The heterogeneity of dermal mesenchymal cells reproduced in skin equivalents regulates barrier function and elasticity

  1. Shun Kimura
  2. Sachiko Sekiya
  3. Sawa Yamashiro
  4. Tetsutaro Kikuchi
  5. Masatoshi Haga
  6. Tatsuya Shimizu

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

The heterogeneity of dermal mesenchymal cells, including perivascular mesenchymal cells and papillary and reticular fibroblasts, plays critical roles in skin homeostasis. Herein, we present human skin equivalents (HSEs), in which pericytes, papillary fibroblasts, and reticular fibroblasts are spatially organized through autonomous three-cell interactions among epidermal keratinocytes, dermal fibroblasts, and vascular endothelial cells. The replication of dermal mesenchymal cell heterogeneity enhances skin functions, including epithelialization, epidermal barrier formation, and dermal elasticity, enabling in vitro evaluation of drug efficacy using methodologies that are identical to those used in human clinical studies. Furthermore, ascorbic acid-induced epidermal turnover and synthesis of well-aligned extracellular matrix via perivascular niche cells play crucial roles in improving skin barrier function and elasticity. Therefore, HSEs with heterogeneous dermal mesenchymal cells may improve our understanding of the mechanisms underlying skin homeostasis through cell-to-cell communication and serve as a model to animal experiments for developing precision medicine.

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

    Major points

      • The introduction describes the effects of different environmental cues and aging on fibroblast phenotype, but it would be good to note the developmental origins of dermal fibroblasts, which specifies their fate and function (Driskell et al, Nature 2013).* Our response:

    In accordance with the reviewers' suggestions, we have incorporated a summary of prior research regarding the developmental origins of dermal fibroblasts into lines 53–56 of the Introduction.

    • In Fig 2, how do TEWL measurements compare to constructs without an epidermal layer or human skin? It may seem obvious that barrier function would be negligible in these models, but it would be a helpful negative control for interpreting the relative effects of vasculature on barrier function.*

    We appreciate your valuable comments regarding the accurate interpretation of TEWL measurements. Estimated TEWL values for human skin have been reported in a systematic review and meta-analysis by Kottner et al. Specifically, the estimated TEWL (95% CI) for individuals aged 18–64 years varies by anatomical site: 15.4 (13.9–17.0) g/m²h for the right cheek, 6.5 (6.2–6.8) g/m²h for the midvolar right forearm, and 36.3 (29.5–43.1) g/m²h for the right palm. In comparison, the TEWL of our EDV model was 9.68 g/m²h, a value relatively close to that of human skin.

    We also considered measuring TEWL in artificial skin models lacking epidermis. However, we found that such models remain moist due to culture medium, and pressing the measurement probe against them risks water droplets adhering to the sensor and causing damage. Although we recognize the significance of this measurement as a negative control, we refrained from conducting it due to the limitations of the equipment.

    This information has been added to the Results section, lines 178–182.

    • The mechanical measurements in Fig 2 are a nice idea, but it is a bit difficult to interpret without comparison to other conditions (e.g. human skin) or by reporting more universal mechanical parameters (e.g. Young's modulus).*

    We greatly appreciate your insightful comments regarding the interpretation of skin viscoelasticity measurements using the Cutometer. The Cutometer is a device that applies negative pressure to the skin to elevate its surface, allowing for the calculation of biomechanical properties based on the temporal changes in skin displacement. Notably, the R7 parameter—defined as the ratio of immediate retraction after pressure release to the maximum deformation during suction—has been shown to correlate significantly with age.

    In this study, we evaluated HSEs under the same measurement conditions as those used in previous human clinical studies. Accordingly, we have cited past Cutometer data for human skin and discussed the relationship between those findings and our HSE measurements. These revisions have been made to lines 205–215.

    We determined that performing Cutometer measurements on human skin would be impractical due to the ethical committee procedures and associated costs. Although evaluating Young’s modulus using techniques such as AFM to assess the mechanical properties of collagen fibers is a fascinating and informative approach, we have opted not to pursue this analysis due to the substantial time and cost required for sample preparation.

    • The induction of region-specific fibroblast markers is interesting and a bit unexpected since all the fibroblasts came from the same source before seeding into HSEs. The conclusions require additional support from quantification of the IF staining in Fig 3.*

    Our response:

    Thank you for your valuable advice on strengthening the conclusion of our manuscript. We are currently conducting quantitative analysis through manual counting across multiple fields for all mesenchymal cell markers and Vimentin immunostaining data presented in Fig. 3.

    • Likewise, could the authors clarify whether the cells were passaged before seeding into the HSE, and if so, what passage number. Could passaging affect the responses observed? Please add a discussion point about this.*

    Our response:

    For all cell types, passage 4 or 5 cells were utilized for the reconstitution of human skin equivalents (HSE). Indeed, Philippeos et al. demonstrated that while CD39, CD90, and CD36 are detectable in primary CD31⁻CD45⁻Ecad⁻ dermal cells, the expression of CD39 is lost after a single passage. In contrast, CD90 and CD36 remain detectable for up to four passages. These findings underscore the impact of in vitro culture on the depletion of fibroblast marker expression. Since we employed NHDFs that had undergone four to five passages for HSE reconstruction, it is reasonable to assume that these cells had already lost specific fibroblast subpopulations, including CD39⁺ cells. Consistent with this, our scRNA-seq analysis revealed that most fibroblasts cultured in 2D formed an artificial population comprising cells in the S and G2M phases, along with secretory-reticular fibroblasts. Additionally, immunohistochemical analysis confirmed a near-complete absence of CD39⁺, CD90⁺, FAP⁺, NG2⁺, and αSMA⁺ cells in the dermis of both D and DV models, further indicating that serial passaging significantly reduces the expression of markers associated with papillary fibroblasts, reticular fibroblasts, and pericytes. Interestingly, the introduction of vascular endothelial cells into the HSE appears to facilitate a partial restoration of fibroblast heterogeneity in cells passaged four to five times. However, whether this effect can be replicated in more extensively passaged fibroblasts remains to be verified. It is well established that excessive passaging induces cellular senescence, leading to reduced proliferative and differentiation capacities in mesenchymal stem cells. Therefore, it is conceivable that fibroblasts beyond a certain passage number may fail to recapitulate dermal mesenchymal cell heterogeneity, even in the presence of endothelial cells.

    We have added this discussion to the revised manuscript on lines 372-385, 391–397, and 470-471. However, due to the prolonged culture period required, we regret that we are unable to perform the additional validation experiments at this time.

    • The scRNA-seq suggests that the in vitro populations do not discriminate between secretory papillary and pro-inflammatory fibroblasts. Could the authors add some further analysis or discussion regarding this point?*

    Our response:

    We are currently conducting an additional enrichment analysis on fibroblast subpopulations #0, 1, 2, 6, 8, and 11, identified through UMAP analysis integrating HSE and human skin datasets. We believe that this analysis will elucidate the functional characteristics of each in vitro subpopulation and enable us to speculate on the underlying factors contributing to the observed differences from the human skin analysis results.

    • In Fig 6, it will be important to add quantification of epidermal thickness and differentiation marker expression to support the conclusions.*

    Our response:

    Thank you for your valuable advice regarding quantitative analysis. We are currently measuring the thickness of the entire epidermal layer, the CK5-positive cell layer, and the CK10-positive cell layer based on HE-stained and IHC-stained images.

    • A key question is how NP and AA conditions affect the fibroblast populations as this seems to be a key factor in HSE maturation and would then link back to the previous sections. It would be good to stain for fibroblast markers in these samples.*

    Our response:

    We are grateful for your insightful comments, which are crucial for a more precise understanding of the physiological relevance of the NP culture model. In response, we are currently undertaking additional analyses to investigate the expression patterns of dermal mesenchymal markers under both NP and AA conditions.

    • As noted above, the ability of the vasculature to direct differentiation of a common fibroblast population into different phenotypes is one of the key findings of the study. To strengthen these observations, could additional analysis of the transcriptional data be possible. For example, would trajectory analysis potentially show how the different populations are evolving or related? In addition, could the CellChat analysis be performed between the vasculature and the different populations in Fig 5, which are mapped to in vivo populations? This might be a more relevant analysis than the populations in Fig 4.*

    Our response:

    As pointed out by reviewers, we acknowledge that elucidating the process and underlying mechanisms by which fibroblasts, whose heterogeneity is compromised in 2D culture, re-differentiate into distinct dermal mesenchymal subtypes constitutes a critical additional analysis to strengthen our findings. Accordingly, we are currently conducting trajectory analysis using Monocle3. This includes identifying branch points that regulate the differentiation of dermal mesenchymal clusters shown in Fig. 4b, as well as predicting transcription factors and cell signaling pathways playing pivotal roles at those branch points. Furthermore, we are planning a CellChat analysis between vascular endothelial cells and dermal mesenchymal cells. We anticipate that integrating the results of these two analyses will provide valuable insights into the differentiation processes of dermal mesenchymal cells, particularly the induction of perivascular cell differentiation.

    Reviewer #1

    Minor points

      • The abstract states that enabling in vitro evaluation of drug efficacy using methodologies that are identical to those used in human clinical studies. This seems to be an over interpretation of the study and not well supported by the data. Please consider revising or removing.*

    Our Response:

    Upon thorough consideration, we have deleted the statements that may be regarded as exaggerated (line 26-28 and 346-348).

    • Check referencing formatting in lines 118-121*

    Our Response:

    We appreciate your attention to the reference format error. The necessary revisions have been completed.

    Reviewer #2 Major comments:

      • Despite its strengths, the study has several limitations that warrant further investigation. The authors describe a "senescent-like" phenotype under nutrient-poor (NP) conditions, yet do not provide direct evidence of cellular senescence using canonical markers such as SA-β-gal staining, p16^INK4a or p21 expression, or SASP profiling-weakening their aging-related conclusions.*

    Our Response

    Thank you for your valuable advice, which has helped clarify the physiological phenomena modeled by the NP condition. We are planning additional experiments involving histological analysis, including SA-β-gal staining and the detection of p16^INK4a and/or p21.

    • The 500 μM dose of ascorbic acid (AA), while within the reported range for skin models, is at the higher end compared to commonly used concentrations (100-300 μM) and lacks justification via dose/response data. Normal physiological levels and changes in aging dermis should be referenced in discussion. AA is also an additive in their standard HSE media, but this was not sufficiently emphasized to draw attention. Would its removal from the baseline media make a difference?*

    Our Response

    We sincerely appreciate the important comment regarding the rationale behind the ascorbic acid concentration used in the culture medium. As Reviewer 3 rightly pointed out, concentrations around 100–300 μM are commonly employed in general in vitro assays. In our artificial skin model, we opted for a concentration of 500 μM AA in the growth medium based on two considerations: (1) the model contains a high cell density of approximately 4 × 10⁶ cells immediately after reconstruction, which is expected to result in substantial AA consumption, and (2) AA is not sufficiently stable in culture medium. Given the relatively long medium exchange interval of 48–72 hours, we deemed it necessary to maintain a certain AA level throughout this period. While no rigorous dose–response validation has been conducted, we have confirmed that this concentration does not induce toxicity or abnormalities in skin morphogenesis.

    As part of the revision, we considered revisiting the basal medium formulation; however, due to the significant time and resource demands, we have decided to forgo further optimization at this stage.

    As described on lines 307–311, the NP medium was formulated to evaluate the potential impact of age-related declines in plasma component transport. We apologize for any confusion regarding the relationship between the HSE growth medium and the NP medium. In response to the reviewer’s suggestion, we have added clarifying explanations and cautionary notes regarding the composition and rationale of these two media in both the Results and Methods sections (line 307-311 and 634-636).

    • Mechanistically, fibroblast heterogeneity is attributed to keratinocyte and vascular signals, but the signaling pathways involved (e.g., Wnt, TGF-β, VEGF) are not directly examined. Validating which paracrine factors (VEGF, PDGF, LAMA5, KGF) are mediating fibroblast transitions using inhibitors or RNA profiling could shed more light.*

    Our response:

    As pointed out by reviewers, we acknowledge that elucidating the process and underlying mechanisms by which fibroblasts, whose heterogeneity is compromised in 2D culture, re-differentiate into distinct dermal mesenchymal subtypes constitutes a critical additional analysis to strengthen our findings. Accordingly, we are currently conducting trajectory analysis using Monocle3. This includes identifying branch points that regulate the differentiation of dermal mesenchymal clusters shown in Fig. 4b, as well as predicting transcription factors and cell signaling pathways playing pivotal roles at those branch points. Furthermore, we are planning a CellChat analysis between vascular endothelial cells and dermal mesenchymal cells. We anticipate that integrating the results of these two analyses will provide valuable insights into the differentiation processes of dermal mesenchymal cells, particularly the induction of perivascular cell differentiation. We fully recognize that validation using specific inhibitors is crucial to substantiate the mechanisms suggested by the scRNA-seq analysis. However, given that the reconstruction and reanalysis of the artificial skin model requires more than three months, we have decided not to include these experiments in the current revision and instead consider them as important subjects for future investigation.

    Minor comments: 1. The role of pericytes is also underexplored; while their presence is confirmed, functional assays or transcriptomic analyses to elucidate their contribution to ECM remodeling or vascular stability are not fully explored. The origin of pericyte-like cells remains uncertain without lineage tracing or barcoding to distinguish whether they derive from fibroblasts, endothelial cells, or culture artifacts. Since they observe induced differentiation of fibroblast-like cells in 3D culture, it would be compelling to reconstruct differentiation trajectories (pseudotime analysis) from progenitor states to papillary/reticular/pericyte-like states from their scRNAseq data.

    Our respnse:

    This point will be addressed and validated through our response to Major Comment 3 from Reviewer #2.

    • Although AA enhanced collagen production and elasticity in the vascularized EDV model, the lack of response in the ED model is not addressed mechanistically.*

    Our response

    We have planned additional experiments to examine two hypotheses regarding the mechanism underlying the improved responsiveness of the EDV model to AA. The first hypothesis posits that the behavior of ascorbic acid uptake in the cells constituting the EDV model differs from that in the ED model. To investigate this, we plan to analyze the expression patterns of transporter genes potentially involved in the uptake and efflux of ascorbic acid, such as SVCT1 (SLC23A1), SVCT2 (SLC23A2), GLUT1 (SLC2A1), GLUT3 (SLC2A3), GLUT4 (SLC2A4), and MRP4, using scRNA-seq data. The second hypothesis suggests that the absence of bFGF signaling and low FBS treatment under NP conditions may affect subpopulations of dermal mesenchymal cells in the HSEs. To test this, we plan to analyze the expression patterns of dermal mesenchymal cell markers by IHC under NP and AA conditions, following the same approach as shown in Fig. 3.

    • The omission of immune cells which are key players in skin aging and homeostasis could increase physiological relevance of the model.*

    Our response:

    As rightly noted by Reviewer 2, immune cells are integral to skin aging and the maintenance of tissue homeostasis, underscoring the necessity of incorporating them into future research models. Nonetheless, the primary aim of the present study is to elucidate the influence of vascular endothelial cells on dermal mesenchymal cell heterogeneity and to establish an in vitro research model specifically addressing this heterogeneity, with particular emphasis on perivascular cells. Accordingly, we would prefer to consider the analysis of immune cells as a subject for future investigation.

    • The exclusive use of standard HUVECs may not fully capture the behavior of tissue-specific microvascular endothelial cells, potentially limiting the fidelity of the vascular niche.*

    In this study, we opted to use HUVECs as vascular endothelial cells due to their relative ease of expansion in culture. Consequently, we acknowledge the potential limitation in fully recapitulating the functions of tissue-specific endothelial cells. To address this concern, we have revised and expanded the Discussion section on lines 352–356.

    Reviewer #3 Major comments:

      • Are the key conclusions convincing? The core claim-that tricellular interactions recapitulate dermal mesenchymal heterogeneity and enhance skin functionality-is well-supported by histology, immunohistochemistry, functional assays (TEWL, elasticity), and scRNA-seq.
    2. Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? The assertion that HSEs enable "identical" methodology to clinical studies (p. 2, line 29) is exaggerated. While elasticity was measured via Cutometer (used clinically), the model lacks immune/neural components and long-term stability for full translational equivalence.* Our Response:

    Upon thorough consideration, we have deleted the statements that may be regarded as exaggerated (line 26-28 and 346-348).

    • 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. Adequacy of Experimental Evidence & Need for Additional Experiments: No essential control appears to be missing: the authors include conditions {plus minus}ascorbic acid and {plus minus}vascular cells to isolate those effects. One could suggest a few additional experiments to further bolster the conclusions, but they are not strictly required for the main message. For example, to pinpoint the contribution of each mesenchymal subset, the authors could engineer HSE variants lacking one component at a time (omit pericytes or use only papillary vs. only reticular fibroblasts) to see how each omission affects barrier or elasticity. This would directly confirm each cell type's role. However, such experiments may be technically involved (especially isolating pure papillary vs. reticular fibroblast populations and ensuring viability in 3D culture) and might be beyond the scope of a single study. Another possible extension could be mechanistic assays, such as examining specific molecular signals: e.g., testing if blocking known paracrine factors from pericytes or fibroblast subsets diminishes the observed improvements. Given that pericytes can secrete laminin-511 and other factors that promote keratinocyte growth, the authors might, in future work, explore whether such factors mediate the enhanced epidermal proliferation seen with the vascularized HSE. Overall, the current data are sufficiently convincing that additional experiments are not absolutely necessary for publication.
    1. 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-*

    Our response

    We are deeply grateful for the reviewer’s constructive feedback. As rightly pointed out, cell ablation and mechanistic assays utilizing signaling inhibitors to assess the contribution of individual mesenchymal subsets are indispensable for reinforcing our findings and claims. However, as the reviewer has also indicated, these experiments would require no less than four months to complete. Consequently, we have opted to forgo high-cost additional experiments such as the optimization of HSE construction protocols and inhibitor-based assays. Instead, we are proactively conducting mechanism-oriented analyses using our existing scRNA-seq and histological datasets. Specifically, we are currently implementing an integrated approach combining Monocle3 and CellChat to pinpoint critical branch points in dermal mesenchymal cell differentiation and to elucidate the signaling pathways orchestrating these bifurcations.

    • Are the experiments adequately replicated and statistical analysis adequate? The manuscript's data are presented in a manner that generally supports reproducibility. The authors state that all data are presented as "mean {plus minus} SD" (Methods, p.36). This is acceptable and clearly reported. However, I suggest that the authors consider using mean {plus minus} SEM for specific datasets where the primary goal is to assess statistical significance between groups - for example, for the Ki67-positive cell proliferation data (Fig. 6c) - as SEM better reflects the precision of the group mean for inferential comparisons. In contrast, for functional measures that inherently exhibit biological variation across samples (e.g., TEWL, skin elasticity), using mean {plus minus} SD remains fully appropriate, as SD reflects true inter-sample variability. To improve clarity and reproducibility, I encourage the authors to briefly state in the Methods or figure legends why SD or SEM is used in each case, in line with best practice guidelines.*

    Our Response:

    We appreciate your guidance regarding appropriate statistical analysis and data presentation. We planned to revise the depiction of error margins in accordance with best practice guidelines.

    * *

    Reviewer #3 Minor comments:*

    1. For Figure 4e, it would be helpful if the authors could clarify in the figure legend or Methods whether the heatmap shows log-normalized expression values (as derived from the Seurat object) or z-scored expression across cells or samples. This distinction affects the interpretation of relative versus absolute expression levels of the collagen and elastic fiber-related genes, which are central to the study's conclusions about ECM remodeling.*

    Our response:

    Thank you for pointing out the inconsistency in data representation. We have revised the manuscript to clearly indicate that Fig. 4e presents the Z-score normalized average expression levels.

    * *

    • Typos: "factr" → "factor" (p. 16, line 244); "severl" → "several" (p. 22, line 367).

    Our response

    Thanks for pointing out the typo, we have corrected it.

    *Reviewer #4 *

    Minor Points:

      • The human skin control in Fig. 1c seems thinner than normal and would suggest that the ED and EDV models are hyperproliferative. Replacing the control with one that shows normal thickness would prevent incorrect conclusions of the data.* Our response:

    In accordance with the reviewer’s suggestion, the display area of the human skin image in Fig. 1c has been modified.

    KI67 and TEWL readings for human skin as controls for Fig. 2b-c would help gauge how the organoids perform and whether they are abnormal. What is the elasticity index for facial sagging?

    Thank you for your valuable advice, which has deepened our understanding of the evaluation results of HSEs. We are currently planning and conducting an additional analysis by including the quantification of Ki67-positive cells in human skin samples. Regarding the assessment of skin barrier and viscoelasticity using TEWL and Cutometer measurements, we have reffered data from previous clinical studies and added an explanation of the functional differences between HSEs and human skin.

    • Ascorbic acid utilizes SLC23A1 and SLC23A2 to transport across cell membranes. Are their expression more pronounced in cluster 14 fibroblasts? This would help connect the scRNA-seq data to the ascorbic acid experiments.

    Our response:

    We appreciate the valuable suggestions provided to investigate the mechanisms underlying the altered VC responsiveness observed in the EDV model. We plan to analyze the expression patterns of transporter genes potentially involved in the uptake and efflux of ascorbic acid, such as SVCT1 (SLC23A1), SVCT2 (SLC23A2), GLUT1 (SLC2A1), GLUT3 (SLC2A3), GLUT4 (SLC2A4), and MRP4, using scRNA-seq data.

    There seems to be quite a bit of variability between replicant immunostains, in particular, vimentin in Fig. 3. Can the authors discuss this variability and whether any of the HSE organoid combinations reduced this variability?

    Our response:

    Thank you for your comments regarding the immunostaining. A reanalysis of the data, including newly acquired immunostaining images during the revision process, is planned.

    • Please provide number of replicates throughout figure legends.*

    Our response:

    Thank you for your valuable advice. We have added the number of replicates to all figure legends.

    • Line 148 states "E and EV models were transparent and extremely soft", should read "E and ED models".*

    Our response:

    The photographic data for the EV and ED models in Fig. 1b was incorrect and has therefore been corrected. We sincerely apologize for our oversight. As it was actually the E and EV models that appeared transparent, the description in the text remains unchanged.

    • Line 150-151 states "In the E and EV models, an abnormal epidermis lacking a basal cell layer formed". The Krt5 staining in Figure 2 clearly shows a basal cell layer in these models, albeit abnormal. Stating that this the abnormal epidermis displayed a disrupted basal cell layer or columnar shape of basal cells were disrupted is more appropriate. In addition, these results do not show "crosstalk between NHEKs and NHDFs is essential for epithelialization" as the E and EV organoid models show epithelial stratification.*

    Our response:

    We sincerely appreciate your insightful guidance regarding the accurate presentation of the histological analysis results. Accordingly, we have revised lines 154–156 in the Results section in line with your recommendations.

  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 #4

    Evidence, reproducibility and clarity

    The manuscript by Kimura et al. define how epidermal morphogenesis in human skin equivalents (HSE) differ by combining vascular endothelial cells, epidermal keratinocytes, and dermal fibroblasts using staining and single-cell RNA-sequencing (scRNA-seq). The three cell system (EDV) displayed higher levels of Ki67+ cells, decreased levels of TEWL, and higher elasticity in comparison to the keratinocyte and fibroblast HSE system (ED). The overall structural morphology between the two systems is quite similar, though the expression of cytokeratin markers varies. EDV organoids specifically express COL1 and COL4 collagen markers surrounding the blood vessels. VEGF-VEGFR1 signaling between endothelia-fibroblasts seems to be pronounced in the EDV organoids according to scRNA-seq, suggesting active signaling between these two cell types. And ascorbic acid appeared to help nutrient poor ED and EDV organoids proliferate compared to controls. This work is well detailed and interesting, helping to define how endothelial cells function to make HSE organoids more faithfully mimic in vivo human skin. Only minor clarifications detailed below are needed.

    1. The human skin control in Fig. 1c seems thinner than normal and would suggest that the ED and EDV models are hyperproliferative. Replacing the control with one that shows normal thickness would prevent incorrect conclusions of the data.
    2. KI67 and TEWL readings for human skin as controls for Fig. 2b-c would help gauge how the organoids perform and whether they are abnormal. What is the elasticity index for facial sagging?
    3. Ascorbic acid utilizes SLC23A1 and SLC23A2 to transport across cell membranes. Are their expression more pronounced in cluster 14 fibroblasts? This would help connect the scRNA-seq data to the ascorbic acid experiments.
    4. There seems to be quite a bit of variability between replicant immunostains, in particular, vimentin in Fig. 3. Can the authors discuss this variability and whether any of the HSE organoid combinations reduced this variability?
    5. Please provide number of replicates throughout figure legends.
    6. Line 148 states "E and EV models were transparent and extremely soft", should read "E and ED models".
    7. Line 150-151 states "In the E and EV models, an abnormal epidermis lacking a basal cell layer formed". The Krt5 staining in Figure 2 clearly shows a basal cell layer in these models, albeit abnormal. Stating that this the abnormal epidermis displayed a disrupted basal cell layer or columnar shape of basal cells were disrupted is more appropriate. In addition, these results do not show "crosstalk between NHEKs and NHDFs is essential for epithelialization" as the E and EV organoid models show epithelial stratification.

    Significance

    This work is well detailed and interesting, helping to define how endothelial cells function to make HSE organoids more faithfully mimic in vivo human skin. Only minor clarifications detailed below are needed.

  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 #3

    Evidence, reproducibility and clarity

    The study develops a tricellular human skin equivalent (HSE) model incorporating epidermal keratinocytes (NHEKs), dermal fibroblasts (NHDFs), and vascular endothelial cells (HUVECs). This model autonomously organizes pericytes, papillary fibroblasts, and reticular fibroblasts, mimicking in vivo dermal mesenchymal heterogeneity. The EDV model (all three cell types) demonstrates enhanced epidermal barrier function (reduced TEWL), dermal elasticity, collagen deposition, and vascular organization compared to simpler models. Single-cell RNA-seq confirms the emergence of pericyte-like and fibroblast subpopulations resembling in vivo counterparts. Nutrient-poor (NP) culture replicates aging phenotypes (reduced proliferation, barrier dysfunction, disordered collagen), rescued by ascorbic acid (AA), highlighting vascular cells' role in skin homeostasis. However, several key methodological clarifications (e.g., heatmap normalization, statistical reporting), more precise qualification of certain claims, and enhanced contextualization within the literature are needed before the work can be considered suitable for publication; I therefore recommend major revision.

    Major comments:

    1. Are the key conclusions convincing?
      The core claim-that tricellular interactions recapitulate dermal mesenchymal heterogeneity and enhance skin functionality-is well-supported by histology, immunohistochemistry, functional assays (TEWL, elasticity), and scRNA-seq.
    2. Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? The assertion that HSEs enable "identical" methodology to clinical studies (p. 2, line 29) is exaggerated. While elasticity was measured via Cutometer (used clinically), the model lacks immune/neural components and long-term stability for full translational equivalence.
    3. 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. Adequacy of Experimental Evidence & Need for Additional Experiments: No essential control appears to be missing: the authors include conditions {plus minus}ascorbic acid and {plus minus}vascular cells to isolate those effects. One could suggest a few additional experiments to further bolster the conclusions, but they are not strictly required for the main message. For example, to pinpoint the contribution of each mesenchymal subset, the authors could engineer HSE variants lacking one component at a time (omit pericytes or use only papillary vs. only reticular fibroblasts) to see how each omission affects barrier or elasticity. This would directly confirm each cell type's role. However, such experiments may be technically involved (especially isolating pure papillary vs. reticular fibroblast populations and ensuring viability in 3D culture) and might be beyond the scope of a single study. Another possible extension could be mechanistic assays, such as examining specific molecular signals: e.g., testing if blocking known paracrine factors from pericytes or fibroblast subsets diminishes the observed improvements. Given that pericytes can secrete laminin-511 and other factors that promote keratinocyte growth, the authors might, in future work, explore whether such factors mediate the enhanced epidermal proliferation seen with the vascularized HSE. Overall, the current data are sufficiently convincing that additional experiments are not absolutely necessary for publication.
    4. 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
    1. Are the data and the methods presented in such a way that they can be reproduced? Yes
    2. Are the experiments adequately replicated and statistical analysis adequate? The manuscript's data are presented in a manner that generally supports reproducibility. The authors state that all data are presented as "mean {plus minus} SD" (Methods, p.36). This is acceptable and clearly reported. However, I suggest that the authors consider using mean {plus minus} SEM for specific datasets where the primary goal is to assess statistical significance between groups - for example, for the Ki67-positive cell proliferation data (Fig. 6c) - as SEM better reflects the precision of the group mean for inferential comparisons. In contrast, for functional measures that inherently exhibit biological variation across samples (e.g., TEWL, skin elasticity), using mean {plus minus} SD remains fully appropriate, as SD reflects true inter-sample variability. To improve clarity and reproducibility, I encourage the authors to briefly state in the Methods or figure legends why SD or SEM is used in each case, in line with best practice guidelines.

    Minor comments:

    1. For Figure 4e, it would be helpful if the authors could clarify in the figure legend or Methods whether the heatmap shows log-normalized expression values (as derived from the Seurat object) or z-scored expression across cells or samples. This distinction affects the interpretation of relative versus absolute expression levels of the collagen and elastic fiber-related genes, which are central to the study's conclusions about ECM remodeling.
    2. Typos: "factr" → "factor" (p. 16, line 244); "severl" → "several" (p. 22, line 367).

    Significance

    The study innovatively reconstructs dermal mesenchymal heterogeneity using commercially available cells and autonomous tricellular interactions, bypassing costly cell-sorting approaches. This democratizes complex HSE models for broader labs. This study demonstrates that vascularization is critical not only for nutrient supply but for instructing fibroblast/pericyte differentiation and ECM organization. The NP+AA paradigm (Fig. 6) offers a facile in vitro model for skin aging interventions, highlighting AA's efficacy via perivascular mechanisms.

    Audience: Tissue engineers, dermatologists, cosmetic/pharma researchers (anti-aging screening), and developmental biologists studying mesenchymal niche regulation.

    Placement in existing literature: Recent advances in skin tissue engineering have highlighted the importance of dermal fibroblast heterogeneity in skin homeostasis and regeneration. Single-cell transcriptomic studies (Tabib et al., J Invest Dermatol 2018; Solé-Boldo et al., Commun Biol 2020) have established that papillary and reticular fibroblasts exhibit distinct gene expression and functional roles. Prior engineered skin models incorporating fibroblast subtypes (Moreira et al., Biomater Sci 2023) or pericytes (Paquet-Fifield et al., J Clin Invest 2009) demonstrated improvements in vascularization or epidermal differentiation. However, a unified 3D human skin equivalent integrating vascular cells, pericytes, and spatially organized fibroblast subpopulations has not been systematically achieved. The present work by Kimura et al. advances the field by demonstrating that autonomous interaction among keratinocytes, endothelial cells, pericytes, and heterogeneous fibroblasts significantly enhances both barrier function and dermal elasticity, thus bringing engineered skin models closer to physiological skin. This addresses a key gap between prior single-cell descriptive studies and functional tissue engineering.

    Define your field of expertise with a few keywords: experimental dermatology, skin cancer, tissue engineering and 3D skin models, cell biology, tumor microenvironment, and the skin microbiome and barrier function.

  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 #2

    Evidence, reproducibility and clarity

    In this study, the authors present a novel and well-executed approach to reconstructing human skin equivalents (HSEs) that more faithfully replicate the functional complexity of native skin by incorporating the natural heterogeneity of dermal mesenchymal cells, including spatially organized pericytes, papillary fibroblasts, and reticular fibroblasts. Through autonomous interactions among keratinocytes, fibroblasts, and vascular endothelial cells, the fully tricellular EDV model emerged as the most functionally complete among seven engineered HSE variants, demonstrating enhanced epithelialization, barrier integrity, dermal elasticity, and angiogenic architecture. The study's strengths lie in its realistic aging induction via nutrient deprivation by mimicking aspects of vascular insufficiency in the papillary dermis, and its integration of diverse and rigorous evaluation methods, including histological and molecular analyses (Ki67, ECM markers), barrier function (TEWL), and mechanical testing. Notably, ascorbic acid treatment improved epidermal turnover and extracellular matrix organization, particularly through effects on perivascular niche cells, highlighting its translational relevance for anti-aging interventions. Although the EDV model showed superior elasticity via suction testing, more comprehensive mechanical characterization and longitudinal ECM analysis could further elucidate how mesenchymal heterogeneity supports biomechanical resilience. Overall, this work underscores the importance of multicellular crosstalk in skin physiology and positions the EDV model as a robust in vitro platform with high relevance for regenerative medicine, aging research, and therapeutic screening, offering the potential to eliminate animal models in skin biology.

    Major comments:

    Despite its strengths, the study has several limitations that warrant further investigation. The authors describe a "senescent-like" phenotype under nutrient-poor (NP) conditions, yet do not provide direct evidence of cellular senescence using canonical markers such as SA-β-gal staining, p16^INK4a or p21 expression, or SASP profiling-weakening their aging-related conclusions.

    The 500 μM dose of ascorbic acid (AA), while within the reported range for skin models, is at the higher end compared to commonly used concentrations (100-300 μM) and lacks justification via dose/response data. Normal physiological levels and changes in aging dermis should be referenced in discussion. AA is also an additive in their standard HSE media, but this was not sufficiently emphasized to draw attention. Would its removal from the baseline media make a difference? Mechanistically, fibroblast heterogeneity is attributed to keratinocyte and vascular signals, but the signaling pathways involved (e.g., Wnt, TGF-β, VEGF) are not directly examined. Validating which paracrine factors (VEGF, PDGF, LAMA5, KGF) are mediating fibroblast transitions using inhibitors or RNA profiling could shed more light.

    Minor comments:

    The role of pericytes is also underexplored; while their presence is confirmed, functional assays or transcriptomic analyses to elucidate their contribution to ECM remodeling or vascular stability are not fully explored. The origin of pericyte-like cells remains uncertain without lineage tracing or barcoding to distinguish whether they derive from fibroblasts, endothelial cells, or culture artifacts. Since they observe induced differentiation of fibroblast-like cells in 3D culture, it would be compelling to reconstruct differentiation trajectories (pseudotime analysis) from progenitor states to papillary/reticular/pericyte-like states from their scRNAseq data. Although AA enhanced collagen production and elasticity in the vascularized EDV model, the lack of response in the ED model is not addressed mechanistically. The omission of immune cells which are key players in skin aging and homeostasis could increase physiological relevance of the model. The exclusive use of standard HUVECs may not fully capture the behavior of tissue-specific microvascular endothelial cells, potentially limiting the fidelity of the vascular niche.

    Significance

    This study presents a robust and innovative approach to human skin equivalent (HSE) reconstruction by integrating pericyte-like and endothelial cells with dermal fibroblast subtypes, using only commercially available cell types. A key strength lies in its ability to recapitulate aspects of in vivo fibroblast heterogeneity, including papillary, reticular, and perivascular populations, and to demonstrate functional consequences on tissue architecture, barrier integrity, ECM dynamics, and mechanical properties under aging-like, nutrient-poor conditions. The spontaneous emergence of a pericyte-like population without relying on freshly isolated primary pericytes or complex sorting protocols represents a methodological advance that increases the model's accessibility and scalability. Furthermore, the use of ascorbic acid to reverse aging-associated features in a vascular cell-dependent manner adds a compelling functional dimension, linking cell composition with therapeutic response.

    Compared to existing models that either lack vascular cell compartments or do not account for dermal fibroblast heterogeneity, this study fills an important gap at the intersection of skin aging, vascular biology, and mesenchymal-epithelial interactions. The advance is both conceptual by elucidating the role of vascular and perivascular cells in shaping fibroblast identity and function and methodological, through the generation of a human skin model that approximates in vivo complexity without requiring animal models or ethically limited human tissue. The work will be of strong interest to basic science researchers in dermatology, tissue engineering, and aging, and has potential influence in regenerative medicine, cosmetic science, and drug screening, especially in the context of skin repair and anti-aging therapies. The audience is broad but most relevant to specialized communities in skin biology, mesenchymal cell biology, vascular biology, and organoid modeling, and may also attract attention from those developing non-animal testing platforms in applied and translational settings.

    As a reviewer with expertise in inflammatory skin disease modeling using both animal systems and 3D organoid cultures, I bring a critical understanding of how cellular composition, microenvironmental cues, and co-culture conditions influence skin physiology and pathology. My interest in developing advanced co-culture systems to recapitulate human skin complexity positions me well to evaluate the relevance, innovation, and translational potential of this vascularized HSE model. I am especially qualified to assess the biological fidelity of the reconstructed skin architecture, the functional outcomes of introducing pericyte-like populations, and the implications of nutrient deprivation and ascorbic acid supplementation as aging-relevant perturbations.

  5. 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

    The manuscript by Kimura et al investigates the role of different cell populations in the development of human skin equivalents (HSEs). The observe that the addition of vascular endothelial cells to HSEs improves epidermal differentiation and barrier function, alongside differentiation of fibroblasts into papillary, reticular, and pericyte like mesenchymal cells. The authors also use single-cell transcriptomics to characterise the gene signatures and putative signalling pathway in the fibroblasts. Finally, the authors use nutrient poor medium and ascorbic acid to modulate HSE develop.

    One of the most significant questions arising from the findings is how the presence of vasculature can induce differentiation of fibroblasts from a common population, especially given that previous studies have shown that fibroblast identity is programmed during development. Some specific comments and suggestions for improving the manuscript are listed below.

    Major points:

    1. The introduction describes the effects of different environmental cues and aging on fibroblast phenotype, but it would be good to note the developmental origins of dermal fibroblasts, which specifies their fate and function (Driskell et al, Nature 2013).
    2. In Fig 2, how do TEWL measurements compare to constructs without an epidermal layer or human skin? It may seem obvious that barrier function would be negligible in these models, but it would be a helpful negative control for interpreting the relative effects of vasculature on barrier function.
    3. The mechanical measurements in Fig 2 are a nice idea, but it is a bit difficult to interpret without comparison to other conditions (e.g. human skin) or by reporting more universal mechanical parameters (e.g. Young's modulus).
    4. The induction of region-specific fibroblast markers is interesting and a bit unexpected since all the fibroblasts came from the same source before seeding into HSEs. The conclusions require additional support from quantification of the IF staining in Fig 3.
    5. Likewise, could the authors clarify whether the cells were passaged before seeding into the HSE, and if so, what passage number. Could passaging affect the responses observed? Please add a discussion point about this.
    6. The scRNA-seq suggests that the in vitro populations do not discriminate between secretory papillary and pro-inflammatory fibroblasts. Could the authors add some further analysis or discussion regarding this point?
    7. In Fig 6, it will be important to add quantification of epidermal thickness and differentiation marker expression to support the conclusions.
    8. A key question is how NP and AA conditions affect the fibroblast populations as this seems to be a key factor in HSE maturation and would then link back to the previous sections. It would be good to stain for fibroblast markers in these samples.
    9. As noted above, the ability of the vasculature to direct differentiation of a common fibroblast population into different phenotypes is one of the key findings of the study. To strengthen these observations, could additional analysis of the transcriptional data be possible. For example, would trajectory analysis potentially show how the different populations are evolving or related? In addition, could the CellChat analysis be performed between the vasculature and the different populations in Fig 5, which are mapped to in vivo populations? This might be a more relevant analysis than the populations in Fig 4.

    Minor points:

    1. The abstract states that enabling in vitro evaluation of drug efficacy using methodologies that are identical to those used in human clinical studies. This seems to be an over interpretation of the study and not well supported by the data. Please consider revising or removing.
    2. Check referencing formatting in lines 118-121

    Significance

    Overall, the study represents a systematic analysis of how vasculature contributes to skin model development, and the impact on fibroblast differentiation is an interesting observation. It would have been more impactful if some of the pathways and genes were followed up with mechanistic studies, but the findings are still useful to the field. Likewise, further insight into exactly how the vasculature regulates fibroblast phenotype would add to the impact as this is an unexpected but important finding.

  6. Version published to 10.1101/2024.12.08.627431 on bioRxiv