggested topic in the first paragraph under section 6.3.3.

13. Most of the sections contain long paragraphs, authors are requested to divide them into separate paragraphs to improve the flow.

Thanks to the reviewer for the suggestions. We divided most of the sections into separate paragraphs.

Minor comments

1. Please abbreviate (ECM; Line number 35, FGF2; Line number 82,) at their first occurrence in the manuscript. Please check for others as well throughout the manuscript.

Thanks to the reviewer for the suggestions. The title of the manuscript already introduced the extracellular matrix abbreviation as ECM. Still, we took you suggestions and introduced the ECM abbreviation in line 35. We also took care of FGF2 and others in the manuscript.

2. Please avoid re-abbreviating (MSCs; line number 364, EBs; line number 635).

Thanks to the reviewer for the suggestions. We removed re-abbreviations, wherever applicable.

3. Please abbreviate “Retinal pigment epithelial cells” as RPE in 327 and use the abbreviation consistently and check in the table 2 as well.

Thanks to the reviewer for the suggestion. We introduced the abbreviations in the text and table.

4. In lines 582 and 627, authors mentioned “numerous studies”, but no references are provided.

Thanks to the reviewer for the suggestion. With respect to line 582, we have now included the references. For line 627, the sentence is rewritten.

5. A minor typographical correction- a full stop is missing at the end of line 458.

Thanks to the reviewer for pointing it out. The full stop is now added.

6. The reference number 64 seems to be somewhat outdated. Please replace it with more recent reference.

Thanks to the reviewer for the suggestion. Though the reference 64 is from many years ago, it is an appropriate citation to support the scientific findings depicted in context.

Reviewer 3 Report

Comments and Suggestions for Authors

We read with great interest the manuscript submitted by Drs. Subhajit Giri and Pratyush Rajesh, entitled “The synthetic extracellular matrix (ECM) as a maestro of the stem cell niche: orchestrating fate and function.” This work provides a strong and timely overview of the current use of synthetic compounds to create ECM‑like environments capable of guiding stem cell differentiation and function. The topic of maintaining stem cells in culture and achieving proper differentiation—particularly in relation to mechanobiology—remains insufficiently highlighted in the literature despite its crucial importance, and this manuscript contributes valuably to that discussion.

A few minor points could nevertheless be addressed by the authors. First, since the manuscript focuses primarily on synthetic compounds, the title could better reflect this by specifying, for example, in vitro stem cell niche rather than simply stem cell niche, in order to avoid potential ambiguity. Additionally, Table 2—although particularly interesting—is difficult to read in its current form. The authors may consider splitting it into several tables (Table 1 is, in our opinion, an appropriate length), which could be distributed across the different sections describing the use of synthetic compounds in the differentiation of stem or progenitor cells derived from the various germ layers.

The manuscript could also benefit from a few additions. In the first section, the authors discuss Matrigel exclusively, but other “natural” supports for differentiation exist, such as cell‑culture–derived matrices. Although, similarly to Matrigel, these matrices can suffer from limited reproducibility, they offer a more explicitly organ‑specific character. Moreover, tissues such as urological or pulmonary tissues, which derive from endodermal germ layers, could also be mentioned given their significant relevance in research, particularly in the case of pulmonary tissues.

On a very minor note, a few residual formatting issues appear to originate from the use of different versions of word‑processing software. Aside from this, the manuscript reads smoothly, and the figures are very well designed. The authors might also consider adding one additional figure illustrating the relationship between the mechanical properties of synthetic ECMs, native in vivo niches, and stem cell differentiation.

 

 

Author Response

We read with great interest the manuscript submitted by Drs. Subhajit Giri and Pratyush Rajesh, entitled “The synthetic extracellular matrix (ECM) as a maestro of the stem cell niche: orchestrating fate and function.” This work provides a strong and timely overview of the current use of synthetic compounds to create ECM‑like environments capable of guiding stem cell differentiation and function. The topic of maintaining stem cells in culture and achieving proper differentiation—particularly in relation to mechanobiology—remains insufficiently highlighted in the literature despite its crucial importance, and this manuscript contributes valuably to that discussion.

A few minor points could nevertheless be addressed by the authors. First, since the manuscript focuses primarily on synthetic compounds, the title could better reflect this by specifying, for example, in vitro stem cell niche rather than simply stem cell niche, in order to avoid potential ambiguity.

Thanks to the reviewer for the suggestion. We included the ‘in-vitro’ part in the title of this manuscript.

Additionally, Table 2—although particularly interesting—is difficult to read in its current form. The authors may consider splitting it into several tables (Table 1 is, in our opinion, an appropriate length), which could be distributed across the different sections describing the use of synthetic compounds in the differentiation of stem or progenitor cells derived from the various germ layers.

Thanks to the reviewer for the suggestion. We now split the Table 2 into Table 3 (Ectodermal lineage), Table 4 (Endodermal lineage), and Table 5 (Mesodermal lineage) and updated the in-text citations of these table accordingly.

The manuscript could also benefit from a few additions. In the first section, the authors discuss Matrigel exclusively, but other “natural” supports for differentiation exist, such as cell‑culture–derived matrices. Although, similarly to Matrigel, these matrices can suffer from limited reproducibility, they offer a more explicitly organ‑specific character.

We appreciate the suggestion from the reviewer. However, as the focus of this manuscript is to discuss and consolidate the information on the applications of synthetic ECM in different lineage specified hPSC derived 2D cell culture and 3D organoid engineering and discuss how these synthetic ECM can effectively replace the usage of Matrigel. We think discussion on ‘Decellularized ECM’ or ‘Natural ECM’ is scope of a different review article and inclusion of such information will shift the focus of this manuscript. I hope reviewer will understand our perspective.

Moreover, tissues such as urological or pulmonary tissues, which derive from endodermal germ layers, could also be mentioned given their significant relevance in research, particularly in the case of pulmonary tissues.

Thanks to the reviewer for the suggestion. We have now included a subsection ‘6.2.4. Pulmonary lineage’ and discussed synthetic ECM usage in iPSC derived lung organoid development. There are very limited numbers of studies have been done on the fabrication and usage of synthetic ECM scaffold for hPSC derived bladder and urethra cell types and organoid culture. Due to this limitation, we didn’t include this tissue type. The mesodermal origin of renal tissue is discussed in the subsection ‘6.3.2. Renal Lineages’ already. We hope the reviewer will appreciate our efforts.

 

On a very minor note, a few residual formatting issues appear to originate from the use of different versions of word‑processing software. Aside from this, the manuscript reads smoothly, and the figures are very well designed. The authors might also consider adding one additional figure illustrating the relationship between the mechanical properties of synthetic ECMs, native in vivo niches, and stem cell differentiation.

Thanks to the reviewer for the suggestion. We have fixed the formatting related issues. We have now included another figure (Figure 3: Mechanical properties of extracellular matrix and stem cell differentiation) illustrating the mechanical properties of synthetic ECMs, native in vivo niches, and stem cell differentiation.

 

Reviewer 4 Report

Comments and Suggestions for Authors

This review may be of some interest to researchers. However, I have several concerns.

1. The authors extensively discuss and highlight the negative aspects of using Matrigel for further translational research due to the nature of its components. However, they do not discuss or provide examples of other matrices in use, such as GelTrex, which does not share the same limitations as Matrigel
2. The review is written in rather broad strokes. While the introduction and abstract discuss the application of ECM in the context of translational medicine, the subsequent details and examples are primarily focused on animal models. Therefore, the entire focus of the review should be adjusted
3. Furthermore, there are very few examples provided involving patient-specific cell cultures or human cells for 3D modeling of various organoids. For instance, when discussing processes related to osteodifferentiation, the authors limit themselves to examples from studies conducted on rat cells. This significantly constrains their conclusions.
4. Additionally, the authors primarily cite articles from around 2013. Unless this is intended as a historical overview, they should be discussing the latest scientific advancements.
5. In their review, the authors include several useful tables. The section Synthetic bioengineered polymeric ECM materials and their application in lineage-specific differentiation of hPSCs is undoubtedly of great interest to modern researchers. I would suggest focusing the review on this section by expanding and deepening its discussion, while condensing the "introductory/educational" part, which explains what human ESCs are, why they are needed, and types of matrices—topics already extensively covered in many other reviews.
6. The Conclusion and Future Perspectivessection is currently underdeveloped and lacks a structured discussion regarding the existing research limitations and the directions future studies should take. Additionally, many current studies now include the analysis of various Omics data obtained from organoid research. This data provides crucial insights for determining the necessary future research directions.
7. By enhancing the discussion of recent advancements in the field, the authors will undoubtedly improve the scientific quality and relevance of the review, thereby making it more valuable for contemporary researchers.

Author Response

This review may be of some interest to researchers. However, I have several concerns.

1. The authors extensively discuss and highlight the negative aspects of using Matrigel for further translational research due to the nature of its components. However, they do not discuss or provide examples of other matrices in use, such as GelTrex, which does not share the same limitations as Matrigel.

Thanks to the reviewer for the suggestion. We have now included a separate paragraph on Geltrex and Cultrex in the section 2.

2. The review is written in rather broad strokes. While the introduction and abstract discuss the application of ECM in the context of translational medicine, the subsequent details and examples are primarily focused on animal models. Therefore, the entire focus of the review should be adjusted.

Thanks to the reviewer for making this comment. We would like to draw the reviewer’s attention that majority of the examples been cited in the manuscript are about human PSC and adult stem cell derived cellular types and organoids. This is evident in every section and consolidated in the Tables. In this context, we believe the focus of the review article is well aligned with translational capabilities of synthetic ECM for human relevant disease research and disease modeling.

3. Furthermore, there are very few examples provided involving patient-specific cell cultures or human cells for 3D modeling of various organoids. For instance, when discussing processes related to osteodifferentiation, the authors limit themselves to examples from studies conducted on rat cells. This significantly constrains their conclusions.

Thanks to the reviewer for making this comment. Again, we would like to draw attention of the reviewer to the fact that in the ‘6.3.4. Osteoprogenitor lineage’, we primarily provide examples of human osteoprogenitor differentiation either from hESCs, hiPSCs, hBMSCs, or hDPSCs. So, we believe, our effort is well aligned with the focus of this manuscript and conclusion. We hope, the reviewer will agree with our counter argument.

4. Additionally, the authors primarily cite articles from around 2013. Unless this is intended as a historical overview, they should be discussing the latest scientific advancements.

In the revised version, we have included many articles and examples which have been published in last 5 years. We hope the reviewer will find it useful.

5. In their review, the authors include several useful tables. The section Synthetic bioengineered polymeric ECM materials and their application in lineage-specific differentiation of hPSCs is undoubtedly of great interest to modern researchers. I would suggest focusing the review on this section by expanding and deepening its discussion, while condensing the "introductory/educational" part, which explains what human ESCs are, why they are needed, and types of matrices—topics already extensively covered in many other reviews.

Thanks to the reviewer for making these comments. Based on other reviewer’s suggestion, we have now separated the Table 2 into Table 3-5 based on ectodermal, endodermal, and mesodermal lineage specified examples of synthetic scaffold usage. In the text, we also added extra examples and deep discussion as per the reviewer’s suggestion.

In the “Introductory/educational” part, we laid some background information in a context dependent manner to make the manuscript more engaging to the readers. We believe this is necessary to provide readers a landing pad to the subject. We hope the reviewer will find it useful.

6. The Conclusion and Future Perspectives section is currently underdeveloped and lacks a structured discussion regarding the existing research limitations and the directions future studies should take. Additionally, many current studies now include the analysis of various Omics data obtained from organoid research. This data provides crucial insights for determining the necessary future research directions.

Thanks to the reviewer for such a constructive comment. We have significantly changed the writing of the ‘Conclusion and Future Perspective’ and tried to address the shortcomings as the reviewer pointed out. We hope the reviewer will find it useful.

7. By enhancing the discussion of recent advancements in the field, the authors will undoubtedly improve the scientific quality and relevance of the review, thereby making it more valuable for contemporary researchers.

Thanks to the reviewer for this constructive comment. We have now included a separate ‘Section 8: Recent advances and emerging trends in synthetic ECM development’. Here we discussed the recent advances in ECM synthesis chemistries, ligand choices, bioprinting strategies, and AI/ML based synthetic ECM design. We hope the reviewer will find it impactful.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all the comments. I have no further comments at this time.

Thank you.