Epidermal stem cells: Interplay with the skin microenvironment during wound healing

Abstract

Skin undergoes everyday turnover while often challenged by injuries. The wound healing process in the skin is a dynamic sequence of events that involves various cell types and signaling pathways. Epidermal stem cells (EpdSCs), the tissue-resident stem cells in the skin tissue, are at the center of this complicated process due to their special ability to self-renew and differentiate. During this process, EpdSCs interact actively with the tissue microenvironment, which is essential for proper re-epithelialization and skin barrier restoration. This review describes the intricate interplays between EpdSCs and various components of their surroundings, including extracellular matrix/fibroblasts, vasculature/endothelial cells, and immune cells, as well as their roles in tissue repair.

INTRODUCTION

Overview of the Wound Healing Process in Skin

While the skin undergoes everyday turnover and renewal, it also frequently encounters wounding and tissue injury. Wound healing involves multiple distinct yet overlapping phases: hemostasis, inflammation, proliferation, and remodeling (reviewed in Gurtner et al., 2008).

Hemostasis

The first phase is hemostasis, which lasts a couple of hours and involves the recruitment of platelets to the wound site shortly after tissue injury (Opneja et al., 2019). Platelets then form a scab to create a temporary barrier against infection by external microbes and to prevent blood and fluid loss from the tissue. In addition, these platelets also secrete signaling molecules, including platelet-derived growth factor, to engage macrophages and fibroblasts, preparing them for the following steps (Carmeliet, 2003, Peña and Martin, 2024).

Inflammation

Following hemostasis, active inflammatory responses occur, governed by both recruited immune cells and tissue-resident immune cells. Neutrophils are among the first group of cells recruited to the wound site, reaching their peak numbers in the wound bed at 24 to 48 hours after injury (Boothby et al., 2020, Kim et al., 2008). They are followed by monocytes, which then differentiate into macrophages. These macrophages are especially important during the early inflammatory phase of wound healing; they phagocytose debris, break down necrotic tissue, and serve as a source of cytokines and growth factors, such as transforming growth factor-β (TGF-β), to attract endothelial cells and fibroblasts to the wound site, promoting angiogenesis and dermal regeneration (Lucas et al., 2010).

Proliferation

As the inflammatory responses diminish, the tissue moves on to the next stage, the step characterized by the proliferation and migration of a variety of different cell types. This proliferation phase typically lasts 1 to 2 weeks in human skin (Boothby et al., 2020, Opneja et al., 2019). During the proliferation phase, new tissues are formed to replace the damaged ones. A representative step in this phase is re-epithelialization, where epidermal keratinocytes migrate over the injured dermis, separating the scab from viable tissue. At the leading wound edge, epidermal cells undergo a transient mesenchymal transition, adopting an undifferentiated phenotype. Behind this leading edge, cells proliferate to restore the epithelial layer. Re-epithelialization is essential in tissue repair, as it regenerates the epidermis, which tentatively restores the skin barrier function and helps prevent infection and fluid loss until further tissue remodeling occurs (Hsu and Fuchs, 2022, Pastar et al., 2014). At the same time, the dermal area is regenerated through angiogenesis by endothelial cells and the production of extracellular matrix (ECM), primarily collagen, by fibroblasts (Fuchs and Blau, 2020).

Remodeling

The final step, referred to as the remodeling phase, begins approximately 2 to 3 weeks after an injury and can extend for months or even years. During this phase, all the wound healing–related signals gradually wane, and granulation tissue is resolved. This is followed by the removal of dermal cells that have undergone apoptosis, as well as the reorganization and reformation of the ECM, including collagen fibers. The production of additional collagen fibers leads to the maturation and strengthening of the new tissues formed during the previous proliferation phase, while ultimately resulting in the formation of scar tissues (Hsu and Fuchs, 2022; Yang et al., 2019, Yang et al., 2020).

The Role of Epidermal Stem Cells in Skin Homeostasis and Tissue Repair

Each phase of wound healing is organized by several different cell types. Among them, stem cells play a particularly important role thanks to their unique ability to self-renew and differentiate. While different tissues possess distinct kinds of stem cells, in the context of skin, epidermal stem cells (EpdSCs), which reside in the basal layer of the epidermis, the outermost layer of the skin, are in charge of maintaining tissue integrity. These tissue-resident stem cells are responsible for the maintenance of skin homeostasis, in addition to the repair processes upon tissue injury.

Under homeostatic conditions, EpdSCs oversee the everyday renewal of the epidermis. Located on the external surface of the body, skin is constantly exposed to threats that can potentially breach its barrier. To maintain and ensure the skin barrier functions, EpdSCs proliferate and differentiate into keratinocytes. These keratinocytes migrate upward, undergo terminal differentiation, and ultimately form the protective outer layer of the skin tissue (Blanpain and Fuchs, 2009, Sada et al., 2016).

When the tissue is injured, EpdSCs are activated by injury-induced signals from the damaged tissue microenvironment. These signals cause EpdSCs to proliferate and migrate to the wound site, where they mainly govern the re-epithelialization, close the wound, and finally restore the skin integrity (Fuchs and Blau, 2020). Given the significant impact of their interactions with the components of the tissue microenvironment, it is important to investigate how EpdSCs communicate with their surroundings, including ECM, fibroblasts, vasculatures, and immune cells, as well as the signaling pathways involved, which collectively influence the function of EpdSCs.

THE INTERPLAY BETWEEN ECM/FIBROBLASTS AND EPIDERMAL STEM CELLS DURING WOUND HEALING

The Role of Extracellular Matrix on EpdSC Behaviors

Within the skin microenvironment, EpdSCs balance between various responses, such as self-renewal, proliferation, and differentiation, which dynamically change in response to the alterations in the surrounding environment. The ECM contributes to this by providing structural support and biological signals (Humphrey et al., 2014) (Fig. 1). A good example is the role of ECM components of basement membrane in balancing between keeping stem cells in an undifferentiated state versus inducing their differentiation. The basement membrane, which is composed of various ECM components including collagen, laminin, and fibronectin, is situated right beneath the epidermis, separating the epidermis from the dermis (Driskell et al., 2013, Hsu et al., 2014). When EpdSCs are in direct contact with the basement membrane, they remain undifferentiated; however, they initiate differentiation upon losing contact with the membrane and migrating upward (Jones and Watt, 1993).

Fig. 1.

How ECM/fibroblasts interact with EpdSCs during wound healing. (A) Attachment of EpdSCs to the basement membrane via hemidesmosomes helps maintain these cells in an undifferentiated state. (B) Example of the reciprocal interactions between EpdSCs and fibroblasts that promote the wound healing process. KGF, keratinocyte growth factor.

One mechanism by which basal epidermal cells interact with the basement membrane is via α6β4-induced hemidesmosomes. These structures make keratinocytes firmly attach to the basement membrane, maintaining their self-renewal characteristics and skin barrier function (Molder et al., 2021). Besides, hemidesmosomes also serve as a signaling platform; when the subunits of hemidesmosome are activated by the epidermal growth factor receptor, they engage the extracellular signal-regulated kinase/mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling cascades to support the epidermal cell growth and survival. In this manner, the ECM of the basement membrane controls the EpdSC fate and epidermal homeostasis (Liu et al., 2019; Molder et al., 2021; Simpson et al., 2011).

Beyond the basement membrane, several other instances illustrate how the ECM affects EpdSC responses. For example, mice lacking the ECM protein fibulin 7 showed an imbalance between EpdSC differentiation and proliferation, which resulted in impaired wound healing, potentially owing to upregulated interleukin (IL)-6 production (Raja et al., 2022). Similarly, spatial quantitative proteomics performed on skin tissues suggested that TGF-β-induced protein ig-h3, an ECM glycoprotein, is involved in EpdSC growth and promotes wound healing (Li et al., 2022).

The Role of Fibroblasts on EpdSC Behaviors

During skin tissue repair, the main role of fibroblasts is to remodel ECM, ensuring the ideal environment for EpdSC function. However, these fibroblasts not only actively produce ECM components but also detect the changes in tissue microenvironment following injury and secrete regulatory molecules.

One such regulatory molecule is the protease enzyme referred to as metalloproteinase (MMP). MMPs produced by fibroblasts trigger ECM degradation and remodel the wound stroma, including fibronectin, laminin, collagen, and proteoglycan. Under homeostatic conditions, MMP expression remains minimal; however, its levels surge following skin tissue injury in response to signaling cues, such as collagen fragmentation (Fisher et al., 2009) and reactive oxygen species (Fu et al., 2004), which necessitate tissue remodeling. MMP activity is particularly important for proper re-epithelialization, as it facilitates the loosening of tight contacts between epidermal cells and ECM, providing cells with the mobility required for migration. While different subtypes of MMP play subtly distinct roles, the migration of primary keratinocytes on collagen was blocked when treated with an inhibitor of MMP-1 or when placed on mutant collagen lacking the MMP-1 cleavage site, suggesting that the catalytic activity of MMP-1 is necessary for keratinocyte migration (Pilcher et al., 1997). Likewise, topical treatment with active recombinant MMP-8 mediated the complete re-epithelialization and enhanced angiogenesis, resulting in accelerated wound healing in diabetic db/db mice (Gao et al., 2015), reinforcing the role of MMP in epidermal cell phenotypes.

Among other signaling molecules secreted by fibroblasts are a diverse array of cytokines, including keratinocyte growth factor (KGF), also named as fibroblast growth factor-7. Known for promoting epithelial cell growth and differentiation, KGF is rapidly induced in fibroblasts within the wounded dermis after injury (Werner et al., 1992). By binding to its receptor FGFR2IIIb on epidermal keratinocytes, KGF enhances their proliferation (Marchese et al., 1990) and migration (Karvinen et al., 2003, Putnins et al., 1999) through various signaling pathways. For instance, the TGF-β1/Suppressor of mothers against decapentaplegic (SMAD) pathway is notably activated in primary keratinocytes in vitro and in diabetic rat wounding models in vivo treated with KGF-1 (Peng et al., 2019), a pathway involved in balancing active proliferation and reversible cell cycle exit in the tissue-resident stem cells (Oshimori and Fuchs, 2012).

Reciprocal Interactions Between EpdSCs and Fibroblasts

While fibroblasts influence EpdSCs via ECM remodeling and cytokine production, as shown, EpdSCs likewise modulate the behaviors of fibroblasts through various signaling pathways, establishing a reciprocal relationship. For example, epidermal keratinocytes activated by KGF, which is produced by fibroblasts during tissue injury, secrete TGF-β1. Keratinocyte-derived TGF-β1 then activates downstream SMAD signaling pathways in fibroblasts, causing them to differentiate into myofibroblasts in a paracrine manner. These alpha-smooth muscle actin–expressing myofibroblasts display both the secretory phenotype of fibroblasts and contractile functions reminiscent of smooth muscle cells. Upon transitioning, they subsequently produce collagen-I and promote wound contraction, creating a feedback loop that mediates wound closure (Peng et al., 2019, Sime et al., 1997). Furthermore, when their activation is sustained, these myofibroblasts can serve as a main driver in inducing fibrosis and subsequent scarring (Tabib et al., 2021). As a part of the normal tissue repair response upon acute damage, skin fibrosis by myofibroblasts is contributed by diverse factors, including interactions between myofibroblasts and neighboring immune cells such as macrophages (Shook et al., 2018), signaling pathways like Yes-associated protein (YAP) activity (Brewer et al., 2021), and cytokines such as chemokine (C-X-C motif) ligand 4 (CXCL4) (Affandi et al., 2022).

In addition, Liu and Hur et al. have recently shed light on another cytokine that EpdSCs produce to facilitate wound repair in not only the epidermis but also the dermis involving fibroblasts. Specifically, EpdSCs located at the wound edge were found to produce IL-24 to act on dermal fibroblasts to enhance their proliferation and collagen deposition, which, in turn, promotes EpdSC proliferation and re-epithelialization, again forming the reciprocal loop. While the EpdSC-derived IL-24 directly binds to fibroblasts and mediates this outcome, it can also stimulate metabolic changes within EpdSCs, which then indirectly triggers fibroblasts to proliferate and synthesize collagen (Liu et al., 2023).

THE INTERPLAY BETWEEN VASCULATURE/ENDOTHELIAL CELLS AND EPIDERMAL STEM CELLS DURING WOUND HEALING

Another major process for proper wound healing is angiogenesis, where new blood vessels grow from existing vasculature. While the exchange of nutrients and metabolites via capillaries occurs even under homeostatic conditions, it becomes especially important during tissue repair, when there is a higher demand for these substances for the growth and repair of regenerating tissues (Fig. 2).

Fig. 2.

Diverse examples of reciprocal interactions between vasculature and EpdSCs during wound healing. In addition to exchanging oxygen and nutrients, EpdSCs and the neighboring vasculature interact actively with each other via various cytokines to mediate angiogenesis and immune cell recruitment.

Influence of EpdSCs on Vasculatures During Tissue Injury

Since blood vessels are the main route for oxygen delivery to tissues, wounded tissue often experiences hypoxic conditions due to severed blood vessels. Hypoxia upregulates hypoxia-inducible factor-1α (HIF-1α), a transcription factor stabilized by low oxygen levels (Taylor and Scholz, 2022). HIF-1α is a well-known upstream regulator of canonical angiogenic factors, such as vascular endothelial growth factor-α (Ciarlillo et al., 2017, Pugh and Ratcliffe, 2003), platelet-derived growth factor-B (Kelly et al., 2003), and TGF-β (Carmeliet, 2003). These factors are produced by various cell types in the skin, including fibroblasts (Shams et al., 2022), macrophages (Lucas et al., 2010), platelets (Andrae et al., 2008), as well as epithelial cells (Brown et al., 1992), and mediate the proliferation, recruitment, migration, and differentiation of endothelial cells to regulate angiogenesis (Coultas et al., 2005).

Interestingly, recent studies have shown that HIF-1α can also mediate angiogenesis via EpdSCs, independently of the conventional angiogenic vascular endothelial growth factor-α or platelet-derived growth factor signaling pathways. Specifically, wound-induced HIF-1α in EpdSCs promotes the production of IL-24, which binds to its receptor on endothelial cells, activating downstream signal transducer and activator of transcription 3 (STAT3) to induce dermal angiogenesis and recruit newly regenerated blood vessels near the epidermis to support re-epithelialization. EpdSC-derived IL-24 is upregulated by tissue hypoxia just like other angiogenic factors, but its downstream effects do not involve traditional angiogenic pathways. Instead, glucose transporter 1 (GLUT1), a protein responsible for glucose transport across the cell membrane, has been identified as a key effector upregulated by IL-24, resulting in blood vessel regeneration (Liu et al., 2023).

Potential Differentiation of EpdSCs Into Endothelial Cells

Beyond affecting endothelial cells, recent studies suggest that EpdSCs might also differentiate directly into vascular endothelial cells under certain conditions (Huang et al., 2020). In the study by Huang et al., EpdSCs were isolated from rat skin tissue, transfected with enhanced green fluorescence protein, and then applied to the full-thickness wounds in rat back skin. These enhanced green fluorescence protein–positive EpdSCs were found to colonize and actively proliferate in the subcutaneous tissue layer of the wound bed. It is noteworthy that these colonized EpdSCs, particularly those near the blood vessels, expressed the vascular endothelial marker, namely cluster of differentiation 31 (CD31), and even some formed structures resembling vascular tubes.

While these findings are intriguing, the evidence for the transdifferentiation of EpdSCs into endothelial cells remains controversial. The phenomenon observed could potentially reflect vascular mimicry, a process often observed in tumor vasculature, where cells adopt endothelial cell–like phenotypes without genuinely being transdifferentiated. Further investigations will be necessary to clarify the direct differentiation potential of EpdSCs into endothelial cells.

Regulation of EpdSCs by Angiogenesis and Angiocrine Factors

It is evident that endothelial cells support the increased oxygen and metabolic demands of EpdSCs during the wound repair process through angiogenesis. Additionally, the expression of endothelial markers in cell populations near blood vessels implies that endothelial cells possibly secrete factors that influence EpdSC behavior. Although the exact mechanisms are yet to be elucidated, endothelial cells release numerous signaling molecules, such as IL-7 (Vranova et al., 2019) and IL-6 (Kang et al., 2020), which have the potential to affect EpdSCs during wound healing.

Moreover, endothelial cells have an interesting role in recruiting and regulating immune cells, which can further impact EpdSCs (Amersfoort et al., 2022). This aspect of immune cells is discussed more in detail in the following sections. Specifically, endothelial cells participate in immunomodulation by interacting with circulating immune cells, controlling their adhesion to the vasculature, and facilitating their extravasation into the tissue parenchyma (Ley et al., 2007, Nourshargh et al., 2010). For instance, an in vitro transmigration assay using the human umbilical vein endothelial cells with enhanced intercellular adhesion molecule-1 expression demonstrated that elevated intercellular adhesion molecule-1 levels in endothelial cells promoted the transmigration of polymorphonuclear leukocytes and CD3⁺ lymphocytes across the endothelial layer (Yang et al., 2005). Further studies on the transendothelial migration of lymphocytes suggested that one mechanism mediating this process is to activate Rac through its activator, Tiam1 (Gérard et al., 2009). These observations demonstrate that endothelial cells are not merely components of the vasculature but also act as a signaling center.

THE INTERPLAY BETWEEN IMMUNE CELLS/CYTOKINES AND EPIDERMAL STEM CELLS DURING WOUND HEALING

When the tissue is injured, the immune system is not only responsible for the inflammatory response but also involved in each phase of the wound healing process, including proliferation and tissue remodeling. In other words, while immune cells clear pathogens and debris at the wound site, they also produce a variety of cytokines and growth factors that coordinate the re-epithelialization and the final restoration of the skin barrier. This section discusses the bidirectional interactions between two main immune cells, (1) macrophages and (2) T cells, and EpdSCs during wound healing, highlighting how they influence each other’s phenotypes (Fig. 3).

Fig. 3.

Immune cell interactions with EpdSCs during wound healing. During different phases of the wound healing process, a distinct set of immune cells sends signals to EpdSCs to ensure proper tissue repair.

T Cell-EpdSC Interaction

In the early stages of wounding, epidermal keratinocytes, including EpdSCs, become activated and release a variety of cytokines and chemokines, which facilitate immune responses by recruiting immune cells. One such secreted factor is CC-chemokine ligand 20 (CCL20), a ligand that binds to CC chemokine receptor-6 (CCR6) (Furue et al., 2019, Kennedy-Crispin et al., 2012). Notably, a subset of T cells, including skin resident γδ T cells and Th17 cells, expresses CCR6, enabling them to be recruited to the epidermis via the CCL20-CCR6 axis (Campbell et al., 2017, Hedrick et al., 2009, Mabuchi et al., 2013, Paradis et al., 2008). While the migration of CCR6⁺ T cells to the epidermis and their roles have primarily been studied in the context of psoriatic inflammation, increasing evidence suggests that the same pathways are used for their epidermal recruitment during tissue injury and that these recruited CCR6⁺ T cells are involved in the wound healing process as well. For example, CCR6^−/− mice exhibit defective γδ T cells trafficking to the epidermis upon tissue injury as well as impaired dermal wound healing; this impairment was restored by adoptive transfer of γδ T cells from WT mice (Anderson et al., 2019).

T cells that migrate to the epidermis can reversely influence the phenotypes of EpdSCs, influencing re-epithelialization and overall wound healing. Specifically, recent studies have highlighted the direct impact of T cells, including retinoic acid–related orphan receptor γt+ γδ T cells, on EpdSCs during re-epithelialization. Although the upregulation of HIF-1α during tissue injury has been typically attributed to injury-induced hypoxia, a study by Konieczny et al. showed that IL-17A derived from RORγt+ γδ T cells is both necessary and sufficient to activate HIF-1α in epidermal cells (Konieczny et al., 2022). The IL-17-HIF-1α axis then drives glycolysis in the wound front epithelium, whose importance during tissue repair has been demonstrated in several studies (Eming et al., 2021; Liu et al., 2023).

Interactions between EpdSCs and dendritic epithelial T cells have also been implicated in delayed re-epithelialization in aged skin. Besides the intrinsic changes of aged keratinocytes (ie, decreased migration and proliferation), the impaired re-epithelialization is associated with the compromised capacity of aged EpdSCs to transcriptionally activate the crosstalk between epithelial cells and dendritic epithelial T cells. This activation occurs via upregulating Skints or activating STAT3. Indeed, silencing Skints or STAT3 in the epidermis of young mice disrupted re-epithelialization following tissue injury, suggesting the importance of these pathways (Keyes et al., 2016).

Macrophage-EpdSC Interaction

Macrophages play two seemingly contradictory roles during different phases of wound healing. Initially, M1 macrophages secrete proinflammatory cytokines including IL-1, IL-6, and tumor necrosis factor-alpha (TNF-α) to create an inflammatory environment, which will be followed by healing phases.

For example, IL-1α is significantly increased in M1 macrophages upon tissue injury and binds to its receptor on epidermal keratinocytes, promoting their proliferation (Chen et al., 1995). CCL20 is then subsequently expressed in keratinocytes, attracting additional immune cells to the wound site (Kennedy-Crispin et al., 2012). Similarly, IL-1β causes keratinocytes to secrete chemokines that engage IL-17-producing T cells, further amplifying the inflammatory response (Cai et al., 2019). Additionally, macrophage-derived IL-1 stimulates fibroblasts to release KGF, generating epidermal keratinocyte–fibroblast crosstalk in a paracrine signaling loop.

M1 macrophages also produce TNF-α, which directly triggers the phosphorylation of protein kinase B (AKT) in EpdSCs, activating downstream β-catenin signaling independently of wingless-related integration site (WNT) ligand binding (Wang et al., 2017). TNF-α additionally causes the secretion of MMP 9 to remodel the ECM (Olsson-Brown et al., 2023), again, further preparing the microenvironment for the following proliferative steps.

As the healing progresses and enters the proliferation and remodeling phases, macrophages transition to the M2 phenotype, secreting anti-inflammatory signaling molecules, such as IL-10 and TGF-β. TGF-β signaling, well-known for its influence on the self-renewal and differentiation of various tissue-resident stem cells, is particularly interesting since it plays diverse roles depending on the context, dosage, and wound healing phase, sometimes exerting opposing cellular effects (Oshimori and Fuchs, 2012, Watabe and Miyazono, 2009, Wu and Hill, 2009). For example, the treatment of TGF-β1 has been shown to improve healing rates (Mustoe et al., 1987) and stimulate ECM production (Chakravarthy et al., 2018), suggesting the TGF-β1’s positive role in promoting wound healing. However, contradictorily, transgenic mice expressing TGF-β1 transgene in keratinocytes exhibited rather delayed healing after burn injury (Yang et al., 2001). Moreover, knocking out TGF-β1 or its receptor in mice accelerated re-epithelialization (Amendt et al., 2002, Ashcroft et al., 1999, Koch et al., 2000). These opposing observations imply the importance of delicate regulation of signaling pathways during different wound healing phases, underscoring the need for an inducible gene modification approach to better understand the precise role of TGF-β signaling.

CONCLUSION AND FUTURE DIRECTIONS

EpdSCs interact with various components of the surrounding environment during tissue injury. Even though significant progress has been made in the field over the past decades, several limitations still need to be addressed when understanding these interactions. Specifically, many of the previous studies manipulated only individual cell components or pathways, overlooking complicated interplay between different elements within the tissue microenvironment. Furthermore, the fact that one signaling pathway can play contradictory roles (ie, TGF-β) depending on the contexts makes analysis and interpretation even more challenging. Given such complexity, more comprehensive approaches, such as inducible models allowing gene manipulation within a specific time frame, are necessary.

In conclusion, thorough investigations regarding the communication between EpdSCs and their surroundings will raise potential therapeutic targets for enhancing overall wound healing. This will ultimately lead to more effective treatments for acute and chronic wounds.

AUTHOR CONTRIBUTIONS

Y.H.H. reviewed the previously published literature, wrote the manuscript, and made the figures.

DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS

During the preparation of this work, the author used ChatGPT solely for proofreading to check for grammatical errors. Following this, the author carefully reviewed and edited the content as needed and takes full responsibility for the content of the publication.

DECLARATION OF COMPETING INTERESTS

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.