Keratinocyte Populations Research Articles

Defining the transcriptome of PIK3CA-altered cells in a human capillary malformation using single cell long-read sequencing

PIK3CA-related overgrowth spectrum (PROS) disorders are caused by somatic mosaic variants that result in constitutive activation of the phosphatidylinositol-3-kinase/AKT/mTOR pathway. Promising responses to molecularly targeted therapy have been reported, although identification of an appropriate agent can be hampered by the mosaic nature and corresponding low variant allele frequency of the causal variant. Moreover, our understanding of the molecular consequences of these variants—for example how they affect gene expression profiles—remains limited. Here we describe in vitro expansion of a human capillary malformation followed by molecular characterization using exome sequencing, single cell gene expression, and targeted long-read single cell RNA-sequencing in a patient with clinical features consistent with Megalencephaly-Capillary Malformation Syndrome (MCAP, a PROS condition). These approaches identified a targetable PIK3CA variant with expression restricted to PAX3+ fibroblast and undifferentiated keratinocyte populations. This study highlights the innovative combination of next-generation single cell sequencing methods to better understand unique transcriptomic profiles and cell types associated with MCAP, revealing molecular intricacies of this genetic syndrome.

Tick extracellular vesicles undermine epidermal wound healing during hematophagy.

Wound healing has been extensively studied through the lens of inflammatory disorders and cancer, but limited attention has been given to hematophagy and arthropod-borne diseases. Hematophagous ectoparasites, including ticks, subvert the wound healing response to maintain prolonged attachment and facilitate blood-feeding. Here, we unveil a strategy by which extracellular vesicles (EVs) ensure blood-feeding and arthropod survival in three medically relevant tick species. We demonstrate through single cell RNA sequencing and murine genetics that wildtype animals infested with EV-deficient Ixodes scapularis display a unique population of keratinocytes with an overrepresentation of pathways connected to wound healing. Tick feeding affected keratinocyte proliferation in a density-dependent manner, which relied on EVs and dendritic epidermal T cells (DETCs). This occurrence was linked to phosphoinositide 3-kinase activity, keratinocyte growth factor (KGF) and transforming growth factor β (TGF-β) levels. Collectively, we uncovered a strategy employed by a blood-feeding arthropod that impairs the integrity of the epithelial barrier, contributing to ectoparasite fitness.

Langerhans cells orchestrate apoptosis of DNA-damaged keratinocytes upon high-dose UVB skin exposure.

Ultraviolet (UV) irradiation of the skin causes mutations that can promote the development of melanoma and nonmelanoma skin cancer. High-dose UVB exposure triggers a vigorous skin reaction characterized by inflammation resulting in acute sunburn. This response includes the formation of sunburn cells and keratinocytes (KC) undergoing programmed cell death (apoptosis) when repair mechanisms of DNA damage are inadequate. The primary objective of this research was to clarify the involvement of Langerhans cells (LC) in the development of acute sunburn following intense UVB skin irradiation. To address this, we subjected the dorsal skin of mice to a single high-dose UVB exposure and analyzed the immediate immune response occurring within the skin tissue. Acute sunburn triggered an activation of LC, coinciding with a rapid influx of neutrophils that produced TNF-α. Furthermore, our investigation unveiled a marked increase in DNA-damaged KC and the subsequent induction of apoptosis in these cells. Importantly, we demonstrate a crucial link between the inflammatory cascade, the initiation of apoptosis in DNA-damaged KC, and the presence of LC in the skin. LC were observed to modulate the chemokine response in the skin following exposure to UVB, thereby affecting the trafficking of neutrophils. Skin lacking LC revealed diminished inflammation, contained fewer TNF-α-producing neutrophils, and due to the prevention of apoptosis induction, a lingering population of DNA-damaged KC, presumably carrying the risk of enduring genomic alterations. In summary, our results underscore the pivotal role of LC in preserving the homeostasis of UVB-irradiated skin. These findings contribute to a deeper understanding of the intricate mechanisms underlying acute sunburn responses and their implications for UV-induced skin cancer.

The Efficacy of Imiquimod-Induced Psoriasis Model on Murine Cells.

Keratinocytes are an essential component of the epidermis that undergoes constant proliferation and differentiation. However, the dysregulation of keratinocyte differentiation has been implicated in various skin disorders such as psoriasis. Imiquimod, otherwise known as IMQ, is a topical immunomodulator often used to induce psoriasis-like lesions in murine models for research purposes. This study focuses on the efficacy of using IMQ to induce a psoriasis-like model on murine skin cells by analyzing single-cell RNA sequencing and trajectory analysis. The results indicate a few differences between IMQ-induced and control murine cells, primarily the increased keratinocyte and immune cell populations, which reflects the cell identity found on psoriatic skin. However, trajectory analysis reveals that IMQ-induced cells have quite a linear differentiation pattern compared to the branched pattern found in control cells. As a result, further research must be conducted to explore differing factors between psoriatic cells and IMQ-induced cells to determine its usefulness in mimicking psoriasis-like conditions for research.

Abstract 2102: Understanding the mechanism of action of rigosertib in recessive dystrophic epidermolysis bullosa-associated squamous cell carcinoma through single cellanalysis

Abstract The allosteric inhibitor rigosertib (an experimental therapeutic) was previously identified as an effective inducer of G2M arrest and apoptosis in squamous cell carcinoma (SCC) keratinocytes isolated from patients with the rare genetic disease recessive dystrophic epidermolysis bullosa (RDEB), both in vitro and in vivo. Early clinical trial data treating RDEB-associated SCC with rigosertib has shown remarkable efficacy in a sub-set of patients and understanding the mechanism of action of rigosertib in this lethal cancer would provide rationale for patient stratification. Multiple mechanisms of action were previously attributed to rigosertib, including polo-like kinase-1 (PLK1), PI3-kinase, activated RAS and microtubule polymerization inhibition. Our published data comparing PLK1 siRNA with PI3-Kinase inhibitors, MAPK inhibitors and microtubule disruption favored PLK1 as a mechanism of action without definitive conclusion. We therefore sought to determine the mechanism of action using transcriptomic profiling. Flow cytometry experiments identified three main categories of time to increase G2M in nine separate RDEB SCC keratinocyte populations in culture ranging from 1 to 6 hours after exposure to 1uM rigosertib. Bulk-RNA sequencing of representative populations from time to G2M groupings failed to identify consistent transcriptional change after exposure to rigosertib. However, single cell sequencing was able to identify increases in sub-populations of cells associated with G2M as well as transient emergence of unique populations that were specific to individual patient populations after exposure to rigosertib in culture. Of note a transient population of RDEB SCC keratinocytes only present after 1 hour of exposure to rigosertib was characterized by increased expression of the potassium regulator KCNRG which has previously been associated with apoptosis in chronic lymphocytic leukemia. Ongoing work is determining the role this gene may play in the mechanism of apoptosis induction in RDEB SCC keratinocytes after exposure to rigosertib. Citation Format: Andrew P. South, Suhao Han, Paolo Fortina. Understanding the mechanism of action of rigosertib in recessive dystrophic epidermolysis bullosa-associated squamous cell carcinoma through single cellanalysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2102.

Abstract 5415: Fibroblast mediated head and neck tumor cell invasion in 3D culture is independent of partial epithelial to mesenchymal transition

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with a five-year-survival of 61-64%. Recent studies have demonstrated tumor keratinocytes with features of a partial transition from epithelial to a mesenchymal phenotype, also known as partial-EMT (pEMT), at the edge of invading tumor islands associates with metastatic disease. As such, pEMT likely results from direct interactions between tumor cells and their surrounding microenvironment. To test if specific secreted factors or cell interactions drive the acquisition of pEMT, we first analyzed a series of primary and established HNSCC cell populations to determine TGF-β1 responsiveness since TGF-β1 is a known driver of pEMT. Of the eight populations tested, six displayed an increased level of pSMAD3 upon incubation with 5 ng/mL of TGF-β1 (p < 0.0001). To determine if this TGF-β1 response leads to an increase in pEMT phenotype, a pEMT-score (derived from mRNA expression of 9 pEMT markers) for each HNSCC population was determined following TGF-β1 treatment. As expected, the two non-responsive HNSCC populations showed no increase in pEMT-score, whilst five of the six TGF-β1-responsive HNSCC populations demonstrated a significant increase in pEMT-score (p = 0.006). To assess if TGF-β induced pEMT phenotype correlates with increased invasive potential, we produced 3D skin equivalent models using the eight HNSCC keratinocyte populations, in the presence or absence of primary fibroblasts and/or TGF-β1 treatment. TGF-β1 treatment alone caused a significant increase in invasion of all five TGF-β1-induced pEMT HNSCC populations (r2 = 0.449, p = 0.0045), whilst the three populations previously shown to be pEMT unresponsive to TGF-β1 treatment showed no increase. Interestingly, addition of primary fibroblasts without TGF-β1 treatment caused a universal dramatic increase in invasion in 3D organ culture (p = 0.0064, 34-fold increase), regardless of the previously determined TGF-β1-induced pEMT status. Unexpectedly, TGF-β1 treatment in the presence of fibroblasts caused no significant increase in invasion in 3D cultures (r2 = 0.00001, p = 0.9906). We next compared levels of pEMT markers in 3D cultures using immunofluorescence and observed a significant increase in PDPN in six of the eight HNSCC populations (p = 0.0136) with the addition of fibroblasts suggesting that factors independent of TGF-β1 contributes to pEMT marker expression and invasion in 3D culture. Overall, these data demonstrate fibroblasts induce HNSCC keratinocyte invasion regardless of the population’s ability to respond to TGF-β1-induced pEMT and that pEMT may be driven by factors independent of TGF-β1 in 3D culture. Citation Format: Pyung Hun Park, Michael M. Alexander, Joseph M. Curry, Adam J. Luginbuhl, Andrew P. South. Fibroblast mediated head and neck tumor cell invasion in 3D culture is independent of partial epithelial to mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5415.

Short cell cycle duration is a phenotype of human epidermal stem cells

BackgroundA traditional view is that stem cells (SCs) divide slowly. Meanwhile, both embryonic and pluripotent SCs display a shorter cell cycle duration (CCD) in comparison to more committed progenitors (CPs).MethodsWe examined the in vitro proliferation and cycling behavior of somatic adult human cells using live cell imaging of passage zero keratinocytes and single-cell RNA sequencing.ResultsWe found two populations of keratinocytes: those with short CCD and protracted near exponential growth, and those with long CCD and terminal differentiation. Applying the ergodic principle, the comparative numbers of cycling cells in S phase in an enriched population of SCs confirmed a shorter CCD than CPs. Further, analysis of single-cell RNA sequencing of cycling adult human keratinocyte SCs and CPs indicated a shortening of both G1 and G2M phases in the SC.ConclusionsContrary to the pervasive paradigm, SCs progress through cell cycle more quickly than more differentiated dividing CPs. Thus, somatic human adult keratinocyte SCs may divide infrequently, but divide rapidly when they divide. Additionally, it was found that SC-like proliferation persisted in vitro.

Epigenetic switch reshapes epithelial progenitor cell signatures and drives inflammatory pathogenesis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a complex inflammatory skin disease with undefined mechanistic underpinnings. Here, we investigated HS epithelial cells and demonstrated that HS basal progenitors modulate their lineage restriction and give rise to pathogenic keratinocyte clones, resulting in epidermal hyperproliferation and dysregulated inflammation in HS. When comparing to healthy epithelial stem/progenitor cells, in HS, we identified changes in gene signatures that revolve around the mitotic cell cycle, DNA damage response and repair, as well as cell-cell adhesion and chromatin remodeling. By reconstructing cell differentiation trajectory and CellChat modeling, we identified a keratinocyte population specific to HS. This population is marked by S100A7/8/9 and KRT6 family members, triggering IL1, IL10, and complement inflammatory cascades. These signals, along with HS-specific proinflammatory cytokines and chemokines, contribute to the recruitment of certain immune cells during the disease progression. Furthermore, we revealed a previously uncharacterized role of S100A8 in regulating the local chromatin environment of target loci in HS keratinocytes. Through the integration of genomic and epigenomic datasets, we identified genome-wide chromatin rewiring alongside the switch of transcription factors (TFs), which mediated HS transcriptional profiles. Importantly, we identified numerous clinically relevant inflammatory enhancers and their coordinated TFs in HS basal CD49fhigh cells. The disruption of the S100A enhancer using the CRISPR/Cas9-mediated approach or the pharmacological inhibition of the interferon regulatory transcription factor 3 (IRF3) efficiently reduced the production of HS-associated inflammatory regulators. Our study not only uncovers the plasticity of epidermal progenitor cells in HS but also elucidates the epigenetic mechanisms underlying HS pathogenesis.

Immunomodulatory Effects of Histone Variant H2A.J in Ionizing Radiation Dermatitis

Histone variant H2A.J is associated with premature senescence after ionizing radiation (IR) and modulates senescence-associated secretory phenotype (SASP). Using constitutive H2A.J knock-out mice, the role of H2A.J was investigated in radiation dermatitis. H2A.J wild-type (WT) and knock-out (KO) mice were exposed to moderate or high IR doses (≤20 Gy, skinfold IR). Radiation-induced skin reactions were investigated up to 2 weeks post-IR at macroscopic and microscopic levels. H2A.J and other senescence markers, as well as DNA damage and proliferation markers, were studied by immunohistochemistry, immunofluorescence, and electron microscopy. After high-dose IR, protein-coding transcriptomes were analyzed by RNA sequencing, immune cell infiltration by flow cytometry, and gene expression by reverse transcription polymerase chain reaction in (non-) irradiated WT versus KO skin. In WT skin, epidermal keratinocytes showed time- and dose-dependent H2A.J accumulation after IR exposure. Unexpectedly, stronger inflammatory reactions with increased epidermal thickness and progressive hair follicle loss were observed in irradiated KO versus WT skin. Clearly more radiation-induced senescence was observed in keratinocyte populations of KO skin after moderate and high doses, with hair follicle stem cells being particularly badly damaged, leading to follicle atrophy. After high-dose IR, transcriptomic analysis revealed enhanced senescence-associated signatures in irradiated KO skin, with intensified release of SASP factors. Flow cytometric analysis indicated increased immune cell infiltration in both WT and KO skin; however, specific chemokine-mediated signaling in irradiated KO skin led to more neutrophil recruitment, thereby aggravating radiation toxicities. Increased skin damage in irradiated KO skin led to hyperproliferation, abnormal differentiation, and cornification of keratinocytes, accompanied by increased upregulation of transcription-factor JunB. Lack of radiation-induced H2A.J expression in keratinocytes is associated with increased senescence induction, modulation of SASP expression, and exacerbated inflammatory skin reactions. Hence, epigenetic H2A.J-mediated gene expression in response to IR regulates keratinocyte immune functions and plays an essential role in balancing the inflammatory response during radiation dermatitis.

Distinguishing Keratoacanthoma from Well-Differentiated Cutaneous Squamous Cell Carcinoma Using Single-Cell Spatial Pathology

Keratoacanthoma (KA) is a common keratinocyte neoplasm that is regularly classified as a type of cutaneous squamous cell carcinoma (cSCC) despite demonstrating benign behavior. Differentiating KA from well-differentiated cSCC is difficult in many cases due to the substantial overlap of clinical and histological features. Currently, no reliable discriminating markers have been defined, and consequently, KAs are often treated similarly to cSCC, creating unnecessary surgical morbidity and healthcare costs. In this study, we used RNA sequencing to identify key differences in transcriptomes between KA and cSCC, which suggested divergent keratinocyte populations between each tumor. Imaging mass cytometry was then used to identify single-cell tissue characteristics, including cellular phenotype, frequency, topography, functional status, and interactions between KA and well-differentiated cSCC. We found that cSCC had significantly increased proportions of Ki67+ keratinocytes among tumor keratinocytes, which were dispersed significantly throughout non-basal keratinocyte communities. In cSCC, regulatory T-cells were more prevalent and held greater suppressive capacity. Furthermore, cSCC regulatory T-cells, tumor-associated macrophages, and fibroblasts had significant associations with Ki67+ keratinocytes as opposed to avoidances with KA, indicating a more immunosuppressive environment. Our data suggest that multicellular spatial features can serve as a foundation to enhance the histological discrimination of ambiguous KA and cSCC lesions.

Single cell transcriptomic analysis of HPV16-infected epithelium identifies a keratinocyte subpopulation implicated in cancer

Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.

372 HMGB1 Localization as a Driver of Carcinogenesis in RDEB-Associated Squamous Cell Carcinoma

OBJECTIVES/GOALS: This study investigates whether localization of high mobility group box 1 (HMGB1) controls inflammatory signaling and DNA damage response in human keratinocytes, the cell of origin for squamous cell carcinoma (SCC). SCC is especially metastatic in chronic wounds, burns, and in patients with recessive dystrophic epidermolysis bullosa (RDEB). METHODS/STUDY POPULATION: We used CRISPR/Cas9 gene-editing to knock out HMGB1 in a keratinocyte line, p16INK4a-negative keratinocytes immortalized by ectopic hTERT expression (N/TERT-2G [46,XY]). Following gene editing, clonal keratinocyte populations were screened for knockout by PCR followed by TIDE analysis (Tracking of Indels by Decomposition) to identify indels that would result in a frameshift mutation. Total cell lysates for each clonal population were analyzed by immunoblot and immunofluorescence for HMGB1 protein. These cells will be used to assay for DNA damage sensitivity in the presence of genotoxic agents (etoposide, ultraviolet radiation, γ-irradiation). A lentiviral vector will then be used to express mutant forms of HMGB1 that localize to the nucleus (C23/45S) or cytoplasm (C106S) and DNA damage assays repeated. RESULTS/ANTICIPATED RESULTS: We have confirmed by sequencing, immunoblot, and immunofluorescence that HMGB1 is knocked out in a clonal population of N/TERT-2G human keratinocyte cells. We anticipate that cells with a complete absence of HMGB1 will have high sensitivity to DNA damaging agents, but little change in inflammatory signaling. We also expect that cells expressing mutant HMGB1 that localizes exclusively to the cytoplasm will demonstrate an increased sensitivity to DNA damage relative to wild-type controls, while mutant HMGB1 that localizes exclusively to the nucleus will be protected from DNA damage caused by exposure to genotoxic agents. DISCUSSION/SIGNIFICANCE: HMGB1 is a nuclear protein and damage associated molecular pattern that is elevated systemically in patients with RDEB, many of whom will go on to develop metastatic squamous cell carcinoma. The experiments described here investigate whether HMGB1 plays a mechanistic role in skin carcinogenesis via regulation of the DNA damage response.

Differential cell composition and split epidermal differentiation in human palm, sole, and hip skin

Palmoplantar skin is structurally and functionally unique, but the transcriptional programs driving this specialization are unclear. Here, we use bulk and single-cell RNA sequencing of human palm, sole, and hip skin to describe the distinguishing characteristics of palmoplantar and non-palmoplantar skin while also uncovering differences between palmar and plantar sites. Our approach reveals an altered immune environment in palmoplantar skin, with downregulation of diverse immunological processes and decreased immune cell populations. Further, we identify specific fibroblast populations that appear to orchestrate key differences in cell-cell communication in palm, sole, and hip. Dedicated keratinocyte analysis highlights major differences in basal cell fraction among the three sites and demonstrates the existence of two spinous keratinocyte populations constituting parallel, site-selective epidermal differentiation trajectories. In summary, this deep characterization of highly adapted palmoplantar skin contributes key insights into the fundamental biology of human skin and provides a valuable data resource for further investigation.

579 Clinical grade human pluripotent stem cells derived engineered skin substitutes promote keratinocytes wound closure in vitro

Chronic wounds, such as leg ulcers associated with sickle cell disease, occur as consequence of a prolonged inflammatory phase during the healing process. They are extremely hard to heal and persist as a significant health care problem due to the absence of effective treatment and the uprising number of patients. Indeed, there is a critical need to develop novel cell- and tissue-based therapies to treat these chronic wounds. Development in skin engineering leads to a small catalogue of available substitute manufactured in Good Manufacturing Practices compliant (GMPc) conditions. Those substitutes are produced using primary cells that could limit their use due to restricted sourcing. Here, we propose GMPc protocols to produce functional populations of keratinocytes and fibroblasts derived from pluripotent stem cells to reconstruct the associated dermo-epidermal substitute with plasma based fibrin matrix. In addition, this manufactured composite skin is biologically active and enhances in vitro wounding of keratinocytes. The proposed composite skin open new perspectives for skin replacement using allogeneic substitute.

560 Isolation of an “early” transit amplifying keratinocyte population in interfollicular human epidermis: a role for CD271 receptor

In the interfollicular epidermis stem cells (KSC) generate transit amplifying (TA) cells that, after symmetric divisions, produce differentiating daughters. We isolated and characterized the highly proliferative interfollicular epidermal basal cell population “early” TA (ETA) cells, based on its capacity to adhere to type IV collagen. Proliferation and colony forming efficiency in ETA cells are lower than in KSC, but higher than in “late” TA (LTA). Stem and differentiation markers confirmed that ETA cell display a unique phenotype. Skin reconstructs derived from ETA cells present different features (epidermal thickness, Ki67 and survivin expression), as compared to skin equivalents generated from either KSC or LTA cells. The low affinity neurotrophin receptor CD271, that regulates the KSC to TA cells transition in human epidermis through an on/off switch control mechanism, is predominantly expressed in ETA cells. Skin equivalents generated from siRNA CD271 ETA cells display a more proliferative and less differentiated phenotype, as compared to mock-derived reconstructs. Consistently, CD271 overexpression in LTA cells generates a more proliferative skin equivalent than mock LTA cells. Finally, CD271 expression declines with ageing and cellular senescence. In addition, CD271 overexpressing cells show lower p16ink4a levels than mock cells during replicative senescence and CD271 also appears to delay senescence in ETA in comparison to LTA cells. We conclude that ETA cells represent the first KSC progenitor with exclusive features. CD271 identifies and modulates ETA cells, thus participating in the early differentiation and regenerative capacity of human epidermis.

Skin programming of inflammatory responses to Staphylococcus aureus is compartmentalized according to epidermal keratinocyte differentiation status.

Acute cutaneous inflammation causes microbiome alterations as well as ultrastructural changes in epidermis stratification. However, the interactions between keratinocyte proliferation and differentiation status and the skin microbiome have not been fully explored. Hypothesizing that the skin microbiome contributes to regulation of keratinocyte differentiation and can modify antimicrobial responses, we examined the effect of exposure to commensal (Staphylococcus epidermidis, SE) or pathogenic (Staphylococcus aureus, SA) challenge on epidermal models. Explant biopsies were taken to investigate species-specific antimicrobial effects of host factors. Further investigations were performed in reconstituted epidermal models by bulk transcriptomic analysis alongside secreted protein profiling. Single-cell RNA sequencing analysis was performed to explore the keratinocyte populations responsible for SA inflammation. A dataset of 6391 keratinocytes from control (2044 cells), SE challenge (2028 cells) and SA challenge (2319 cells) was generated from reconstituted epidermal models. Bacterial lawns of SA, not SE, were inhibited by human skin explant samples, and microarray analysis of three-dimensional epidermis models showed that host antimicrobial peptide expression was induced by SE but not SA. Protein analysis of bacterial cocultured models showed that SA exposure induced inflammatory mediator expression, indicating keratinocyte activation of other epidermal immune populations. Single-cell DropSeq analysis of unchallenged naive, SE-challenged and SA-challenged epidermis models was undertaken to distinguish cells from basal, spinous and granular layers, and to interrogate them in relation to model exposure. In contrast to SE, SA specifically induced a subpopulation of spinous cells that highly expressed transcripts related to epidermal inflammation and antimicrobial response. Furthermore, SA, but not SE, specifically induced a basal population that highly expressed interleukin-1 alarmins. These findings suggest that SA-associated remodelling of the epidermis is compartmentalized to different keratinocyte populations. Elucidating the mechanisms regulating bacterial sensing-triggered inflammatory responses within tissues will enable further understanding of microbiome dysbiosis and inflammatory skin diseases, such as atopic eczema.

Isolation of an "Early" Transit Amplifying Keratinocyte Population in Human Epidermis: A Role for the Low Affinity Neurotrophin Receptor CD271.

In the interfollicular epidermis (IFE), stem cells (KSC) generate transit amplifying (TA) cells that, after symmetric divisions, produce differentiating daughters. Here, we isolated and characterized the highly proliferative interfollicular epidermal basal cell population "early" TA (ETA) cells, based on their capacity to adhere to type IV collagen. Proliferation and colony-forming efficiency in ETA cells are lower than in KSC but higher than in "late" TA (LTA). Stemness, proliferation, and differentiation markers confirmed that ETA cells display a unique phenotype. Skin reconstructs derived from ETA cells present different features (epidermal thickness, Ki67, and Survivin expression), as compared to skin equivalents generated from either KSC or LTA cells. The low-affinity neurotrophin receptor CD271, which regulates the KSC to TA cell transition in the human epidermis through an on/off switch control mechanism, is predominantly expressed in ETA cells. Skin equivalents generated from siRNA CD271 ETA cells display a more proliferative and less differentiated phenotype, as compared to mock-derived reconstructs. Consistently, CD271 overexpression in LTA cells generates a more proliferative skin equivalent than mock LTA cells. Finally, the CD271 level declines with cellular senescence, while it induces a delay in p16INK4 expression. We conclude that ETA cells represent the first KSC progenitor with exclusive features. CD271 identifies and modulates ETA cells, thus participating in the early differentiation and regenerative capacity of the human epidermis.

Thy1 marks a distinct population of slow-cycling stem cells in the mouse epidermis

The presence of distinct stem cells that maintain the interfollicular epidermis is highly debated. Here, we report a population of keratinocytes, marked by Thy1, in the basal layer of the interfollicular epidermis. We find that epidermal cells expressing differential levels of Thy1 display distinct transcriptional signatures. Thy1+ keratinocytes do not express T cell markers, express a unique transcriptional profile, cycle significantly slower than basal epidermal progenitors and display significant expansion potential in vitro. Multicolor lineage tracing analyses and mathematical modeling reveal that Thy1+ basal keratinocytes do not compete neutrally alike interfollicular progenitors and contribute long-term to both epidermal replenishment and wound repair. Importantly, ablation of Thy1+ cells strongly impairs these processes, thus indicating the non-redundant function of Thy1+ stem cells in the epidermis. Collectively, these results reveal a distinct stem cell population that plays a critical role in epidermal homeostasis and repair.

448 Physiological function of krox20 (Egr2) in epithelial stem cells

Krox20, a zinc finger-containing transcription factor, is well known for mediating stem and progenitor cell activation and differentiation in a variety of tissues. In a recent study, we reported for the first time a population of epithelial-derived Krox20-expressing keratinocytes in the hair follicle that ultimately terminally differentiate to form the structural component of the hair shaft. These Krox20-lineage cells in the hair follicle also mediate melanocyte differentiation via Stem Cell Factor production for hair pigmentation.

133 Multimodal analyses of vitiligo skin identifies tissue characteristics of stable disease

Vitiligo is an autoimmune skin disease that is characterized by the progressive destruction of melanocytes by autoreactive CD8+ T cells. Melanocyte destruction in active vitiligo is mediated by CD8+ T cells but why white patches in stable disease persist is poorly understood. The interaction between immune cells, melanocytes, and keratinocytes in situ in human skin has been difficult to study due to the lack of proper tools. We combine non-invasive multiphoton microscopy (MPM) imaging and single-cell RNA sequencing (scRNA-seq) to identify distinct subpopulations of keratinocytes in lesional skin of stable vitiligo patients. We show that these keratinocytes are enriched in lesional vitiligo skin and differ in metabolism, an observation corroborated by both MPM and scRNA-seq. Systematic investigation of cell-cell communication show that CXCL is the prominent signaling change in this small population of keratinocytes, which secrete CXCL9 and CXCL10 to create local inflammatory cytokine loops with T cells to drive stable vitiligo persistence. Further MPM imaging of patients undergoing punch grafting treatment showed that metabolically altered keratinocytes persist in non-responders but normalize in treatment responders. In summary, we couple advanced imaging with transcriptomics and bioinformatics to discover cell-cell communication networks and keratinocyte cell states that persist in stable disease and potentially prevent repigmentation.