Mimicking Senescence Factors to Characterize the Mechanisms Responsible for Hair Regression and Hair Loss: An In Vitro Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comment to manuscript ID: organoids-3575664 titled "Mimicking senescence factors to characterize the mechanisms responsible for hair regression and hair loss”

The authors propose that dermal papilla (DP) spheroid systems provide a valuable platform for studying hair cycle dynamics and for the development of new therapeutic strategies for hair loss. I have some concerns regarding this study.

 

1. Please explain the connection between TGF-β1, FGF-18, and the genes they measured (FGF7, CCND1, and WNT5B).
2. Table 1 needs to describe the RQ study clearly in the results 3.1.
3. There is insufficient explanation regarding retinol's role in the hair cycle.

Author Response

Comment 1: Please explain the connection between TGF-β1, FGF-18, and the genes they measured (FGF7, CCND1, and WNT5B).

Response 1: Transforming Growth Factor Beta 1 (TGF-β1) and Fibroblast Growth Factor 18 (FGF-18) are critical modulators of the hair cycle, particularly in the transition between the anagen (growth), catagen (regression), and telogen (resting) phases. Both factors exert their effects in part by regulating the expression of downstream genes involved in follicular proliferation, differentiation, and quiescence. In the context of hair cycle regulation, FGF7, CCND1, and WNT5B are key genes linked to epithelial cell proliferation and follicle maintenance. FGF7 (also known as keratinocyte growth factor) is primarily i0nvolved in promoting epithelial cell proliferation and anagen induction. CCND1 (Cyclin D1) is a cell cycle regulator essential for G1/S phase progression and is often upregulated during active hair follicle proliferation. WNT5B, a non-canonical Wnt ligand, has been implicated in the regulation of hair follicle morphogenesis and cycling, often associated with promoting follicular quiescence and maintaining telogen. TGF-β1 is known to induce catagen and suppress epithelial proliferation by inhibiting pro-growth signals, which may include downregulation of CCND1 and FGF7, thereby enforcing cell cycle arrest and transitioning follicles out of the anagen phase. Conversely, FGF-18 has been shown to play a role in maintaining the telogen phase and preventing premature anagen entry. Its action is believed to stabilize quiescent states, potentially by modulating WNT5B expression to maintain follicular dormancy, while also repressing cell cycle activators such as CCND1(more ref. and details in the text were added).

Comment 2: Table 1 needs to describe the RQ study clearly in the results 3.1.

Response 2: Stimulation with TGF-β1 accelerated the transition from the anagen (growth) phase to the catagen (regression) phase in dermal papilla (DP) spheroids. This transition was marked by a significant downregulation of FGF7 expression to 0.341, indicating a reduction in proliferative signaling and confirming the initiation of the catagen phase. This observation aligns with in vivo findings where TGF-β1 is known to induce catagen by inhibiting keratinocyte proliferation and promoting  apoptosis . Subsequent exposure of the TGF-β1-treated spheroids to FGF18 for 24 hours further advanced the hair cycle into a telogen-like (resting) phase. This progression was evidenced by significant downregulation of CCND1 (from 0.701 to 0.361) and WNT5B (from 0.971 to 0.601), genes associated with cell cycle progression and follicular maturation, respectively. These findings are consistent with previous studies demonstrating that FGF18 maintains the telogen phase by inducing cell cycle arrest, thereby preventing premature entry into the anagen phase. Collectively, these results suggest that sequential treatment with TGF-β1 and FGF18 effectively models the transition from anagen to catagen and subsequently to telogen in DP spheroids (more ref. and details in the text were added).

Comment 3: There is insufficient explanation regarding retinol's role in the hair cycle.

Response 3: Retinol, through its active metabolite all-trans retinoic acid (RA), plays a pivotal role in regulating hair follicle cycling. RA promotes the transition from telogen to anagen by activating hair follicle stem cells (HFSCs) via the Wnt/β-catenin signaling pathway, thereby enhancing proliferation and hair shaft elongation. In ex vivo organ cultures of androgenetic alopecia (AGA) hair follicles, treatment with 10⁻¹² M RA significantly increased hair shaft growth and maintained follicles in the anagen VI stage, surpassing the effects observed with Minoxidil. However, elevated concentrations of RA can induce premature catagen by upregulating TGF-β2 expression in dermal papilla cells, leading to apoptosis through the Smad2/3 pathway. This biphasic effect underscores the necessity of precise RA dosing to harness its therapeutic potential in hair growth modulation (more ref. and details in the text were added).

Reviewer 2 Report

Comments and Suggestions for Authors

Review for manuscript entitled “Mimicking senescence factors to characterize the mechanisms responsible for hair regression and hair loss: an in vitro study” by Giacomo Masi, Camilla Guiducci and Francesca Rescigno

 

General notes

The manuscripts describes the characterization of an in vitro test system based on spheroids which is commercially available (VitroScreenORA) and can be used to investigate mechanisms of hair cycle, especially with regards to hair cycle regression and treatments to address this condition. The authors conducted experiments to evaluate the response of their models to several treatments with actives that modulate hair aging over time, namely minoxidil, doxorubicin and retinol-based products.

 

Specific notes

• Based on the authors’ affiliation, it looks like the models described in this manuscript are commercially available. If this statement is true, I encourage the authors to detail in the Materials and Methods section on how well established are these models and if they are in any way validated or how are they characterized to ensure their lot to lot reproducibility.
• Are the models available for shipping overseas to other laboratories that might be interested to use them? Do the authors have goals to make the models available for experiments conducted in other labs and if so, could they discuss these goals or future directions in this regard? If the models will be made available to other labs, do the authors intend to conduct shipping studies to ensure their transferability and performance in labs across the world?
• The authors conducted multiple experiments in which the spheroids were treated with various agents like TGF-b1, FGF18, minoxidil, doxorubicin and retinol. It would be useful to include a brief discussion as of how did they determine the right concentrations to use, the right exposure, etc. It is important to know that the toxicity of these compounds was assessed and the right parameters were selected with a demonstrated scientific rationale. Also, what solvents were used to prepare these treatments and were there any solubility (and subsequently bioavailability) concerns?
• Do the authors envision this model being used for studies of large scales investigating actives that could address various hair conditions? Is there any drive from industry or regulators to validate this model by comparison with maybe clinical studies or historical animal data? Given the complexity of the model and length of the assays, do the authors see the use of the model possible financially by labs?
• As a personal scientific curiosity, I would like to ask if the spheroids are pigmented in culture and if so, do they maintain the melanin production? If so, the spheroids may be a consideration for studies focused on pigmentation of the hair (and loss with age) and also for vitiligo studies that target the hair roots as reservoir of stem cells capable to restore the pigmentation of the skin in vitiligo patients. There is no need to include this direction in the paper unless you consider it worthy depending on the performance of the spheroids when it comes to the pigmentation. Thank you.

 

Author Response

Comment 1: Based on the authors’ affiliation, it looks like the models described in this manuscript are commercially available. If this statement is true, I encourage the authors to detail in the Materials and Methods section on how well established are these models and if they are in any way validated or how are they characterized to ensure their lot to lot reproducibility.

Response 1: The model described in our manuscript is not commercially available, although it is possible to commission the assay directly through our facility. While the model’s production is protected by patent, its generation and characterization are fully and transparently detailed in the Materials and Methods section.

Comment 2: Are the models available for shipping overseas to other laboratories that might be interested to use them? Do the authors have goals to make the models available for experiments conducted in other labs and if so, could they discuss these goals or future directions in this regard? If the models will be made available to other labs, do the authors intend to conduct shipping studies to ensure their transferability and performance in labs across the world?

Response 2: No, the model is not available for other labs. We are still considering this option for the future so we will not provide any additional information. 

Comment 3: The authors conducted multiple experiments in which the spheroids were treated with various agents like TGF-b1, FGF18, minoxidil, doxorubicin and retinol. It would be useful to include a brief discussion as of how did they determine the right concentrations to use, the right exposure, etc. It is important to know that the toxicity of these compounds was assessed and the right parameters were selected with a demonstrated scientific rationale. Also, what solvents were used to prepare these treatments and were there any solubility (and subsequently bioavailability) concerns?

Response 3: The concentrations and exposure times for TGF-β1, FGF18, minoxidil, doxorubicin, and retinol were selected based on published studies demonstrating their capacity to modulate distinct phases of the hair‐follicle cycle and on our internal dose-finding screening perfomed by ATP assays (for tissue viability). All primary references supporting these choices are cited in our bibliography; we will additionally incorporate several key citations and details on products preparation in the Materials and Methods section to clarify the scientific rationale.

Comment 4: Do the authors envision this model being used for studies of large scales investigating actives that could address various hair conditions? Is there any drive from industry or regulators to validate this model by comparison with maybe clinical studies or historical animal data? Given the complexity of the model and length of the assays, do the authors see the use of the model possible financially by labs?

Response 4: Sure, the model can be used on a large scale to test different substances associated with the treatment of potential pathological conditions. We can simulate senescence and we can influence the hair cycle. We have conducted several studies for many potential customers interested in evaluating the effectiveness of ingredients or finished products on hair vitality and hair cycle. At the moment we are the first to provide this type of in vitro model and we have not had the opportunity to make comparisons on in vivo models because our aim is not to develop a possible implantable device, but only to provide an alternative testing platform for in vitro tests, especially from the cosmetic point of view (for which European legislation prohibits in vivo experiments on animals). The development of the model itself does not take long time to be produced: about 3 weeks are sufficient before proceeding with treatments.

Comment 5: As a personal scientific curiosity, I would like to ask if the spheroids are pigmented in culture and if so, do they maintain the melanin production? If so, the spheroids may be a consideration for studies focused on pigmentation of the hair (and loss with age) and also for vitiligo studies that target the hair roots as reservoir of stem cells capable to restore the pigmentation of the skin in vitiligo patients. There is no need to include this direction in the paper unless you consider it worthy depending on the performance of the spheroids when it comes to the pigmentation. Thank you.

Response 5: Thank you so much for your kind interest on our tissue systems. Yes, our spheroids represent a simplified dermal-papilla model (the structural compartment of the hair bulb): due to the lack of hair-follicle keratinocytes essential for root and fiber formation, they are unable to produce any hair fiber in culture and therefore remain non-pigmented in culture. We have developed a complete hair follicle model based on hair keratinocytes and dermal papilla fibroblasts: although no visible fiber emerged, immunohistochemical analysis confirmed expression of key keratins and differentiation markers characteristic of follicular maturation. 

Reviewer 3 Report

Comments and Suggestions for Authors

This is an interesting but confusing report by Masi, Guiducci & Rescigno who apparently are employed by VitroScreen srl but state at the end of this manuscript that they have no conflict of interest.

1. Introduction: the opening first 2 paragraphs have NO references
2. line 65 with a stop of growing, please re-write
3. lines 83-84 transition to new in vitro models…..

At this point there are no figures, diagrams, etc. to aid the reader in understanding what the new invitro models look like or what are the components of these systems.  Please provide graphic illustrations…..

4. line 98 is a scalable, correct
5. hypothesis line 104, with no diagrams to follow how is the reader to follow what is being tested?
6. lines 98-109 and 118 – 123 No references
7. Figure 3 difficult to follow
8. Figure 4 difficult to see
9. Discussion lines 517-539; 561-568 and 584-602 No references

10.This discussion section is poor, difficult to follow, a repeat of the results and provides no clear explanation as to the value or conclusions of this study

This significance of the report is unknown based upon the poor presentation, description of the in vitro models, lack of references and unclear discussion of the results.

Comments on the Quality of English Language

 

This report needs major revisions due to poor presentation, lack of references and figures, poor discussion section, etc.

Author Response

Comment 1: Introduction: the opening first 2 paragraphs have NO references

Response 1: more reference were added 

Comments 2: line 65 with a stop of growing, please re-write

Response 2: Done.

Comment 3: lines 83-84 transition to new in vitro models…..At this point there are no figures, diagrams, etc. to aid the reader in understanding what the new invitro models look like or what are the components of these systems. Please provide graphic illustrations…..

Response 3: new illustrations are added. 

Comments 4: line 98 is a scalable, correct.

Response 4: Done.

Comment 5: hypothesis line 104, with no diagrams to follow how is the reader to follow what is being tested? 

Response 5: a new figure was added. 

Comment 6: ines 98-109 and 118 – 123 No references

Response 6: more references were added. 

Comment 7: Figure 3 difficult to follow

Response 7:  Improved description.

Comment 8: Figure 4 difficult to see

Response 8: Improved description and description.

Comment 9: Discussion lines 517-539; 561-568 and 584-602, LINE 561-568 The relative ref is n°59, LINE 584-602

Response 9: additional references were added. 

Comment 10: This discussion section is poor, difficult to follow, a repeat of the results and provides no clear explanation as to the value or conclusions of this study. This significance of the report is unknown based upon the poor presentation, description of the in vitro models, lack of references and unclear discussion of the results.

Response 10: Discussion improved. 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

This version of the manuscript is improved and acceptable for publication as is. I thank the authors for responding in detail to my questions. 

Author Response

Thank you once again for your availability and the professionalism with which you assisted us in revising our manuscript. We truly appreciate your support and constructive feedback throughout the process.
Please do not hesitate to contact us should you have any further comments or suggestions.
Wishing you a pleasant day.