Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

The manuscript entitled "Human Liver Organoids as an Experimental Tool to Investigate Lipocalin-2 in Hepatic Inflammation"  presents a well-designed and technically sound study establishing patient-derived human liver organoids as a relevant in vitro model to investigate Lipocalin-2 (LCN2) regulation under inflammatory conditions. The work addresses an important gap in the field by moving beyond rodent models and tumor-derived hepatocyte cell lines, providing a human-based 3D system that more closely recapitulates liver architecture. The experimental approach is comprehensive, combining ultrastructural validation, protein and transcriptomic analyses, cytokine stimulation, pathway inhibition, and imaging. Overall, the study is clearly written, the data are convincing, and the conclusions are largely supported by the results. Below you can find my specific comments. Please repond to all of them in a stepwise manner.

Major comments

1. Characterization of organoid cellular identity and maturity
   While the organoids clearly display hepatocyte-like ultrastructure and HNF4α expression, the lack of albumin and CYP3A4 expression indicates an immature or progenitor-like phenotype. This is acknowledged in the Discussion, but the implications for LCN2 biology should be discussed more explicitly. Since hepatocyte maturation status can significantly affect inflammatory signaling and acute-phase protein production, the authors should clarify whether LCN2 induction is expected to differ between progenitor-like versus fully differentiated hepatocytes. If available, inclusion of additional maturation markers (e.g. ALB mRNA, CYP enzymes at transcript level, or bile acid transporters) would strengthen the model characterization.

2. Cellular heterogeneity of liver organoids
   The detection of SOX9 and KRT19 suggests the presence of cholangiocyte-like or bipotent progenitor populations. Given that LCN2 can be expressed by multiple hepatic cell types, it is important to clarify whether LCN2 induction is hepatocyte-restricted or potentially contributed by non-hepatocyte populations within the organoids. The authors should discuss this limitation and, if possible, comment on whether immunofluorescence suggests uniform or heterogeneous LCN2 expression within the organoid structures.

3. Interpretation of IL-6 and LPS unresponsiveness
   The lack of LCN2 induction by IL-6 and LPS contrasts with several mouse and hepatoma cell line studies. Although this discrepancy is discussed, the authors should further elaborate on potential mechanistic explanations, such as limited IL-6 receptor expression, impaired STAT3 signaling, or absence of Kupffer/macrophage-like cells in the organoid system. If STAT3 activation was examined but not shown, this should be clarified. Otherwise, this represents an important limitation of the current model that should be more explicitly stated.

4. Signaling pathway inhibition and partial mRNA suppression
   In Figure 6C, NF-κB inhibition completely blocks LCN2 protein expression but only partially reduces mRNA levels. This discrepancy deserves deeper discussion. Possible explanations include post-transcriptional regulation, mRNA stability, or delayed transcriptional kinetics. The authors should clarify whether mRNA was measured at a single time point and whether earlier or later time points were considered.

5. Donor variability and statistical robustness
   Although the authors state that at least five donors were used, donor-to-donor variability is not clearly shown in the figures. Given the clinical relevance of patient-derived organoids, it would be valuable to comment on the extent of inter-individual variability in LCN2 induction. Even if pooled analyses were performed, this should be clarified in the Methods or Results.

6. Translational relevance and disease modeling
   The study proposes organoids as a platform for patient-specific predictions, yet all experiments are performed using tissue described as “healthy.” The authors should better delineate how this model could be extended to disease-specific organoids (e.g. MASLD, MASH, HCC) and whether LCN2 induction patterns are expected to differ. This would strengthen the translational impact of the work.

Author Response

Dear Reviewer 1,

Thank you for taking the time to review our paper. Please find our response to your valuable comments in the attached PDF-file.

Regards

Ralf Weiskirchen

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors' manuscript data indicated creating a model of 3-D organoid from patients' hepatic liver cells and investigating LCN-2 in hepatic inflammation. These models can be useful for aiding disease progression understanding and facilitating patient-specific treatment predictions.  The represented data did not consistently represent, especially the main topic of LCN2 expression in the organoid. The manuscript does not sufficiently support conclusions. 

 

Main Comments

• In Figure 4, LCN2 western blot A and C., the organoid expressed LCN2 and remained stable over 6 passages. However, the control group did not detect LCN2 protein in Figure C.

 

• In Figure 5, the authors LCN2 expressed in the organoid and were stable. However, IF stain did not support, and IL-1b was well stained in the cytoplasm, but the other group's data was difficult to judge. If authors have a sample of organoids, they can process a section of fresh frozen or paraffin-embedded (EM sample). It will be better to have the whole organoid staining of LCN2.

 

Author Response

Dear Reviewer 2,

Thank you for taking the time to review our paper. Please find our response to your valuable comments in the attached PDF-file.

Regards

Ralf Weiskirchen

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Human Liver Organoids as an Experimental Tool to Investigate Lipocalin-2 in Hepatic Inflammation” is well structured, methodologically sound, and scientifically robust. The objectives are clearly stated, the experimental approach is appropriate, and the results are consistent with current knowledge. The use of human liver organoids represents a modern and relevant model system that aligns well with the scope of Cells.

Overall, the manuscript is suitable for publication; however, several minor issues related to clarity, presentation, and editorial refinement should be addressed prior to acceptance.

Minor comments requiring revision

1. Abstract: density and readability

Although scientifically accurate, the abstract is relatively dense and could benefit from improved readability. It is recommended to.

• Divide long sentences into shorter ones • More clearly separate background, objectives, main findings, and conclusions

This would facilitate rapid comprehension by readers and editors.

2. Consistency of verb tense

In several sections of the Results and Discussion, verb tenses alternate inconsistently between past and present. For clarity and editorial consistency:

• Past tense should be used to describe experimental results • The present tense should be reserved for interpretation and references to the figure

 

3. Figures: size and visual clarity

Some figures, particularly Figures 2,3, and 6, contain multiple panels and detailed information that is difficult to interpret at the current scale. The authors are encouraged to:

• Increase figure size • Enlarge fonts and labels • Split complex figures into additional subpanels

Improved figure readability would significantly enhance data accessibility

4. Emphasis and visibility of figures within the text

All figures are appropriately cited; however, their visibility within the manuscript could be improved. Enhancing figure and table references in bold (e.g., Figure 4A, Figure 6B) would facilitate visual navigation, in line with MDPI editorial practices.

 

5. Minor terminology and formatting inconsistencies

Several small inconsistencies were noted, including:

• Variable notation of NF-kB versus NF-kB • Inconsistent spacing between numerical values and units (e.g., µm, ng/mL) • Occasional repetition of abbreviations already defined

These should be harmonized throughout the manuscript

6. Discussion: finals synthesis

The discussion is thorough and well-referenced; however, some repetition occurs in the concluding paragraphs. A more concise final synthesis linking:

• Liver organoid relevance • The role of lipocalin-2 • Translational implications

Would strengthen the overall impact and provide a clearer conceptual closure.

Author Response

Dear Reviewer 3,

Thank you for taking the time to review our paper. Please find our response to your valuable comments in the attached PDF-file.

Regards

Ralf Weiskirchen

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

You have addressed the majority of my comments.

Reviewer 2 Report

Comments and Suggestions for Authors

The author's response is acceptable.  The data presentation and analysis are well organized and conclusive.

Reviewer 3 Report

Comments and Suggestions for Authors

 Accept in present form.