Epidermal Stem Cell Population Research Articles

Transcription factor KROX20 marks epithelial stem cells for hair follicle formation.

Epidermal stem cells control homeostasis and regeneration of skin and hair. In the hair follicle (HF) bulge of mammals, populations of slow-cycling stem cells regenerate the HF during cyclical rounds of anagen (growth), telogen (quiescence), and catagen (regression). Multipotent epidermal cells are also present in the HF above the bulge area, contributing to the formation and maintenance of sebaceous gland and upper and middle portions of the HF. Here, we report that the transcription factor Krox20 is enriched in an epidermal stem cell population located in the upper/ middle HF. Expression analyses and lineage tracing using inducible Krox20-CreERT showed that Krox20-lineage cells migrate out of this HF region and contribute to the formation of bulge in the HF, serving as ancestors of bulge stem cells. In vivo depletion of these cells arrests HF morphogenesis. This study identifies a novel marker for an epidermal stem cell population that is indispensable for hair homeostasis.

Human Skin Aging and the Anti-Aging Properties of Retinol.

The skin is the most-extensive and -abundant tissue in the human body. Like many organs, as we age, human skin experiences gradual atrophy in both the epidermis and dermis. This can be primarily attributed to the diminishing population of epidermal stem cells and the reduction in collagen, which is the primary structural protein in the human body. The alterations occurring in the epidermis and dermis due to the aging process result in disruptions to the structure and functionality of the skin. This creates a microenvironment conducive to age-related skin conditions such as a compromised skin barrier, slowed wound healing, and the onset of skin cancer. This review emphasizes the recent molecular discoveries related to skin aging and evaluates preventive approaches, such as the use of topical retinoids. Topical retinoids have demonstrated promise in enhancing skin texture, diminishing fine lines, and augmenting the thickness of both the epidermal and dermal layers.

A History of Mechanical Tension Depletes Lgr6+ Epidermal Stem Cells

PURPOSE: Recent research found the epidermal Lgr6 stem cell population to be progressively depleted under mechanical tension, with a paralleled increase in the Lgr6 descendant population.1 This study aims to determine if there is a reversal of tension-induced skin growth characteristics, specifically if the Lgr6 stem cell population recovers after tension is removed. Our results will guide future research in therapeutic interventions for optimal skin recovery following tension, such as seen with bariatric surgery and the resulting excess skin from rapid weight loss. METHOD: Genetic Lgr6-EGFP-Cre-ERT2;tdTomato mice (EGFP=Lgr6, tdT=Lgr6 descendants) underwent controlled expansion (E) of the back skin. A tissue expander was surgically placed under the back skin on day zero, rested for 7 days, expanded over 10 days with a total of 24 mL saline, rested for 14 days, then deflated of saline at post-injection day 14 (PI-14) or deflation day zero (DF-0). Skin over the expander was resected, embedded in paraffin, and sectioned at 4µm. Cell populations were defined using immunofluorescence and quantified by relative expression and cell counting (ImageJ). Control mice underwent expander surgery but did not undergo saline expansion (non-expanded, NE). NE and E mice (n=3 each) were compared for PI-14, DF-14, and DF-56. We used two-tailed t-test with alpha set at 0.05. RESULTS: Relative expression of EGFP decreased from NE to both DF-14 (p=0.0474) and DF-56 (p=0.0477), while relative expression of tdT increased from NE to DF-56 (p=0.0125). EGFP+ cells decreased from NE to PI-14, DF-14, and DF-56 (p=0.0114 for all), while tdT+ cells increased from NE to PI-14, DF-14, and DF-56 (p=0.0086, p=0.0133, p=0.00002). Proliferating cells (Ki67+) increased from NE to PI-14, DF-14, and DF-56 (p=0.00007, p=0.00001, p=0.0417), but decreased from DF-14 to DF-56 (p=0.0278). Of the proliferating cell population, the [Ki67+tdT+] population increased from NE to DF-56 (p=0.0002). Cytokeratin 5 (K5) relative expression increased from NE to PI-14, DF-14, and DF-56 (p=0.0109, p<0.00001, p<0.00001). The number of epidermal keratinocyte layers increased from NE to PI-14, DF-14, and DF-56 (p<0.00001, p<0.00001, p=0.0043), with a decrease from DF-14 to DF-56 (p=0.0020). CONCLUSION: The epidermal Lgr6 stem cell population remained severely depleted following discontinuation of mechanical tension. Conversely, both the Lgr6 descendent population and K5 stem cell expression continued to increase while under tension and following release of tension. A prolonged history of discontinued tension revealed that the majority of the proliferating cell population was from Lgr6 progeny. Together, these observations suggest that in skin with a history of tension: (1) the Lgr6 population is permanently depleted, (2) the majority of the epidermis will be from Lgr6 progeny, and (3) Lgr6 progeny and K5 stem cells will be preferentially and progressively activated over Lgr6 stem cells to maintain the epidermis. Overall, these results imply that specific stimuli will prioritize responses from specific stem cell populations, and possibly to the detriment of an existing stem cell population. REFERENCES: 1. Xue Y, Lyu C, Taylor A. Mechanical Tension Mobilizes Lgr6+ Epidermal Stem Cells to Drive Skin Growth. [provisionally accepted ahead of print February 23, 2022]. Sci Adv.

Assaying Candidate Human Skin Keratinocyte Stem Cells by Determining Their Long-Term Serial Proliferative Output in Culture.

Stem cells are found in niches around the body, including the epidermis of the skin, and can be distinguished from their more committed progeny by their high long-term proliferative capacity in vitro. Here we describe a technique used to isolate three main epidermal cell fractions from human neonatal foreskin termed early differentiating (ED), transient amplifying (TA) and keratinocyte stem cells (KSC) based on their differential expression of two cell surface markers: CD49f and CD71. These three fractions were cultivated in parallel in a serial proliferation assay to determine their long-term proliferative output. This assay demonstrates that the KSC fraction had the highest proliferative output (total cell yield) over a long experimental timeframe of 2-3months, as well as a higher proliferative rate compared to the other two fractions (P>0.05). This assay can be utilized under similar conditions to determine the proliferative capacity of other putative stem cells using novel stem cell markers for epidermal or other stem cell populations.

Lgr6 marks epidermal stem cells with a nerve-dependent role in wound re-epithelialization.

Stem cells support lifelong maintenance of adult organs, but their specific roles during injury are poorly understood. Here we demonstrate that Lgr6 marks a regionally restricted population of epidermal stem cells that interact with nerves and specialize in wound re-epithelialization. Diphtheria toxin-mediated ablation of Lgr6 stem cells delays wound healing, and skin denervation phenocopies this effect. Using intravital imaging to capture stem cell dynamics after injury, we show that wound re-epithelialization by Lgr6 stem cells is diminished following loss of nerves. This induces recruitment of other stem cell populations, including hair follicle stem cells, which partially compensate to mediate wound closure. Single-cell lineage tracing and gene expression analysis reveal that the fate of Lgr6 stem cells is shifted toward differentiation following loss of their niche. We conclude that Lgr6 epidermal stem cells are primed for injury response and interact with nerves to regulate their fate.

Evidence for a non-stochastic two-field hypothesis for persistent skin cancer risk

With recurring carcinogen exposures, individual tumors develop in a field of genetic mutations through a stepwise process of clonal expansion and evolution. Once established, this “cancer field” persists in the absence of continued carcinogen exposures, resulting in a sustained risk for cancer development. Using a bioimaging approach, we previously demonstrated that a dermal premalignant field characterized by inflammatory angiogenesis persists following the cessation of ultraviolet light exposures and accurately predicts future overlying epidermal tumor formation. Following ultraviolet light treatments, others have observed that patches of p53 immunopositive cells persist stochastically throughout the epidermal stem cell population. However, these studies were done by random biopsies, introducing sampling bias. We now show that, rather than being randomly distributed, p53+ epidermal cells are enriched only in areas overlying this multi-focal dermal field. Moreover, we also show that the dermal field is characterized by a senescent phenotype. We propose that persistence of the overlying epithelial cancerization field in the absence of exogenous carcinogens or promoters requires a two-field composite consisting of a dermal senescent field driving the persistence of the overlying epidermal cancer field. These observations challenge current models that suggest that persistence of cancer risk in the absence of continued carcinogen exposures is simply a function of stochastically arranged, long-lived but dormant epithelial clonal stem cells mutants. The model proposed here could provide new insights into how cancer risk persists following cessation of carcinogenic exposures.

Altering MYC phosphorylation in the epidermis increases the stem cell population and contributes to the development, progression, and metastasis of squamous cell carcinoma

cMYC (MYC) is a potent oncoprotein that is subject to post-translational modifications that affect its stability and activity. Here, we show that Serine 62 phosphorylation, which increases MYC stability and oncogenic activity, is elevated while Threonine 58 phosphorylation, which targets MYC for degradation, is decreased in squamous cell carcinoma (SCC). The oncogenic role of MYC in the development of SCC is unclear since studies have shown in normal skin that wild-type MYC overexpression can drive loss of stem cells and epidermal differentiation. To investigate whether and how altered MYC phosphorylation might affect SCC development, progression, and metastasis, we generated mice with inducible expression of MYCWT or MYCT58A in the basal layer of the skin epidermis. In the T58A mutant, MYC is stabilized with constitutive S62 phosphorylation. When challenged with DMBA/TPA-mediated carcinogenesis, MYCT58A mice had accelerated development of papillomas, increased conversion to malignant lesions, and increased metastasis as compared to MYCWT mice. In addition, MYCT58A-driven SCC displayed stem cell gene expression not observed with MYCWT, including increased expression of Lgr6, Sox2, and CD34. In support of MYCT58A enhancing stem cell phenotypes, its expression was associated with an increased number of BrdU long-term label-retaining cells, increased CD34 expression in hair follicles, and increased colony formation from neonatal keratinocytes. Together, these results indicate that altering MYC phosphorylation changes its oncogenic activity—instead of diminishing establishment and/or maintenance of epidermal stem cell populations like wild-type MYC, pS62-MYC enhances these populations and, under carcinogenic conditions, pS62-MYC expression results in aggressive tumor phenotypes.

A Cell Fate Switch in the Caenorhabditis elegans Seam Cell Lineage Occurs Through Modulation of the Wnt Asymmetry Pathway in Response to Temperature Increase.

Populations often display consistent developmental phenotypes across individuals despite inevitable biological stochasticity. Nevertheless, developmental robustness has limits, and systems can fail upon change in the environment or the genetic background. We use here the seam cells, a population of epidermal stem cells in Caenorhabditis elegans, to study the influence of temperature change and genetic variation on cell fate. Seam cell development has mostly been studied so far in the laboratory reference strain (N2), grown at 20° temperature. We demonstrate that an increase in culture temperature to 25° introduces variability in the wild-type seam cell lineage, with a proportion of animals showing an increase in seam cell number. We map this increase to lineage-specific symmetrization events of normally asymmetric cell divisions at the fourth larval stage, leading to the retention of seam cell fate in both daughter cells. Using genetics and single-molecule imaging, we demonstrate that this symmetrization occurs via changes in the Wnt asymmetry pathway, leading to aberrant Wnt target activation in anterior cell daughters. We find that intrinsic differences in the Wnt asymmetry pathway already exist between seam cells at 20° and this may sensitize cells toward a cell fate switch at increased temperature. Finally, we demonstrate that wild isolates of C. elegans display variation in seam cell sensitivity to increased culture temperature, although their average seam cell number is comparable at 20°. Our results highlight how temperature can modulate cell fate decisions in an invertebrate model of stem cell patterning.

Role of bulge epidermal stem cells and TSLP signaling in psoriasis

Psoriasis is a common inflammatory skin disease involving a cross‐talk between epidermal and immune cells. The role of specific epidermal stem cell populations, including hair follicle stem cells (HF‐SCs) in psoriasis is not well defined. Here, we show reduced expression of c‐JUN and JUNB in bulge HF‐SCs in patients with scalp psoriasis. Using lineage tracing in mouse models of skin inflammation with inducible deletion of c‐Jun and JunB, we found that mutant bulge HF‐SCs initiate epidermal hyperplasia and skin inflammation. Mechanistically, thymic stromal lymphopoietin (TSLP) was identified in mutant cells as a paracrine factor stimulating proliferation of neighboring non‐mutant epidermal cells, while mutant inter‐follicular epidermal (IFE) cells are lost over time. Blocking TSLP in psoriasis‐like mice reduced skin inflammation and decreased epidermal proliferation, VEGFα expression, and STAT5 activation. These findings unravel distinct roles of HF‐SCs and IFE cells in inflammatory skin disease and provide novel mechanistic insights into epidermal cell interactions in inflammation.

Wild-type and SAMP8 mice show age-dependent changes in distinct stem cell compartments of the interfollicular epidermis.

Delayed wound healing and reduced barrier function with an increased risk of cancer are characteristics of aged skin and one possible mechanism is misregulation or dysfunction of epidermal stem cells during aging. Recent studies have identified heterogeneous stem cell populations within the mouse interfollicular epidermis that are defined by territorial distribution and cell division frequency; however, it is unknown whether the individual stem cell populations undergo distinct aging processes. Here we provide comprehensive characterization of age-related changes in the mouse epidermis within the specific territories of slow-cycling and fast-dividing stem cells using old wild-type, senescence-accelerated mouse prone 1 (SAMP1) and SAMP8 mice. During aging, the epidermis exhibits structural changes such as irregular micro-undulations and overall thinning of the tissue. We also find that, in the old epidermis, proliferation is preferentially decreased in the region where fast-dividing stem cells reside whereas the lineage differentiation marker appears to be more affected in the slow-cycling stem cell region. Furthermore, SAMP8, but not SAMP1, exhibits precocious aging similar to that of aged wild-type mice, suggesting a potential use of this model for aging study of the epidermis and its stem cells. Taken together, our study reveals distinct aging processes governing the two epidermal stem cell populations and suggests a potential mechanism in differential responses of compartmentalized stem cells and their niches to aging.

Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing.

Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB.

NRP-1 interacts with GIPC1 and α6/β4-integrins to increase YAP1/∆Np63α-dependent epidermal cancer stem cell survival.

We have identified an epidermal cancer stem (ECS) cell population that drives formation of rapidly growing and highly invasive and vascularized tumors. VEGF-A and neuropilin-1 (NRP-1) are highly expressed in ECS cell tumors and VEGF-A/NRP-1 interaction is required for ECS cell survival and tumor vascularization. We now identify a novel signaling cascade that is triggered by VEGF-A/NRP-1. We show that NRP-1 forms a complex with GIPC1 and α6/β4-integrin to activate FAK/Src signaling, which leads to stabilization of a YAP1/∆Np63α to enhance ECS cell survival, invasion, and angiogenesis. Loss of NRP-1, GIPC1, α6/β4-integrins, YAP1, or ∆Np63α reduces these responses. Moreover, restoration of constituently active YAP1 or ∆Np63α in NRP-1 null cells restores the ECS cell phenotype. Tumor xenograft experiments show that NRP-1 knockout ECS cells form small tumors characterized by reduced vascularization as compared to wild-type cells. The NRP-1 knockout tumors display signaling changes consistent with a role for the proposed signaling cascade. These studies suggest that VEGF-A interacts with NRP-1 and GIPC1 to regulate α6/β4-integrin, FAK, Src, PI3K/PDK1, LATS1 signaling to increase YAP1/∆Np63α accumulation to drive ECS cell survival, angiogenesis, and tumor formation.

Abstract IA06: Stem cells in mammalian epidermis

Abstract Multiple epidermal stem cell subpopulations in adult mouse skin have been identified over the last decade. Although they are all competent to give rise to all the different epidermal lineages following transplantation, wounding or genetic modification, under steady state conditions they maintain the epidermis in a compartmentalized manner. Stem cells in the lower hair follicle express LGR5, while stem cells in the upper follicle express LGR6 and cells in the junctional zone express LRIG1. In addition, a subset of stem cells in the interfollicular epidermis expresses LGR6. In my presentation I will discuss the consequences of selectively expressing activated beta-catenin in the different epidermal stem cell populations. Citation Format: Fiona M. Watt. Stem cells in mammalian epidermis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA06.

In vitro study on proliferation kinetics of oral mucosal keratinocytes

The limited availability of autogenous oral mucosa in oral and maxillofacial surgery for intraoral grafting after trauma or tumor resection can be balanced by the use of tissue-engineered oral mucosa. However, the use of tissue engineering in autologous grafts is still subject to further research. The aim of this study was to evaluate conditions that lead to a rapid proliferation of vital and highly proliferative oral keratinocytes, which can be used in tissue engineering and consequently help improve surgical management of intraoral mucosal defects. Human oral keratinocytes were obtained from oral mucosal specimens and cultivated. According to their affinity to β1-integrin, epidermal stem cell populations were isolated by using collagen type IV and laminin-coated dishes. Cell proliferation and cell viability were measured by using the CASY cell counter, WST-1 assays, and real-time cell analysis (xCELLigence). Measurements on cell proliferation (CASY cell counter) and cell viability (WST-1 assay) showed the characteristic proliferation stages of invitro-cultivated cells. No statistically significant differences could be monitored (P > .05). Real-time cell analysis, as a more direct and precise technique, revealed a steeper growth curve of adherent cells and therefore generally higher proliferation kinetics compared with cells derived from the supernate. Data from real-time cell analysis showed an increased proliferation of adherent cells compared with those derived from the supernate. These results demonstrate the increase of the proliferation capacity by cultivation of keratinocytes derived by adhesion to extracellular matrix proteins.

TLR7-expressing cells comprise an interfollicular epidermal stem cell population in murine epidermis.

Normal interfollicular epidermis (IFE) homeostasis is maintained throughout the entire life by its own stem cells that self-renew and generate progeny that undergo terminal differentiation. However, the fine markers of the stem cells in interfollicular epidermis are not well defined yet. Here we found that TLR7 identified the existence of progenitors and interfollicular epidermal stem cells in murine skin. In vitro, TLR7-expressing cells comprised of two subpopulations that were competent to proliferate and exhibited distinct differentiation potentials. Three-dimensional (3D) organotypic culture and skin reconstitution assays showed that TLR7-expressing cells were able to reconstruct the interfollicular epidermis. Finally, TLR7-expressing cells maintained the intact interfollicular epidermal structures revealed in serial transplantation assays in vivo in mice. Taken together, our results suggest that TLR7-expressing cells comprise an interfollicular epidermal stem cell population.

Epidermal Development in Mammals: Key Regulators, Signals from Beneath, and Stem Cells

Epidermis is one of the best-studied tissues in mammals that contain types of stem cells. Outstanding works in recent years have shed great light on behaviors of different epidermal stem cell populations in the homeostasis and regeneration of the epidermis as well as hair follicles. Also, the molecular mechanisms governing these stem cells are being elucidated, from genetic to epigenetic levels. Compared with the explicit knowledge about adult skin, embryonic development of the epidermis, especially the early period, still needs exploration. Furthermore, stem cells in the embryonic epidermis are largely unstudied or ambiguously depicted. In this review, we will summarize and discuss the process of embryonic epidermal development, with focuses on some key molecular regulators and the role of the sub-epidermal mesenchyme. We will also try to trace adult epidermal stem cell populations back to embryonic development. In addition, we will comment on in vitro derivation of epidermal lineages from ES cells and iPS cells.

P63 and its isoforms as markers of rat oral mucosa epidermal stem cells in vitro

Although techniques for purifying epidermal stem cells (ESCs) have been established, enriching a pure population of viable ESCs is still a challenging task. One approach toward enhancing the purity and viability of ESCs involves cell markers. While evidence suggests that p63 plays a role in maintaining the population of ESCs, whether p63 can function as a specific marker for ESCs is unclear. We isolated and cultured oral ESCs and illustrated the expression of p63 and its isoforms in rat oral mucosa tissues and stem cells before and after differentiation. Semi-reverse transcription PCR detected the TA, DeltaN, alpha and beta isoforms when cells were cultured for 2 days, but only TA and gamma were detected after 14 days. We also found that p63 is expressed in basal and suprabasal epithelial layers of rat oral mucosa, but it was implied p63 does not mark stem cells specifically, while the DeltaNp63alpha and DeltaNp63beta isoforms may be specific markers of rat oral mucosa stem cells.

Are epidermal stem cells unique with respect to aging?

Epidermal stem cells are a population of somatic stem cells responsible for maintaining and repairing the epidermis of the skin. A malfunctioning epidermal stem cell compartment results in loss of the epidermis and death of the whole organism. Since the epidermis continually renews itself by sloughing a layer of cells every day, it is in a constant state of cellular turnover and requires continual cell replacement for life. Thus, maintaining a pristine epidermal stem cell population is of prime importance, even during aging. Unlike stem cells from internal tissues, epidermal stem cells show little response to aging. They do not appear to decrease in number or functionality with age, and do not show changes in gene expression, developmental responsiveness, or age-associated increases of reactive oxygen species. Thus, epidermal stem cells may be a unique somatic stem cell.

Epidermal stem cell diversity and quiescence

Mammalian epidermis is maintained by self-renewal of stem cells and terminal differentiation of their progeny. New data reveal a diversity amongst stem cells that was previously unrecognized. Different stem cell populations have different locations and differ in whether they are quiescent or actively cycling. During normal epidermal homeostasis, each stem cell population feeds a restricted number of differentiated lineages. However, in response to injury or genetic manipulation the different pools of stem cells demonstrate multi-lineage differentiation ability. While it is well established that Wnt signalling promotes hair follicle (HF) differentiation, new observations suggest a role for EGF receptor signalling in promoting differentiation of interfollicular epidermis. NFATc1 maintains quiescence in the HF, while Lrig1 exerts the same function in the junctional zone. The stage is now set for exploring the relationship between the different epidermal stem cell populations and between quiescence and lineage selection.

Isolation and identification of stem cells from adult cashmere goat skin

Continuously renewing epithelia are maintained by stem cells that slowly proliferate and remain in the tissues for life. It has been known for decades that mouse epithelial stem cells can be selected by adherence to specific integrins. The adherence of cashmere goat epidermal cells to collagen type IV for 10 min was used to obtain enriched epidermal stem cells. The characteristics of the rapidly adherent epidermal cells were determined. The rapidly adherent epidermal cells exhibited the stem cell characteristics of immaturity, were quiescent, showed a high colony formation efficiency, and expressed candidate surface markers for epidermal stem cells (keratin 15, keratin 19, p63, CD34, and beta1-integrin). The rapidly adherent epidermal cells represented the epidermal stem cell population.