Fibroblast alignment coordinates epithelial migration and maintains intestinal tissue integrity

  1. Jordi Comelles
  2. Aina Abad-Lázaro
  3. Verónica Acevedo
  4. David Bartolomé-Català
  5. Aitor Otero-Tarrazón
  6. Anna Esteve-Codina
  7. Xavier Hernando-Momblona
  8. Eduard Batlle
  9. Vanesa Fernández-Majada
  10. Elena Martinez

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Fibroblasts reside underneath most epithelial tissues. In the intestine, recent studies have shown that fibroblast migration contributes to tissue morphogenesis and wound healing. Yet, whether physical interactions between epithelial cells and fibroblasts contribute to epithelial movement remains elusive. Here, we show that subepithelial fibroblast alignment enhances directed and persistent migration of organoid-derived intestinal epithelia. Using a reconstituted epithelial–stromal gap-closure model, we demonstrate that direct contact with fibroblasts improves gap closure by promoting cell alignment, sustaining tissue integrity, and synchronizing crypt–villus migration. Fibroblasts undergo long-range ordering to align perpendicularly to the epithelial front and deposit protein paths that act as guidance features to direct epithelial migration. In parallel, epithelial cells acquire a wound-associated epithelial-like phenotype, but insufficient to explain the effects of fibroblast contact. Our findings uncover a dual role for intestinal fibroblasts in epithelial repair, coordinating both biochemical and physical cues to ensure efficient and cohesive migration.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Background and unknown in the field:

    This study investigates how fibroblast alignment influences the migration of intestinal epithelial cells, contributing to tissue integrity and repair. It is well established that intestinal fibroblasts are important regulators in the tissue through their ability to secrete essential paracrine factors for the epithelium. However, it is less well understood if they also play additional structural, tissue architecture instructing role and how the communication between the fibroblasts and the epithelia is regulated.

    Advance over state of the art:

    Here the authors have set-up an elegant three-component system to investigate this. They have gone beyond the recent advances of culturing intestinal and colonic organoids in 2D (in a manner that preserves- and villus-like organization) and bioengineered epithelial-stromal model comprising organoid-derived intestinal epithelial cells (IECs), primary intestinal fibroblasts, and a basement membrane matrix. Using this model, they have uncovered fibroblasts enhancing the directed and persistent migration of intestinal epithelial cells (IECs). They used scRNAseq to carefully analyse the stromal cell populations present in their co-cultures of primary mouse intestinal subepithelial fibroblasts and organoid-derived intestinal mouse epithelial cells. They observed that this reflected well the stromal cell-type composition as well as the paracrine activity previously reported for these cells in tissue. Using a clever system with Matrigel and an elastomeric barrier, the authors were able to induce non-epithelial gaps in different scenarios (IECs alone or with fibroblasts or with conditioned media) and observe the wound-closure as well as the presence of specific cell types. They observed that the epithelial monolayers showed significant gap closure when in direct contact with fibroblasts compared to controls. Interestingly, the enhanced efficiency of epithelial migration and gap closure, in the presence of fibroblasts, was independent of PGE₂-EP4 signaling and was not due to differences in cell proliferation. Instead, the imaging revealed that the fibroblasts were in direct contact with the epithelium. The authors observed that in the absence of fibroblasts the migration properties of cells in the villus and the crypt regions were dramatically different and the fibroblast presence was necessary to efficiently synchronize these to support gap closure. In addition, the presence of fibroblasts enhanced the directionality of the epithelial cell migration. Detailed imaging and image analyses revealed that gap closure involved activation of the fibroblasts and co-ordinated coalignment of IECs and fibroblasts. They also explored matrix deposition of the fibroblasts during the process and found that they deposited aligned ECM fibers that guide epithelial migration. Mere cell-derived matrix (devoid of live fibroblasts) was able to partially recapitulate the fibroblast-coordinated epithelial migration that the fibroblast generated matrix and its alignment are key contributors to the phenotype.

    Comments:

    This is overall a very interesting and well-written study. The imaging and the image analysis are state-of-the art and the bioengineered model is an exciting advancement over current methods developed by these researchers and others. This study meets all the criteria for a publication in the since that all the experiments seem to be carefully conducted, with appropriate controls and sufficient quantifications and statistics. The claims made by the authors are supported by the data. This is currently suitable to be published as a method/protocol and as a descriptive study uncovering interesting cross-talk and co-dependencies of epithelial and stromal cells during injury repair. There are of course aspects that could improve the study further like more mechanistic insight into the underpinnings of the direct epithelia-fibroblast interaction and its involvement in the directed IEC migration. However, these may be topics to investigate in a future study.

    Reviewer #1 (Significance (Required)):

    The strengths of the study are the highly in vivo relevant model system that is amendable to imaging and detailed image analysis of distinct cell populations. This may be adapted by others in in the field and has the potential to transform the way cell dynamics in the intestinal epithelium are visualized and investigated in vitro

    We thank the reviewer for their thoughtful and positive assessment of our work, and their recognition of the relevance of the bioengineered epithelial-stromal model and its potential for quantitative imaging and analysis of epithelial and fibroblast dynamics.

    We agree that further mechanistic insight into epithelial-fibroblast crosstalk would strengthen the study. While the current manuscript establishes this tractable system and identifies a role for fibroblast organization and matrix alignment in coordinating epithelial migration, we also aim to deepen the mechanistic understanding in the revision. As outlined in our response to Reviewer 2, we will perform additional experiments to further investigate the epithelial-fibroblast crosstalk and force-dependent interactions underlying this process.

    We believe that these additions will complement the current findings and strengthen the conceptual contribution of the study beyond its methodological advances.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Please find enclosed my review comments on the manuscript entitled "Fibroblast alignment coordinates epithelial migration and maintains intestinal tissue integrity" by Jordi Comelles et al.

    In this manuscript, the authors use a bioengineered epithelial-stromal system composed of organoid-derived intestinal epithelial cells, primary intestinal fibroblasts, and a basement membrane matrix to show that direct physical interactions between fibroblasts and epithelial cells drive a large-scale organization of the fibroblast network. This spatial reorganization, in turn, promotes persistent and oriented migration of epithelial cells, ultimately enabling restoration of the intestinal epithelium in an in vitro gap-closure assay. Overall, while the authors use an elegant in vitro model to study intestinal wound closure, and more specifically the role of fibroblasts in this context, I find this manuscript not suitable for publication in its present form. The data are overinterpreted, the novelty is limited, and the molecular mechanisms underlying WAE-fibroblast interactions are insufficiently addressed.

    We thank the reviewer for their contribution to the revision process with their valuable assessments. We will address their specific points below.

    Figure 1 - What are the units of the "fraction gap closure" shown in panels d and e? Is it expressed as a percentage?

    We thank the reviewer for pointing this out. The "fraction of gap closed" was calculated as (A(t = 0h)-A(t))/A(t = 0h), where A(t = 0h) corresponds to the initial gap area and A(t) is the area of the gap measured at the time point t. With this definition, the fraction of gap closed is dimensionless, it is 0 at the initial time point, will reach 1 if the gap is fully closed and will have negative values if the gap area increases beyond the initial size, as observed in some replicates of the control condition. To avoid misinterpretation, we will express this quantity as a percentage (i.e., multiplied by 100), as suggested by the reviewer. Moreover, we realized it was ill defined in the methods section. This will be corrected as well in the revised version.

    "Actually, epithelial monolayers achieved the most effective gap closure when cultured in direct physical contact with fibroblasts (Figure 1e and Movies 2 and 3)." From the data shown in panels c, d, and e, it appears that fibroblast-conditioned medium alone promotes efficient gap closure, comparable to the + fibroblast condition.

    We agree with the reviewer that the original closing sentence overstated the effect. While both fibroblast-conditioned medium and direct fibroblast contact promote efficient gap closure compared to control conditions, the data do not support a consistent difference between these two conditions. We will therefore remove this statement in the revised version to more accurately reflect the results.

    Figure 2 - The use of a cell proliferation inhibitor during the gap-closure assay would help determine the contribution of cell proliferation at the migration front.

    We agree with the reviewer that inhibiting proliferation would help assess the contribution of cell proliferation to gap closure. However, in the 2D gap-closure assay, our Ki67 immunostaining showed no significant differences in the proportion of proliferative cells between conditions, either within the monolayer or at the migration front. This suggests that differential proliferation is unlikely to account for the differences in gap closure observed between control and fibroblast-containing conditions.

    We note that, in a separate 3D organoid assay, fibroblast-derived signals induced a WAE-like transcriptional program associated with reduced Ki67 mRNA expression, indicating that fibroblasts can promote a more migratory epithelial state without increasing proliferation. Thus, while proliferation may contribute to epithelial homeostasis and repair, our data do not point it as the main determinant of the differences observed in the 2D gap-closure phenotypes.

    In addition, pharmacological inhibition of proliferation would likely perturb the homeostasis of the organoid-derived epithelial monolayers, in which proliferative crypt compartments are essential, and would be difficult to restrict to epithelial cells without also affecting fibroblasts in co-culture. For these reasons, although such experiments could inform the general contribution of proliferation to gap closure, we do not think they would directly clarify the differences observed between conditions in our system.

    Figure 2f and 2g - Has a dose-dependent effect of PGE2 been tested?

    We thank the reviewer for pointing this out. We did not perform a dose-response analysis of PGE2 in this study, as our aim was to assess the involvement of the PGE2-EP4 axis rather than to characterize its quantitative dynamics. We therefore selected a concentration based on previous work demonstrating dose-dependent induction of the WAE program in 3D organoid systems (Miyoshi et al., 2017). In that study, 1 µM PGE2 was sufficient to induce a significant increase in the WAE marker Cldn4, and we used this concentration as a biologically relevant reference condition. We will clarify this in the methods section.

    Figure 2i - The + fibroblast + EP4i condition (pink) is missing.

    We thank the reviewer for pointing this out. The + fibroblast + EP4i condition is present in the plot but not visually distinguishable because it overlaps with the + fibroblast condition and is therefore masked by it. As shown in Figure S4e, the + fibroblast + EP4i condition falls within the variability range of the + fibroblast condition. To improve clarity, we will revise the figure to ensure that this condition is visually identifiable.

    "This suggests a mechanical or contact-mediated role for fibroblasts in preserving epithelial integrity and promoting coordinated migration beyond their paracrine signaling." While PGE2-EP4 signaling does not appear to be involved in the fibroblast-mediated enhancement of gap-closure efficiency, the conclusion that physical interactions are more important than paracrine effects is overstated. For instance, an experimental condition in which fibroblast-conditioned medium is inactivated (boiling for 5 minutes) would strengthen this conclusion. In addition, inhibition of actomyosin contractility in fibroblasts would be informative.

    Figure 3 - The data presented here do not convincingly support the dismissal of conditioned medium as a contributing factor. The differences between the + fibroblast-conditioned medium and + fibroblast conditions are modest. In both cases, epithelial cells migrate and gaps close.

    We agree with the reviewer that inhibition of actomyosin contractility in fibroblasts would provide valuable insight into the role of force-dependent interactions in epithelial-stromal coupling. However, pharmacological inhibitors of the Rho-ROCK-myosin pathway (e.g., blebbistatin, ML-7, or the ROCK inhibitor Y-27632) would also affect epithelial contractility in our co-culture system, making it difficult to specifically attribute any observed effects to fibroblast mechanics.

    We also agree that paracrine signaling plays an important role in epithelial gap closure. Indeed, supplementation of control media with PGE₂* improves gap closure compared to control conditions, although it does not reach the levels observed with fibroblast-conditioned medium, suggesting that additional soluble factors contribute beyond the PGE₂-EP4 axis. However, time-lapse imaging revealed direct and dynamic interactions between fibroblasts and epithelial cells (Movie 6; Figure S5a-d; Movie 7), which prompted us to further investigate the contribution of physical interactions, as addressed in Figure 3.*

    In Figure 3, we analyzed migration at the single-cell level, in contrast to the tissue-level measurements used for gap closure quantification. In organoid-derived intestinal monolayers, two distinct compartments can be identified: crypt-like and villus-like regions. In vivo, these compartments exhibit different migration behaviors: cells in the crypt are primarily displaced due to crowding, whereas cells in the villus actively migrate, as suggested by the presence of cryptic lamellipodia (Krndija et al., 2019). Consistent with this, tracking individual cells revealed that crypt cells are largely static, while villus cells migrate toward the gap. This compartmentalized behavior was observed in both control and fibroblast-conditioned medium conditions. Strikingly, in the presence of fibroblasts, this differential behavior was reduced, resulting in coordinated migration of both crypt and villus regions.

    This mismatch between compartments in control conditions may contribute to the appearance of discontinuities ("holes") within the epithelial layer during migration. In control experiments, these defects failed to close, whereas in conditioned medium they closed slowly or incompletely. In contrast, in the presence of fibroblasts, these disruptions were rapidly and efficiently resolved, indicating improved tissue integrity.

    Additionally, analysis of individual trajectories near the migration front showed that cells exhibit significantly increased directional persistence (i.e., movement aligned with the direction of gap closure) in the presence of fibroblasts compared to conditioned medium alone.

    Taken together, while paracrine signaling from fibroblasts contributes to epithelial migration and gap closure, the physical presence of fibroblasts induces qualitative changes in epithelial behavior, including coordinated migration across compartments, improved hole closure, and enhanced directional persistence.

    Figure 4a - "Upon removal of the barrier (t = 0 h), fibroblasts at the epithelial front were small and evenly distributed, with no prominent α-SMA fibers present." Here, fibroblasts are α-SMA positive but not elongated. α-SMA may therefore not be the most appropriate marker. What are the levels of phosphorylated MLC2? These may increase during wound closure. Also, fibroblasts culture promotes aSMA expression, therefore, it may be possible that the fibroblasts used in this assay may not represent the healthy fibroblasts found in vivo.

    We agree with the reviewer that fibroblasts are α-SMA positive at early time points but are not yet elongated. In our system, we observe that α-SMA is already present at t = 0 h, while fibroblasts progressively elongate and reorganize α-SMA into prominent fiber structures over time. This suggests that changes in α-SMA organization, rather than its initial presence, are associated with fibroblast activation during gap closure.

    We note that baseline α-SMA expression may be influenced by in vitro culture conditions prior to the assay, which could differ from the state of fibroblasts in vivo. We will clarify this point in the Discussion to better contextualize our observations relative to native fibroblast populations.

    In addition, we agree that assessing phosphorylated myosin light chain 2 (pMLC2) levels would provide complementary information on contractile activity. We will therefore perform pMLC2 staining, as suggested, to further evaluate force generation by fibroblasts during the wound closure process.

    Figure 5 - Fibroblast alignment could also result from paracrine signals secreted by epithelial cells. This possibility should be tested.

    We thank the reviewer for this suggestion. To test whether fibroblast alignment could be driven by epithelial-derived paracrine signals, we will culture fibroblasts in conditioned medium collected from epithelial monolayers undergoing gap closure (control condition without fibroblasts) and quantify their alignment over time. This will be compared to fibroblasts maintained in standard fibroblast medium.

    This experiment will directly assess whether epithelial-derived soluble factors are sufficient to induce fibroblast alignment, or whether direct physical interactions are required.

    In summary, this manuscript demonstrates that epithelial cells migrate more efficiently on extracellular matrix proteins deposited and oriented by fibroblasts. This concept is not novel. Identifying the molecular mechanisms governing interactions between WAE and subepithelial fibroblasts would significantly enhance the novelty and impact of this study.

    Reviewer #2 (Significance (Required)):

    In this manuscript, the authors use a bioengineered epithelial-stromal system composed of organoid-derived intestinal epithelial cells, primary intestinal fibroblasts, and a basement membrane matrix to show that direct physical interactions between fibroblasts and epithelial cells drive a large-scale organization of the fibroblast network. This spatial reorganization, in turn, promotes persistent and oriented migration of epithelial cells, ultimately enabling restoration of the intestinal epithelium in an in vitro gap-closure assay. Overall, while the authors use an elegant in vitro model to study intestinal wound closure, and more specifically the role of fibroblasts in this context, I find this manuscript not suitable for publication in its present form. The data are overinterpreted, the novelty is limited, and the molecular mechanisms underlying WAE-fibroblast interactions are insufficiently addressed.

    *We thank the reviewer for this thorough and critical assessment. We have clarified the overstatements in the rebuttal and we will modify the text to address concerns regarding overinterpretation and clearly acknowledge the limitations of our approach. In particular, we will refine the framing of the study to better distinguish between the contributions of paracrine signaling and physical epithelial-stromal interactions. *

    *To address the reviewer's concerns regarding mechanism and novelty, we will perform additional experiments aimed at further characterizing epithelial-stromal cross-talk, and experiments to assess fibroblast contractility and its contribution to epithelial coordination. *

    We believe that these revisions and proposed experiments will strengthen the manuscript and clarify its conceptual contribution.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Summary:

    - Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    The study by Comelles et al. focuses on how primary intestinal fibroblasts contribute to organoid-derived intestinal epithelial migration in wound healing assays. Using fibroblast-epithelial co-cultures in a 2D in vitro gap closure system, the authors found that direct interaction with fibroblasts drives cohesive and directed migration of intestinal epithelia toward the gap. They further propose that long-range fibroblast alignment promotes the deposition of extracellular matrix (ECM) proteins in an oriented fashion, contributing to directed epithelial migration.

    Major comments:

    - Are the key conclusions convincing?

    Some of the key conclusions of this manuscript are not entirely convincing given the available data. The manuscript would benefit from additional evidence and/or clarifications to support their conclusions. See comments below.

    - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    (Fig 4a) The authors claim that fibroblasts become activated during gap closure as evidenced by the enhanced assembly of a-SMA fibers 24 hours following barrier removal. Yet, long a-SMA fibers are also observed when fibroblasts are cultured in the absence of epithelial cells or barrier removal (Fig. S1b). To support this conclusion, the authors should consider including additional controls to account for potential time-dependent assembly of a-SMA fibers (e.g., fibroblast-only control).

    We thank the reviewer for pointing this out. We agree that a fibroblast-only control would be important to account for potential time-dependent assembly of α-SMA fibers. We will therefore perform additional experiments monitoring α-SMA organization in fibroblasts cultured alone over time, which will allow us to better interpret the dynamics observed in the co-culture conditions.

    (Fig. 5a) The authors conclude that fibroblasts align parallel to the direction of epithelial migration during gap closure. While quantifications are convincing, again, a fibroblast-only control accounting for time-dependent spreading and elongation (as seen in Fig. S1) is missing. Including such a control would strengthen their claim that alignment is specific to the gap closure context rather than a time-dependent phenotype.

    We agree with the reviewer that, given the intrinsic ability of fibroblasts to form ordered domains with long-range alignment, this control would be highly informative. We will therefore quantify fibroblast alignment over time in fibroblast-only cultures, which will allow us to determine to what extent the long-range organization observed in co-culture is specific to the gap closure context.

    (Fig 6) The authors claim that fibroblast-derived aligned ECM drives directional epithelial migration. While fibronectin fibers appear scarce and weakly aligned with the direction of migration, laminin and type IV collagen fibers are barely detectable (Fig. 6f). This may reflect a defect in ECM deposition rather than fiber alignment, which contrasts with Fig. S1, where fibroblasts are shown to deposit and assemble laminin and type IV collagen fibers. One possible explanation is that primary fibroblasts were not cultured long enough to allow robust ECM deposition. Alternatively, the observed effect may be specific to fibronectin, which is consistent with fibroblasts being its major source. The authors should revise their interpretation or provide additional evidence to support their current claim.

    We thank the reviewer for this important point. We agree that differences in ECM signal within the gap may reflect not only fiber alignment but also differences in the amount of protein deposited. In the +fibroblast condition, fibroblasts in the gap have more time to secrete ECM compared to the "empty gap" condition, where fibroblasts remain confined beneath the epithelium.

    In addition, the presence of Matrigel likely masks the contribution of certain ECM components, making laminin or type IV collagen more apparent than fibronectin. We will therefore revise the interpretation of these results to explicitly acknowledge the contribution of ECM abundance in addition to alignment.

    (Fig 6i) The authors propose that the presence of ECM alone within the gap enhances epithelial gap closure compared to empty gap conditions, although gap closure remains less effective than in the presence of primary fibroblasts. From the figure legend and methods, it seems that the decellularized ECM condition is generated using NIH-3T3 fibroblasts cultured for 8 days, whereas the other conditions used primary fibroblasts cultured for 1 day (Fig. 6a-h). This comparison is confounded by differences in cell source and ECM deposition time. If I am misunderstanding this, please clarify, otherwise consider repeating the decellularized ECM condition using primary fibroblasts and matching culture times for a fair comparison. Along these lines, please include images showing that ECM fibers remain intact following decellularization.

    We thank the reviewer for this suggestion. We will include additional staining to confirm that ECM fibers remain intact after decellularization in the revised version.

    Regarding the use of NIH-3T3 fibroblasts for CDM generation, this choice was made to minimize potential residual paracrine signaling from primary intestinal fibroblasts after decellularization. We acknowledge that this introduces differences in cell source.

    *Concerning culture time, we followed established protocols for CDM formation, which recommend extended culture periods ({greater than or equal to}8 days) to allow robust ECM deposition (Cukierman et al., 2001; Franco-Barraza et al., 2016; Godeau et al., 2020). *We will clarify these points in the revised manuscript and discuss the limitations associated with these differences.

    - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    Yes. The additional experiments outlined above would help support the current conclusions of the manuscript, rather than to explore new directions beyond its scope.

    - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    Yes, the additional experiments primarily involve the inclusion of controls and additional immunofluorescence imaging to their existing experimental setups. They should be relatively straightforward to implement (~2-3 months).

    - Are the data and the methods presented in such a way that they can be reproduced?

    Yes.

    - Are the experiments adequately replicated and statistical analysis adequate?

    Overall, yes. But some plot legends should specify the number of replicates analyzed (e.g. Fig. 2b, Fig. 2d, Fig. 3h).

    We will review and correct these issues.

    Minor comments:

    - Specific experimental issues that are easily addressable.

    (Fig. 1c-e) The authors state that intestinal epithelial monolayers exhibit the most effective gap closure when in direct contact with fibroblasts. However, fibroblast-conditioned media and co-cultures show comparable gap closure efficiencies (Fig. 1e). The authors should consider revising this interpretation based on the provided data.

    We thank the reviewer for pointing this out, which was also raised by Reviewer 2. As discussed above, we agree that the original statement overstated the effect. Both fibroblast-conditioned medium and direct fibroblast contact promote efficient gap closure compared to control conditions, and we will revise the text accordingly to reflect that no consistent quantitative difference is observed between these two conditions.

    (Fig. 3b) The authors suggest that crypt-like epithelial cells undergo migration when grown on fibroblasts, but not in conditioned media alone. This is interesting, but it is not clear how they identify crypt-like cells for tracking. The authors should clarify if crypt-like cells are defined based on markers or inferred from their morphology.

    We thank the reviewer for this comment. In these tracking analyses, crypt-like cells were identified based on morphology. As shown in Figure S3 and in Larrañaga et al., 2025, crypt-like cells, defined by specific molecular markers, are significantly smaller than villus-like cells and form high-density regions. These features allow their identification based on morphology in fluorescently labeled monolayers. We will clarify this criterion in the Methods section of the revised manuscript.

    (Fig 3f-h) The authors conclude that fibroblasts promote directed epithelial cell motility based on cell trajectory analysis. Although they state that this analysis is performed on epithelial monolayers, their tdTomato epithelial population appears sparse in some conditions (control and conditioned media; Fig. S6a). Such variability in cell density may bias measurements of migration directionality at the cell-level, unless a mixed population is being used for tracking. The authors should clarify whether this analysis was indeed conducted on confluent monolayers.

    We thank the reviewer for this comment. For trajectory analysis, we used a mixed population of tdTomato-positive and non-fluorescent epithelial cells in some experiments to facilitate individual cell tracking. Importantly, epithelial monolayers were confluent in all conditions analyzed. We will clarify this in the Methods section.

    (Fig 6b) Their gap closure experimental setup indicates that fibroblasts are cultured on a Matrigel-coated surface, which should already contain abundant laminin and type IV collagen. Thus, it is unclear why type IV collagen is not detected underneath fibroblasts. The authors should explain why this is the case for clarity.

    We thank the reviewer for pointing out this observation. Indeed, fibroblasts are cultured on a Matrigel-coated surface which contains laminin and collagen type IV among many other components. We observed thick collagen-rich structures between the fibroblasts and the epithelia that we atributed, not only to fibroblasts' secreted collagen, but also a rearrengement of the collagen available in the coated surface. We will clarify this in the discussion of the revised version for clarity.

    - Are prior studies referenced appropriately?

    Yes

    - Are the text and figures clear and accurate?

    Mostly. Figures 6d and 6g seem to be duplicated by mistake.

    We thank the reviewer for noting this. We will correct this mistake.

    - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    There are some missing frames in Movie 2. If they are not available, it's okay to include black frames, so that the sequence remains consistent with the timestamps.

    The authors may consider using asterisks as significance indicators instead of reporting precise p-values directly on their plots. Having this format would facilitate visual comparison of statistical significance across conditions.

    Displaying single channels of experiments where co-cultures are used would help to better interpret their data.

    We thank the reviewer for pointing out these issues and for their valuable suggestions. We will correct the errors in the movie and improve the presentation as suggested where possible.

    Reviewer #3 (Significance (Required)):

    - Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    This study provides a valuable contribution to understanding how fibroblasts influence intestinal epithelial migration. The main advance lies in the use of a co-culture system combining organoid-derived intestinal epithelial cells that assemble into a crypt-villus organization with primary intestinal fibroblasts in a 2D gap closure system. This approach allows the authors to examine epithelial-fibroblast interactions in a more physiologically relevant context compared to prior work.

    We thank the reviewer for their positive assessment of the significance of our work.

    - Place the work in the context of the existing literature (provide references, where appropriate).

    Addressed above.

    - State what audience might be interested in and influenced by the reported findings.

    Cell and developmental biology, extracellular matrix biology, tissue regeneration.

    - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Tissue morphogenesis, cell motility, extracellular matrix dynamics.

    We thank the reviewer for their positive assessment and for their suggestions to improve the manuscript.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    • Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    The study by Comelles et al. focuses on how primary intestinal fibroblasts contribute to organoid-derived intestinal epithelial migration in wound healing assays. Using fibroblast-epithelial co-cultures in a 2D in vitro gap closure system, the authors found that direct interaction with fibroblasts drives cohesive and directed migration of intestinal epithelia toward the gap. They further propose that long-range fibroblast alignment promotes the deposition of extracellular matrix (ECM) proteins in an oriented fashion, contributing to directed epithelial migration.

    Major comments:

    • Are the key conclusions convincing?

    Some of the key conclusions of this manuscript are not entirely convincing given the available data. The manuscript would benefit from additional evidence and/or clarifications to support their conclusions. See comments below.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    (Fig 4a) The authors claim that fibroblasts become activated during gap closure as evidenced by the enhanced assembly of a-SMA fibers 24 hours following barrier removal. Yet, long a-SMA fibers are also observed when fibroblasts are cultured in the absence of epithelial cells or barrier removal (Fig. S1b). To support this conclusion, the authors should consider including additional controls to account for potential time-dependent assembly of a-SMA fibers (e.g., fibroblast-only control). (Fig. 5a) The authors conclude that fibroblasts align parallel to the direction of epithelial migration during gap closure. While quantifications are convincing, again, a fibroblast-only control accounting for time-dependent spreading and elongation (as seen in Fig. S1) is missing. Including such a control would strengthen their claim that alignment is specific to the gap closure context rather than a time-dependent phenotype. (Fig 6) The authors claim that fibroblast-derived aligned ECM drives directional epithelial migration. While fibronectin fibers appear scarce and weakly aligned with the direction of migration, laminin and type IV collagen fibers are barely detectable (Fig. 6f). This may reflect a defect in ECM deposition rather than fiber alignment, which contrasts with Fig. S1, where fibroblasts are shown to deposit and assemble laminin and type IV collagen fibers. One possible explanation is that primary fibroblasts were not cultured long enough to allow robust ECM deposition. Alternatively, the observed effect may be specific to fibronectin, which is consistent with fibroblasts being its major source. The authors should revise their interpretation or provide additional evidence to support their current claim. (Fig 6i) The authors propose that the presence of ECM alone within the gap enhances epithelial gap closure compared to empty gap conditions, although gap closure remains less effective than in the presence of primary fibroblasts. From the figure legend and methods, it seems that the decellularized ECM condition is generated using NIH-3T3 fibroblasts cultured for 8 days, whereas the other conditions used primary fibroblasts cultured for 1 day (Fig. 6a-h). This comparison is confounded by differences in cell source and ECM deposition time. If I am misunderstanding this, please clarify, otherwise consider repeating the decellularized ECM condition using primary fibroblasts and matching culture times for a fair comparison. Along these lines, please include images showing that ECM fibers remain intact following decellularization.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    Yes. The additional experiments outlined above would help support the current conclusions of the manuscript, rather than to explore new directions beyond its scope.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    Yes, the additional experiments primarily involve the inclusion of controls and additional immunofluorescence imaging to their existing experimental setups. They should be relatively straightforward to implement (~2-3 months).

    • Are the data and the methods presented in such a way that they can be reproduced?

    Yes.

    • Are the experiments adequately replicated and statistical analysis adequate?

    Overall, yes. But some plot legends should specify the number of replicates analyzed (e.g. Fig. 2b, Fig. 2d, Fig. 3h).

    Minor comments:

    • Specific experimental issues that are easily addressable.

    (Fig. 1c-e) The authors state that intestinal epithelial monolayers exhibit the most effective gap closure when in direct contact with fibroblasts. However, fibroblast-conditioned media and co-cultures show comparable gap closure efficiencies (Fig. 1e). The authors should consider revising this interpretation based on the provided data. (Fig. 3b) The authors suggest that crypt-like epithelial cells undergo migration when grown on fibroblasts, but not in conditioned media alone. This is interesting, but it is not clear how they identify crypt-like cells for tracking. The authors should clarify if crypt-like cells are defined based on markers or inferred from their morphology. (Fig 3f-h) The authors conclude that fibroblasts promote directed epithelial cell motility based on cell trajectory analysis. Although they state that this analysis is performed on epithelial monolayers, their tdTomato epithelial population appears sparse in some conditions (control and conditioned media; Fig. S6a). Such variability in cell density may bias measurements of migration directionality at the cell-level, unless a mixed population is being used for tracking. The authors should clarify whether this analysis was indeed conducted on confluent monolayers. (Fig 6b) Their gap closure experimental setup indicates that fibroblasts are cultured on a Matrigel-coated surface, which should already contain abundant laminin and type IV collagen. Thus, it is unclear why type IV collagen is not detected underneath fibroblasts. The authors should explain why this is the case for clarity.

    • Are prior studies referenced appropriately?

    Yes

    • Are the text and figures clear and accurate?

    Mostly. Figures 6d and 6g seem to be duplicated by mistake. - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    There are some missing frames in Movie 2. If they are not available, it's okay to include black frames, so that the sequence remains consistent with the timestamps. The authors may consider using asterisks as significance indicators instead of reporting precise p-values directly on their plots. Having this format would facilitate visual comparison of statistical significance across conditions. Displaying single channels of experiments where co-cultures are used would help to better interpret their data.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    This study provides a valuable contribution to understanding how fibroblasts influence intestinal epithelial migration. The main advance lies in the use of a co-culture system combining organoid-derived intestinal epithelial cells that assemble into a crypt-villus organization with primary intestinal fibroblasts in a 2D gap closure system. This approach allows the authors to examine epithelial-fibroblast interactions in a more physiologically relevant context compared to prior work.

    • Place the work in the context of the existing literature (provide references, where appropriate). Addressed above.

    • State what audience might be interested in and influenced by the reported findings.

    Cell and developmental biology, extracellular matrix biology, tissue regeneration.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Tissue morphogenesis, cell motility, extracellular matrix dynamics.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Please find enclosed my review comments on the manuscript entitled "Fibroblast alignment coordinates epithelial migration and maintains intestinal tissue integrity" by Jordi Comelles et al. In this manuscript, the authors use a bioengineered epithelial-stromal system composed of organoid-derived intestinal epithelial cells, primary intestinal fibroblasts, and a basement membrane matrix to show that direct physical interactions between fibroblasts and epithelial cells drive a large-scale organization of the fibroblast network. This spatial reorganization, in turn, promotes persistent and oriented migration of epithelial cells, ultimately enabling restoration of the intestinal epithelium in an in vitro gap-closure assay. Overall, while the authors use an elegant in vitro model to study intestinal wound closure, and more specifically the role of fibroblasts in this context, I find this manuscript not suitable for publication in its present form. The data are overinterpreted, the novelty is limited, and the molecular mechanisms underlying WAE-fibroblast interactions are insufficiently addressed.

    Figure 1 - What are the units of the "fraction gap closure" shown in panels d and e? Is it expressed as a percentage? "Actually, epithelial monolayers achieved the most effective gap closure when cultured in direct physical contact with fibroblasts (Figure 1e and Movies 2 and 3)." From the data shown in panels c, d, and e, it appears that fibroblast-conditioned medium alone promotes efficient gap closure, comparable to the + fibroblast condition. Figure 2 - The use of a cell proliferation inhibitor during the gap-closure assay would help determine the contribution of cell proliferation at the migration front. Figure 2f and 2g - Has a dose-dependent effect of PGE2 been tested? Figure 2i - The + fibroblast + EP4i condition (pink) is missing. "This suggests a mechanical or contact-mediated role for fibroblasts in preserving epithelial integrity and promoting coordinated migration beyond their paracrine signaling." While PGE2-EP4 signaling does not appear to be involved in the fibroblast-mediated enhancement of gap-closure efficiency, the conclusion that physical interactions are more important than paracrine effects is overstated. For instance, an experimental condition in which fibroblast-conditioned medium is inactivated (boiling for 5 minutes) would strengthen this conclusion. In addition, inhibition of actomyosin contractility in fibroblasts would be informative. Figure 3 - The data presented here do not convincingly support the dismissal of conditioned medium as a contributing factor. The differences between the + fibroblast-conditioned medium and + fibroblast conditions are modest. In both cases, epithelial cells migrate and gaps close. Figure 4a - "Upon removal of the barrier (t = 0 h), fibroblasts at the epithelial front were small and evenly distributed, with no prominent α-SMA fibers present." Here, fibroblasts are α-SMA positive but not elongated. α-SMA may therefore not be the most appropriate marker. What are the levels of phosphorylated MLC2? These may increase during wound closure. Also, fibroblasts culture promotes aSMA expression, therefore, it may be possible that the fibroblasts used in this assay may not represent the healthy fibroblasts found in vivo. Figure 5 - Fibroblast alignment could also result from paracrine signals secreted by epithelial cells. This possibility should be tested. In summary, this manuscript demonstrates that epithelial cells migrate more efficiently on extracellular matrix proteins deposited and oriented by fibroblasts. This concept is not novel. Identifying the molecular mechanisms governing interactions between WAE and subepithelial fibroblasts would significantly enhance the novelty and impact of this study.

    Significance

    In this manuscript, the authors use a bioengineered epithelial-stromal system composed of organoid-derived intestinal epithelial cells, primary intestinal fibroblasts, and a basement membrane matrix to show that direct physical interactions between fibroblasts and epithelial cells drive a large-scale organization of the fibroblast network. This spatial reorganization, in turn, promotes persistent and oriented migration of epithelial cells, ultimately enabling restoration of the intestinal epithelium in an in vitro gap-closure assay. Overall, while the authors use an elegant in vitro model to study intestinal wound closure, and more specifically the role of fibroblasts in this context, I find this manuscript not suitable for publication in its present form. The data are overinterpreted, the novelty is limited, and the molecular mechanisms underlying WAE-fibroblast interactions are insufficiently addressed.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Background and unknown in the field:

    This study investigates how fibroblast alignment influences the migration of intestinal epithelial cells, contributing to tissue integrity and repair. It is well established that intestinal fibroblasts are important regulators in the tissue through their ability to secrete essential paracrine factors for the epithelium. However, it is less well understood if they also play additional structural, tissue architecture instructing role and how the communication between the fibroblasts and the epithelia is regulated.

    Advance over state of the art:

    Here the authors have set-up an elegant three-component system to investigate this. They have gone beyond the recent advances of culturing intestinal and colonic organoids in 2D (in a manner that preserves- and villus-like organization) and bioengineered epithelial-stromal model comprising organoid-derived intestinal epithelial cells (IECs), primary intestinal fibroblasts, and a basement membrane matrix. Using this model, they have uncovered fibroblasts enhancing the directed and persistent migration of intestinal epithelial cells (IECs). They used scRNAseq to carefully analyse the stromal cell populations present in their co-cultures of primary mouse intestinal subepithelial fibroblasts and organoid-derived intestinal mouse epithelial cells. They observed that this reflected well the stromal cell-type composition as well as the paracrine activity previously reported for these cells in tissue. Using a clever system with Matrigel and an elastomeric barrier, the authors were able to induce non-epithelial gaps in different scenarios (IECs alone or with fibroblasts or with conditioned media) and observe the wound-closure as well as the presence of specific cell types. They observed that the epithelial monolayers showed significant gap closure when in direct contact with fibroblasts compared to controls. Interestingly, the enhanced efficiency of epithelial migration and gap closure, in the presence of fibroblasts, was independent of PGE₂-EP4 signaling and was not due to differences in cell proliferation. Instead, the imaging revealed that the fibroblasts were in direct contact with the epithelium. The authors observed that in the absence of fibroblasts the migration properties of cells in the villus and the crypt regions were dramatically different and the fibroblast presence was necessary to efficiently synchronize these to support gap closure. In addition, the presence of fibroblasts enhanced the directionality of the epithelial cell migration. Detailed imaging and image analyses revealed that gap closure involved activation of the fibroblasts and co-ordinated coalignment of IECs and fibroblasts. They also explored matrix deposition of the fibroblasts during the process and found that they deposited aligned ECM fibers that guide epithelial migration. Mere cell-derived matrix (devoid of live fibroblasts) was able to partially recapitulate the fibroblast-coordinated epithelial migration that the fibroblast generated matrix and its alignment are key contributors to the phenotype.

    Comments:

    This is overall a very interesting and well-written study. The imaging and the image analysis are state-of-the art and the bioengineered model is an exciting advancement over current methods developed by these researchers and others. This study meets all the criteria for a publication in the since that all the experiments seem to be carefully conducted, with appropriate controls and sufficient quantifications and statistics. The claims made by the authors are supported by the data. This is currently suitable to be published as a method/protocol and as a descriptive study uncovering interesting cross-talk and co-dependencies of epithelial and stromal cells during injury repair. There are of course aspects that could improve the study further like more mechanistic insight into the underpinnings of the direct epithelia-fibroblast interaction and its involvement in the directed IEC migration. However, these may be topics to investigate in a future study.

    Significance

    The strengths of the study are the highly in vivo relevant model system that is amendable to imaging and detailed image analysis of distinct cell populations. This may be adapted by others in in the field and has the potential to transform the way cell dynamics in the intestinal epithelium are visualized and investigated in vitro

  5. Version published to 10.1101/2021.05.28.446131 on bioRxiv