Neuropeptide Substance P Modulates Stem Cell Fate to Promote Wound Healing and Epidermal Stratification via Asymmetric Divisions

Bioengineering Basic Medical Sciences Cell Biology Biochemistry Life Sciences Medicine
Substance P stem cell fate wound healing epidermal stratification asymmetric division sensory innervation re-epithelialization

Neuropeptide Substance P Regulates Stem Cell Division to Promote Wound Healing and Epidermal Stratification: An In-depth Interpretation of the Latest Study by Khalifa et al.

Research Background and Scientific Questions

Wound healing following skin injury has long been a central focus in both clinical and basic medical sciences. With aging, diabetes, and various neurological diseases, wound healing is frequently impaired, posing significant threats to patient health. In recent years, skin stem cells (SCs), their differentiation mechanisms, and neural regulation in wound repair have become research hotspots. Although clinical observations have repeatedly shown that sensory nerve damage markedly delays wound healing, the specific mechanisms by which neuroregulatory factors—especially neuropeptides—influence skin stem cell behavior and epidermal structural remodeling remain unclear.

This study centers on the regulatory mechanisms by which the neuropeptide Substance P (SP) affects skin stem cell fate and differentiation/stratification. Earlier studies have indicated that loss of sensory innervation not only delays wound healing but also reduces SP levels, while exogenous SP supplementation can markedly accelerate re-epithelialization. In the epidermis, stem cells expand or differentiate through symmetric division (producing two stem cells), asymmetric division (producing one stem cell and one differentiated daughter cell), and committed progenitor (CP) divisions. However, how nerve injury and SP changes affect different cell division types—especially the specific regulatory effects on symmetric/asymmetric SC divisions—lacks in-depth evidence. Breakthroughs in this field are not only of fundamental biological significance but also provide theoretical support and intervention targets for treating chronic, non-healing wounds (such as diabetic ulcers, chronic wounds in the elderly).

Paper Source and Author Information

This original research paper is titled “neuropeptide substance p alters stem cell fate to aid wound healing and promote epidermal stratification through asymmetric stem cell divisions.” The author team includes A Khalifa, T Xiao, B Abegaze, T Weisenberger, A Charruyer, Samia Sanad, Taher Abuelnasr, SW Kashem, M Fassett, and R Ghadially. The authors are from the Department of Zoology, Zagazig University (Egypt), Department of Dermatology at UCSF, VA Medical Center San Francisco, and the Second Affiliated Hospital of Xi’an Jiaotong University (China, Shaanxi). The corresponding author is Professor R. Ghadially. The paper was published in 2024 by Oxford University Press and is in the public domain in the United States.

Research Design and Technical Approach

This paper is a single original experimental study that breaks down the scientific question into the following key experimental steps and logic:

1. Establishment and Grouping of Mouse Models

  • Experimental animals: C57BL/6J mice, 8–12 weeks old.
  • Grouping:
    • Normal control group (normal sensory innervation)
    • Sensory denervation group (sensory denervation induced by capsaicin, simulating nerve injury)
    • SP intervention group (denervated or control mice receiving exogenous SP supplementation)
    • Lapatinib-targeted inhibition group (tyrosine kinase inhibitor blocking EGFR, for mechanistic verification)
  • Wound model: 6mm full-thickness round excisional wounds symmetrically made on the dorsal skin, with no analgesics used postoperatively.

2. Detection of Stem Cell and Progenitor Cell Division Types and Activity

  • Histology and Immunofluorescence Analysis: On day 4 post-injury, tissue sections from the 0–4mm peri-wound region were collected. Sections were stained using primary antibodies to α/γ tubulin and secondary antibodies, the angle between the division axis and the basement membrane was measured—angles <30° as parallel divisions, 60°–90° as perpendicular divisions (the latter representing asymmetric divisions).
  • In vitro Cell Division Fate Detection: Skin within a 4mm radius around the wound was collected, digested enzymatically, and primary keratinocytes were cultured for 48h. Numb (marker for undifferentiated/differentiated cells) and Keratin 1 (differentiation marker) were stained, followed by observation of sister cell pairs. By analyzing double-staining and localization, symmetric/asymmetric SC and CP divisions were identified.

3. Sensory Denervation and SP Intervention

  • Denervation Model: Neonatal mice were injected subcutaneously with capsaicin twice, one week apart. Tail-flick reflex was used to evaluate anesthesia sensitivity and quantify successful denervation.
  • SP Supplementation: Cultures or mice received various concentrations of Substance P (10^-9M, 10^-7M, 10^-5M, 10^-3M) in the medium or by intradermal injections (32μg SP each time, around the wound, every other day, up to 24 days), some combined with the wounding model.
  • Lapatinib Inhibition: Both in vitro and in vivo, murine keratinocytes or mice were treated with Lapatinib (2.5μM) alone or combined with SP to assess effects on SC fate and epidermal regeneration.

4. Wound Healing, Epidermal Thickness, and Cell Density Measurements

  • Wound Area Measurement: Photographed at each time point, wound area calculated using ImageJ software to evaluate healing progress.
  • Epidermal Thickness and Basal Layer Cell Count: H&E-stained sections, 3 equidistant fields per sample, 8 images each, epidermal thickness and basal cell density (per 100 microns) measured using ImageJ.
  • Statistics: Analyzed using Graphpad Prism9—Student’s t-test for two-group means, ANOVA for multi-group, linear regression for correlation, with p<0.05 considered significant.

Main Experimental Results and Data Interpretation

1. Early Proliferative Response to Injury Originates Mainly from the Stem Cell Compartment

  • On day 4 post-injury, a marked increase in dividing cells is found in the 0–4mm peri-wound region.
  • Immunofluorescence showed perpendicular (asymmetric SC division) divisions in the basal layer increased 5-fold, parallel (symmetric) divisions 2-fold; suprabasal divisions did not change significantly.
  • In vitro sister cell pair analysis further confirmed an increase in both symmetric and asymmetric SC self-renewal, with no remarkable rise in CP (progenitor) divisions—suggesting the early proliferative peak is driven by stem cell self-renewal rather than CP expansion.

2. Sensory Denervation Significantly Delays Wound Healing and Impairs SC Function

  • Wound healing in denervated mice is significantly delayed (24.8±0.3 days vs 14.5±0.3 days), with a lower healing rate throughout.
  • Denervated skin, both in injured and intact areas, showed reduced perpendicular and parallel divisions, extreme epidermal thinning, and decreased basal cell density.
  • SC fate analysis revealed marked reductions in both asymmetric SC divisions and CP divisions, supporting that sensory nerves and their mediators play a crucial role in maintaining SC activity and progeny differentiation.

3. Substance P Reverses Effects of Denervation, Promotes SC Asymmetric Division and Wound Healing

  • The optimal SP concentration for promotion is 10^-7M; subcutaneous SP injections in denervated mice shorten healing to 18.8±0.3 days.
  • SP treatment not only restores asymmetric SC division frequency but also directly increases CP symmetric differentiation. Wound areas at all time points are smaller in the SP group.
  • Long-term SP supplementation tests show that SP can induce epidermal thickening and stratification, significantly increasing basal cell density in normal skin regardless of denervation status.

4. Lapatinib Confirms Asymmetric SC Division as the Essential Step for SP-Induced Stratification

  • EGFR inhibition by Lapatinib markedly suppresses asymmetric SC divisions both in vitro and in vivo, regardless of SP supplementation.
  • In both denervated and normal skin, Lapatinib inhibits the SP-induced epidermal thickening, maintaining a thin epidermal state.
  • This indicates that the stratification effect of SP on the epidermis is highly dependent on asymmetric SC divisions.

Conclusions, Academic and Applied Value

This study systematically reveals, for the first time, that in the early phase of mouse skin wound healing, both symmetric and asymmetric stem cell divisions are markedly enhanced, fueling new epidermis formation, while progenitor cell divisions play little role. Sensory nerve injury attenuates SC self-renewal and new epidermal generation and directly or indirectly reduces CP differentiation. SP functions as the “signaling mediator” for sensory nerve integrity; supplementation with SP can compensate for SC dysfunction caused by nerve injury, effectively accelerating wound healing, and through promoting asymmetric SC division, brings about effective epidermal stratification and structural reconstruction. EGFR pathway inhibition confirms that asymmetric division is required for SP-mediated epidermal proliferation and stratification.

This work fills a mechanistic gap in the sensory nerve—neuropeptide—SC fate—skin regeneration axis and provides solid theoretical and experimental support for neuromediator therapy of chronic, non-healing wounds. It implies that targeting SP and its signaling pathways may offer a novel avenue for clinical interventions.

Research Highlights

  1. Mechanistic Innovation: For the first time, it comprehensively reveals that SP-induced epidermal stratification depends on inducing asymmetric division of SCs, advancing our understanding of how division type affects physiological and pathological healing outcomes.
  2. Clinical Translational Value: Demonstrates that SP supplementation significantly improves healing disorders associated with nerve damage and suggests a theoretical pathway for future treatment of chronic wounds in aging and diabetes.
  3. Rigorous and Innovative Methodology: Integrates molecular, cellular, and whole-animal approaches; innovatively quantifies division angles and uses multiplex immunostaining for fate analysis, greatly enhancing the accuracy and conviction of results.
  4. Deepened Theoretical Significance: Presents a novel viewpoint that asymmetric SC divisions dominate epidermal stratification, offering valuable reference for understanding related disease mechanisms and even tumor progression.

Additional Information

All datasets are publicly available via the Figshare platform (DOI: https://doi.org/10.6084/m9.figshare.22280254.v1), enabling further and interdisciplinary research. The authors declare no conflict of interest. All animal procedures and ethical reviews were fully approved and all experimental processes were rigorously documented.

Summary

This international, multicenter study by Khalifa et al. delivers new insights into the basic mechanisms by which nerves regulate skin regeneration and stratification. Through a series of animal models and advanced cellular/molecular experiments, it provides compelling evidence that SP is the key mediator for sensory nerves in regulating skin regeneration, with its effect mechanistically dependent on driving asymmetric SC divisions, ultimately facilitating rapid and complete re-epithelialization and stratification of the wound. This builds a vital bridge between fundamental biology and clinical medicine and opens up a new theoretical pathway for drug interventions and bioengineering therapies for chronic, non-healing wounds.