Identification of microRNA-Related Target Genes for the Development of Otic Organoids

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript, “Identification of microRNA-related Target Genes for the Development of Otic Organoids”, provided valuable insights into EV miRNA dynamics during inner ear organoid development, integrating sequencing data, pathway analysis, and public transcriptomic resources. However, this study was insufficient justification for EV miRNA profiling, lacked the functional validation for pathway regulation, and limited the strength of the conclusions. These issues require substantial clarification and experimental support. A major revision is recommended to enhance scientific rigor and interpretability.

1. The authors tried to use EV-derived miRNA profiling to investigate regulatory mechanisms during organoid development. However, the rationale for selecting EVs over direct organoid miRNA sequencing is not clearly justified. Since organoids themselves represent the cellular context of differentiation, direct miRNA profiling from organoid tissue may offer more immediate insights into intracellular regulatory dynamics. The authors are encouraged to clarify the specific advantages of EV miRNA analysis in this study or consider including organoid-derived miRNA data for comparison.
2. Figures 4a–d suggested that EV miRNAs target genes involved in key developmental pathways. However, Figure 4E only shows differential gene expression from cochlear tissues, without direct evidence that EV miRNAs mediate these changes. The authors should consider functional validation, such as miRNA inhibition or EV depletion experiments, to demonstrate that removal of specific EV miRNAs alters target gene expression and pathway activity.
3. Figures 5C–E present temporal expression profiles of Trp53, Ezh2, and Zbtb4 across multiple datasets, but the time points used are inconsistent, making direct comparison and interpretation challenging. For instance, mouse developmental stages (E16.5–P7), human cochlear organoid differentiation days (D20–D60), and RT-PCR validation time points (D7 and D21) are not temporally aligned. The authors should clarify the rationale behind the selection of these specific time points and, if feasible, harmonize the temporal framework to enhance interpretability and comparative analysis. Alternatively, the authors could explain the developmental equivalence or relationships between these datasets at different time points to justify cross-comparison.

Author Response

The manuscript, “Identification of microRNA-related Target Genes for the Development of Otic Organoids”, provided valuable insights into EV miRNA dynamics during inner ear organoid development, integrating sequencing data, pathway analysis, and public transcriptomic resources. However, this study was insufficient justification for EV miRNA profiling, lacked the functional validation for pathway regulation, and limited the strength of the conclusions. These issues require substantial clarification and experimental support. A major revision is recommended to enhance scientific rigor and interpretability.

1. The authors tried to use EV-derived miRNA profiling to investigate regulatory mechanisms during organoid development. However, the rationale for selecting EVs over direct organoid miRNA sequencing is not clearly justified. Since organoids themselves represent the cellular context of differentiation, direct miRNA profiling from organoid tissue may offer more immediate insights into intracellular regulatory dynamics. The authors are encouraged to clarify the specific advantages of EV miRNA analysis in this study or consider including organoid-derived miRNA data for comparison.

<Response> We appreciate the reviewer’s thoughtful comment. We agree that direct miRNA profiling of organoids can provide important insights into the intracellular regulatory dynamics of differentiation. However, the primary aim of our study was to explore the role of extracellular vesicles as mediators of intercellular communication during organoid development. While organoid-derived miRNA reflects intracellular processes, EV-derived miRNA offers a complementary perspective by capturing signals actively released into the extracellular space, which may influence proliferation, differentiation, and maturation in a paracrine manner.

By focusing on EV-derived miRNA, we aimed to investigate the regulatory molecules that are not only involved within individual organoid cells but also actively participate in shaping the microenvironment and communication among cells during organoid growth. We believe this approach highlights a distinct layer of regulation that may be overlooked by profiling organoid tissue alone.

We’ve added the specific advantages of EV-derived miRNA analysis in the introduction as below;

While organoid-derived miRNA profiling reflects intracellular regulatory dynamics, we focused on extracellular vesicle–derived miRNAs to capture signals mediating intercellular communication during organoid growth, particularly those influencing proliferation and differentiation.

 

2. Figures 4a–d suggested that EV miRNAs target genes involved in key developmental pathways. However, Figure 4E only shows differential gene expression from cochlear tissues, without direct evidence that EV miRNAs mediate these changes. The authors should consider functional validation, such as miRNA inhibition or EV depletion experiments, to demonstrate that removal of specific EV miRNAs alters target gene expression and pathway activity.

<Response> We agree with the reviewer that functional validation is critical to establish causal relationships between EV miRNAs and target gene regulation. While our current study focuses on integrative sequencing and transcriptomic analysis, we are actively planning experiments involving EV depletion and miRNA inhibition to assess the direct effects on target gene expression and pathway activity. In the revised manuscript, we acknowledged this limitation to avoid overinterpretation of correlative findings.

To address your comment, we have inserted the following text into the Discussion section “Our conclusions regarding EV miRNA–mediated regulation are correlative and derived from target prediction, pathway enrichment, and concordant expression patterns across cochlear datasets. We have not yet demonstrated that EV-delivered miRNAs are necessary and/or sufficient to drive the observed pathway changes in recipient otic cells or quantified the contribution of EVs relative to other extracellular miRNA pools. Accordingly, we have tempered causal language and explicitly note this limitation.”

 

3. Figures 5C–E present temporal expression profiles of Trp53, Ezh2, and Zbtb4 across multiple datasets, but the time points used are inconsistent, making direct comparison and interpretation challenging. For instance, mouse developmental stages (E16.5–P7), human cochlear organoid differentiation days (D20–D60), and RT-PCR validation time points (D7 and D21) are not temporally aligned. The authors should clarify the rationale behind the selection of these specific time points and, if feasible, harmonize the temporal framework to enhance interpretability and comparative analysis. Alternatively, the authors could explain the developmental equivalence or relationships between these datasets at different time points to justify cross-comparison.

<Response> We thank the reviewer for highlighting the difficulty of comparing datasets sampled on different absolute time scales (mouse E16.5–P7; human cochlear organoids D20–D60; RT-qPCR D7 and D21). In the revision, we chose not to perform cross-species temporal rescaling, which could imply precision beyond the data. Instead, we clarified the basis of comparison by annotating each panel with developmental phase labels—prosensory/early specification, onset of hair-cell (HC) features, early maturation, and late maturation—and by explicitly stating that our cross-dataset comparisons refer to state equivalence rather than synchronized chronological time.

• Figure 5 (C–E) now shows the original native time units for each dataset and overlays background color shading corresponding to the three developmental phases, with concise descriptors tailored to each dataset’s staging (mouse embryonic/postnatal days; organoid differentiation days; days-in-culture for RT-qPCR). We also clarified in the legends that expression magnitudes are not cross-comparable across panels.

To address your comment, we have inserted the following text into the Results section “We compared datasets from mouse cochlear tissues (E16.5–P0 and E16–P7), human cochlear organoid (D20–D60), and RT-qPCR of mouse cochlear organoid (D7, D21) along a common biological trajectory rather than by absolute time. To aid interpretation without temporal rescaling, Figure 5 annotates each panel with developmental phase labels—prosensory/early specification, onset of HC features, and early and late maturation—and our cross-dataset statements refer to state equivalence across these phases.”

To address your comment, we have inserted the following text into the Methods section “4.10 Phase assignment and cross-dataset comparison

Phase labels were assigned using each dataset's native staging system without re-quantifying canonical markers. Mouse data were staged by embryonic/postnatal day; organoid data by protocol day and cell-type annotations; RT-qPCR data by days-in-culture. Cross-dataset comparisons were based on developmental state equivalence rather than chronological synchronization, with no cross-species temporal normalization applied. Detailed time point-to-phase mappings are provided in Supplementary Table S1.”

To address your comment, we have inserted the following text into the Figure 5 caption “Time is shown in native units for each dataset (E16–P0; E16.5–P7; D20–D60; D7/21), and expression magnitudes are not cross-comparable across panels. Phase overlaid background indicate approximate developmental equivalence used for qualitative comparisons; see Supplementary Table S1 for the time-to-phase mapping.”

 

• We added Supplementary Table S1, which maps each dataset’s time points to the three phases using the dataset-provided annotations (e.g., embryonic vs. postnatal day, organoid protocol day, and, where available in the source data). These updates improve interpretability without imposing cross-species time normalization.

Rationale for the selected time points.

• Mouse (E16.5–P7): spans late embryonic specification to early postnatal maturation, capturing the window in which HC features emerge and consolidate.
• Organoids (D20–D60): follows the standard differentiation schedule in the underlying dataset, with earlier days enriched for progenitors and later days showing HC-like states according to the source annotations.
• RT-qPCR (D7, D21): selected a priori to bracket early induction versus later maturation in our culture paradigm, providing an independent validation readout without asserting day-for-day equivalence to mouse or organoids.

Please refer Supplementary Table S1.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript is by Lee et al.  is well written and presents interesting findings on the role of extracellular vesicle miRNAs in the development of otic organoids. The study addresses an important gap in understanding the molecular mechanisms of inner ear regeneration and identifies key regulatory genes (Trp53, Ezh2, Zbtb4) with potential therapeutic relevance. The experimental design is solid, and the integration of sequencing with public transcriptomic datasets strengthens the results. With some improvements in clarity, organization, and discussion of clinical implications, this work could make a valuable contribution to the field of hearing research.

1. The background is thorough, but the transition from general hair cell regeneration to the specific aim of studying EV miRNAs could be made smoother.
2. Figures should clearly illustrate the differences between proliferation and differentiation stage organoids and also provide high resolution of those image.
3. Do we need to consider adding potential clinical implications of your findings in more detail, especially regarding therapeutic use of EV miRNAs.
Minor points
1. Please check the formatting of author affiliations, references, and keywords.
2. Ensure consistency in gene/protein naming conventions (e.g., Trp53 vs. TP53).

Author Response

Reviewer 2

This manuscript is by Lee et al.  is well written and presents interesting findings on the role of extracellular vesicle miRNAs in the development of otic organoids. The study addresses an important gap in understanding the molecular mechanisms of inner ear regeneration and identifies key regulatory genes (Trp53, Ezh2, Zbtb4) with potential therapeutic relevance. The experimental design is solid, and the integration of sequencing with public transcriptomic datasets strengthens the results. With some improvements in clarity, organization, and discussion of clinical implications, this work could make a valuable contribution to the field of hearing research.

1. 
   The background is thorough, but the transition from general hair cell regeneration to the specific aim of studying EV miRNAs could be made smoother.

<Response> We sincerely thank the reviewer for this valuable comment. We agree that the transition from the general background on hair cell regeneration to the specific focus on EV-derived miRNAs required clarification. As highlighted in our previous response, while organoid-derived miRNA profiling provides important insights into intracellular regulatory mechanisms, our study aimed to explore extracellular vesicles as mediators of intercellular communication during organoid development. EV-derived miRNAs represent signals actively released into the extracellular space, influencing proliferation, differentiation, and maturation in a paracrine manner. This offers a complementary perspective to intracellular profiling.

To address this point, we have revised the introduction for smoother flow and clearer rationale.

While organoid-derived miRNA profiling reflects intracellular regulatory dynamics, we focused on extracellular vesicle–derived miRNAs to capture signals mediating intercellular communication during organoid growth, particularly those influencing proliferation and differentiation.

2. 
   Figures should clearly illustrate the differences between proliferation and differentiation stage organoids and also provide high resolution of those image.

<Response> We thank the reviewer for this helpful suggestion. To better illustrate the differences between proliferation- and differentiation-stage organoids, we have added explanatory details to Figure 2. In addition, we enhanced the image resolution to 600 dpi and incorporated the revised figures into the main text. For further clarity, we have also uploaded the figure files in JPG format.

3. 
   Do we need to consider adding potential clinical implications of your findings in more detail, especially regarding therapeutic use of EV miRNAs.

<Response> We appreciate the reviewer’s thoughtful comment. While the therapeutic implications of EV miRNAs are of great interest, we believe it is premature to draw detailed conclusions at this stage. Our current findings may provide a foundation, but further validation and additional studies will be necessary to clarify their potential clinical relevance. We have mentioned our limitation in the Discussion section.

“Our conclusions regarding EV miRNA–mediated regulation are correlative and derived from target prediction, pathway enrichment, and concordant expression patterns across cochlear datasets. We have not yet demonstrated that EV-delivered miRNAs are necessary and/or sufficient to drive the observed pathway changes in recipient otic cells or quantified the contribution of EVs relative to other extracellular miRNA pools. Accordingly, we have tempered causal language and explicitly note this limitation.”

Minor points
1. Please check the formatting of author affiliations, references, and keywords.

<Response> Thank you for the comment. We’ve check the formatting once again, and updated keywords as below.

Keywords: inner ear organoid, differentiation, development, regeneration, extracellular vesicles, miRNAs

2. 
   Ensure consistency in gene/protein naming conventions (e.g., Trp53 vs. TP53).

<Response> We revised the manuscript to ensure consistent nomenclature, using species-appropriate conventions. For example, to address your comment, we have revised the following text to ensure gene/naming conventions in Result section,

“The directional trends were consistent across datasets. Expression of the human gene TP53 and its mouse ortholog Trp53, as well as the human gene EZH2 and its mouse ortholog Ezh2, decreased from early to later phases. Conversely, expression of the human gene ZBTB4 and its mouse ortholog Zbtb4 increased in the later stages. Temporal analysis of gene expression in mouse inner ear tissues revealed dynamic regulation patterns during maturation (Figure 5C). In these mouse tissues, the expression levels of the mouse genes Trp53 and Ezh2 decreased markedly from embryonic day 16.5 (E16.5) to postnatal day 0 (P0) in both vestibular and cochlear regions. This downregulation pattern of the mouse genes persisted throughout later developmental stages, from embryonic day 16 (E16) to postnatal day 7 (P7), in both hair cells (Pou4f3-GFP positive) and non-hair cells (Pou4f3-GFP negative) of the cochlea and utricle. The mouse gene Zbtb4 showed a more complex pattern, with initial low expression that increased during later developmental stages, particularly in cochlear tissues.”

 

Author Response File: Author Response.docx