RESUMEN
Rationale: Reconstruction of hair follicles (HFs) and eccrine sweat glands (ESGs) is essential for functional skin regeneration. In skin reconstruction research, we found that foreskin-derived epidermal cells reconstructed HF organoids unidirectionally, but not ESG organoids. Methods: To investigate key genes and pathways influencing the fate of ESG and HF, a transcriptome profiling of ESG placode-containing skin and HF placode-containing skin was employed, and key DEGs were identified and validated by RT-qPCR and immunofluorescence staining in mice and rats. Subsequently, adult human epidermal cell-derived organoids were reconstructed to probe functional roles and mechanisms of FGF7 and FGF10 by series of approaches integrating RT-qPCR, immunofluorescence-staining, WB, apoptosis assay, and pathway interference assay. Results: All members of FGF7 subfamily were among the key DEGs screened, the differential expression of FGF7 and FGF10 and their receptors FGFR1/FGFR2 was verified between ESG placode-containing skin and HF placode-containing skin. In vivo and in vitro Matrigel plug models showed that both FGF7 and FGF10 promoted fate transition of human epidermal cell-derived organoids to ESG phenotype organoids, FGF7 and FGF10 had a synergistic effect, and mainly function through the FGFR1/2-MEK1/2-ERK1/2 pathway. Conclusions: Adult epidermal cells can be manipulated to reconstruct personalized HF and ESG to meet different needs.
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Glándulas Ecrinas , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Organoides , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Humanos , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Factor 7 de Crecimiento de Fibroblastos/genética , Organoides/metabolismo , Organoides/citología , Animales , Ratones , Glándulas Ecrinas/metabolismo , Glándulas Ecrinas/citología , Ratas , Células Epidérmicas/metabolismo , Células Epidérmicas/citología , Folículo Piloso/citología , Folículo Piloso/metabolismo , Masculino , FenotipoRESUMEN
In this review we discuss how the mammalian interfollicular epidermis forms during development, maintains homeostasis, and is repaired following wounding. Recent studies have provided new insights into the relationship between the stem cell compartment and the differentiating cell layers; the ability of differentiated cells to dedifferentiate into stem cells; and the epigenetic memory of epidermal cells following wounding.
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Células Madre Adultas , Diferenciación Celular , Células Epidérmicas , Epidermis , Animales , Células Epidérmicas/citología , Células Epidérmicas/metabolismo , Diferenciación Celular/fisiología , Células Madre Adultas/fisiología , Células Madre Adultas/citología , Humanos , Epidermis/metabolismo , Mamíferos , Epigénesis Genética , Cicatrización de Heridas/fisiología , HomeostasisRESUMEN
Epidermal tissues are among the most striking examples of planar polarity. Insect bristles, fish scales, and mammalian fur are all uniformly oriented along an animal's body axis. The collective alignment of epidermal structures provides a valuable system to interrogate the signaling mechanisms that coordinate cellular behaviors at both local and tissue-levels. Here, we provide methods to analyze the planar organization of hair follicles within the mouse epidermis. Hair follicles are specified and bud into the underlying dermis during embryonic development. Shortly after, follicle cells dynamically rearrange to orient each follicle toward the anterior of the animal. When directional signaling is disrupted, hair follicles become misoriented. In this chapter, we describe how to create a spatial map of hair follicle orientations to reveal tissue-scale patterns in both embryonic and postnatal skin. Additionally, we provide a live imaging protocol that can be used to monitor cell movements in embryonic skin explants to reveal the cellular behaviors that polarize the hair follicle itself.
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Polaridad Celular , Epidermis , Folículo Piloso , Animales , Ratones , Folículo Piloso/citología , Folículo Piloso/embriología , Polaridad Celular/fisiología , Epidermis/embriología , Epidermis/metabolismo , Células Epidérmicas/citología , Movimiento CelularRESUMEN
Epidermal stem cells orchestrate epidermal renewal and timely wound repair through a tight regulation of self-renewal, proliferation, and differentiation. In culture, human epidermal stem cells generate a clonal type referred to as holoclone, which give rise to transient amplifying progenitors (meroclone and paraclone-forming cells) eventually generating terminally differentiated cells. Leveraging single-cell transcriptomic data, we explored the FOXM1-dependent biochemical signals controlling self-renewal and differentiation in epidermal stem cells aimed at improving regenerative medicine applications. We report that the expression of H1 linker histone subtypes decrease during serial cultivation. At clonal level we observed that H1B is the most expressed isoform, particularly in epidermal stem cells, as compared to transient amplifying progenitors. Indeed, its expression decreases in primary epithelial culture where stem cells are exhausted due to FOXM1 downregulation. Conversely, H1B expression increases when the stem cells compartment is sustained by enforced FOXM1 expression, both in primary epithelial cultures derived from healthy donors and JEB patient. Moreover, we demonstrated that FOXM1 binds the promotorial region of H1B, hence regulates its expression. We also show that H1B is bound to the promotorial region of differentiation-related genes and negatively regulates their expression in epidermal stem cells. We propose a novel mechanism wherein the H1B acts downstream of FOXM1, contributing to the fine interplay between self-renewal and differentiation in human epidermal stem cells. These findings further define the networks that sustain self-renewal along the previously identified YAP-FOXM1 axis.
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Diferenciación Celular , Células Epidérmicas , Proteína Forkhead Box M1 , Histonas , Células Madre , Humanos , Proteína Forkhead Box M1/metabolismo , Proteína Forkhead Box M1/genética , Células Madre/metabolismo , Células Madre/citología , Células Epidérmicas/metabolismo , Células Epidérmicas/citología , Histonas/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Proliferación Celular , Epidermis/metabolismo , Células CultivadasRESUMEN
In the interfollicular epidermis, keratinocyte stem cells (KSC) generate a short-lived population of transit amplifying (TA) cells that undergo terminal differentiation after several cell divisions. Recently, we isolated and characterized a highly proliferative keratinocyte cell population, named "early" TA (ETA) cell, representing the first KSC progenitor with exclusive features. This work aims to evaluate epidermis, with a focus on KSC and ETA cells, during transition from infancy to childhood. Reconstructed human epidermis (RHE) generated from infant keratinocytes is more damaged by UV irradiation, as compared to RHE from young children. Moreover, the expression of several differentiation and barrier genes increases with age, while the expression of genes related to stemness is reduced from infancy to childhood. The proliferation rate of KSC and ETA cells is higher in cells derived from infants' skin samples than of those derived from young children, as well as the capacity of forming colonies is more pronounced in KSC derived from infants than from young children's skin samples. Finally, infants-KSC show the greatest regenerative capacity in skin equivalents, while young children ETA cells express higher levels of differentiation markers, as compared to infants-ETA. KSC and ETA cells undergo substantial changes during transition from infancy to childhood. The study presents a novel insight into pediatric skin, and sheds light on the correlation between age and structural maturation of the skin.
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Diferenciación Celular , Queratinocitos , Células Madre , Humanos , Lactante , Células Madre/citología , Células Madre/metabolismo , Queratinocitos/metabolismo , Queratinocitos/citología , Preescolar , Proliferación Celular , Células Epidérmicas/metabolismo , Células Epidérmicas/citología , Niño , Piel/citología , Piel/metabolismo , Femenino , Masculino , Epidermis/metabolismo , Células CultivadasRESUMEN
Studying human skin biology can aid in comprehending the pathophysiology of skin diseases and developing novel cell-based therapies, including tissue engineering approaches. This chapter provides a comprehensive guide of methods to determine human skin samples from the perspective of their cellular compositions. We describe as useful technique the histological analysis of tissue sections. We further illustrate the biological characterization of isolated and cultured basal and suprabasal interfollicular keratinocytes by cell sorting, cytospin immunostaining, colony forming efficiency, and long-term dermo-epidermal organotypic cultures.
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Separación Celular , Células Epidérmicas , Queratinocitos , Células Madre , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Separación Celular/métodos , Células Madre/citología , Células Madre/metabolismo , Células Epidérmicas/citología , Células Epidérmicas/metabolismo , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Piel/citología , Epidermis/metabolismo , Ingeniería de Tejidos/métodos , Diferenciación CelularRESUMEN
Transit-amplifying (TA) cells are progenitors that undergo an amplification phase followed by transition into an extinction phase. A long postulated epidermal TA progenitor with biphasic behavior has not yet been experimentally observed in vivo. Here, we identify such a TA population using clonal analysis of Aspm-CreER genetic cell-marking in mice, which uncovers contribution to both homeostasis and injury repair of adult skin. This TA population is more frequently dividing than a Dlx1-CreER-marked long-term self-renewing (e.g. stem cell) population. Newly developed generalized birth-death modeling of long-term lineage tracing data shows that both TA progenitors and stem cells display neutral competition, but only the stem cells display neutral drift. The quantitative evolution of a nascent TA cell and its direct descendants shows that TA progenitors indeed amplify the basal layer before transition and that the homeostatic TA population is mostly in extinction phase. This model will be broadly useful for analyzing progenitors whose behavior changes with their clone age. This work identifies a long-missing class of non-self-renewing biphasic epidermal TA progenitors and has broad implications for understanding tissue renewal mechanisms.
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Células Epidérmicas , Epidermis , Células Madre , Animales , Ratones , Células Madre/citología , Células Madre/metabolismo , Células Epidérmicas/citología , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Proliferación Celular , Linaje de la Célula , Homeostasis , Diferenciación Celular , Autorrenovación de las Células/fisiologíaRESUMEN
Epidermal stem cells (EpSCs) play a vital role in skin wound healing through re-epithelialization. Identifying chemicals that can promote EpSC proliferation is helpful for treating skin wounds. This study investigates the effect of morroniside on cutaneous wound healing in mice and explores the underlying mechanisms. Application of 10â50 µg/mL of morroniside to the skin wound promotes wound healing in mice. In vitro studies demonstrate that morroniside stimulates the proliferation of mouse and human EpSCs in a time- and dose-dependent manner. Mechanistic studies reveal that morroniside promotes the proliferation of EpSCs by facilitating the cell cycle transition from the G1 to S phase. Morroniside increases the expression of ß-catenin via the glucagon-like peptide-1 receptor (GLP-1R)-mediated PKA, PKA/PI3K/AKT and PKA/ERK signaling pathways, resulting in an increase in cyclin D1 and cyclin E1 expression, either directly or by upregulating c-Myc expression. This process ultimately leads to EpSC proliferation. Administration of morroniside to mouse skin wounds increases the phosphorylation of AKT and ERK, the expressions of ß-catenin, c-Myc, cyclin D1, and cyclin E1, as well as the proliferation of EpSCs, in periwound skin tissue, and accelerates wound re-epithelialization. These effects of morroniside are mediated by the GLP-1R. Overall, these results indicate that morroniside promotes skin wound healing by stimulating the proliferation of EpSCs via increasing ß-catenin expression and subsequently upregulating c-Myc, cyclin D1, and cyclin E1 expressions through GLP-1R signaling pathways. Morroniside has clinical potential for treating skin wounds.
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Proliferación Celular , Repitelización , Células Madre , Regulación hacia Arriba , beta Catenina , Animales , beta Catenina/metabolismo , beta Catenina/genética , Proliferación Celular/efectos de los fármacos , Ratones , Regulación hacia Arriba/efectos de los fármacos , Humanos , Repitelización/efectos de los fármacos , Células Madre/metabolismo , Células Madre/efectos de los fármacos , Células Madre/citología , Cicatrización de Heridas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Masculino , Células Epidérmicas/metabolismo , Células Epidérmicas/efectos de los fármacos , Células Epidérmicas/citología , Ciclina D1/metabolismo , Ciclina D1/genéticaRESUMEN
Epidermal stem cells, located in the skin, together with keratinocytes are transplanted in regenerative therapies, e.g., for the treatment of burns or other wounds. Here, we describe the protocol of their enzymatic isolation from human skin. It includes separation of the epidermis form the dermis by incubation with dispase followed by cell isolation for epidermis by digestion with trypsin. Cell isolated with this method can be seeded on collagen IV-coated dishes. The methods of analysis of epidermal stem cells markers (e.g., CD71, CD29) with flow cytometry and RT-PCR are also included.
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Biomarcadores , Separación Celular , Colágeno Tipo IV , Células Epidérmicas , Citometría de Flujo , Células Madre , Humanos , Citometría de Flujo/métodos , Separación Celular/métodos , Células Epidérmicas/metabolismo , Células Epidérmicas/citología , Células Madre/metabolismo , Células Madre/citología , Colágeno Tipo IV/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Antígenos CD/metabolismo , Antígenos CD/genética , EndopeptidasasRESUMEN
One of the major functions of the semaphorin signaling system is the regulation of cell shape. In the nematode Caenorhabditis elegans, membrane-bound semaphorins SMP-1/2 (SMPs) regulate the morphology of epidermal cells via their receptor plexin, PLX-1. In the larval male tail of the SMP-PLX-1 signaling mutants, the border between two epidermal cells, R1.p and R2.p, is displaced anteriorly, resulting in the anterior displacement of the anterior-most ray, ray 1, in the adult male. To elucidate how the intercellular signaling mediated by SMPs regulates the position of the intercellular border, we performed mosaic gene expression analyses by using infrared laser-evoked gene operator (IR-LEGO). We show that PLX-1 expressed in R1.p and SMP-1 expressed in R2.p are required for the proper positioning of ray 1. The result suggests that SMP signaling promotes extension, rather than retraction, of R1.p. This is in contrast to a previous finding that SMPs mediate inhibition of cell extension of vulval precursor cells, another group of epidermal cells of C. elegans, indicating the context dependence of cell shape control via the semaphorin signaling system.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Epidermis , Semaforinas , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Semaforinas/metabolismo , Semaforinas/genética , Epidermis/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Transducción de Señal , Comunicación Celular , Células Epidérmicas/metabolismo , Células Epidérmicas/citología , MasculinoRESUMEN
A 3-dimensional culture system of keratinocytes achieves cornification as a terminal differentiation that can mimic the formation of stratified epidermis. At the onset of keratinocyte differentiation, air-exposure treatment is essential for promotion. We have previously reported that the stimulation of differentiation is accompanied by downregulation of the transcriptional activity of the hypoxia-inducible factor (HIF) and also found that rocking treatment of cultured keratinocytes in the submerged condition restored their differentiation. A comparative study of cultured keratinocytes with and without rocking was then carried out to investigate the characteristics of the recovered differentiation by morphological and biochemical analyses. In addition, transcriptome analysis revealed the expected similar pattern between air-exposed and rocking cultures, including HIF-regulating transcripts. Furthermore, the promotive effect of rocking treatment was impaired under hypoxic culture conditions (1% O2). We showed that the restored promotion of differentiation by rocking culture is mainly due to the abrogation of transcriptional events by hypoxia.
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Diferenciación Celular , Epidermis , Queratinocitos , Queratinocitos/citología , Queratinocitos/metabolismo , Humanos , Epidermis/metabolismo , Hipoxia de la Célula , Células Cultivadas , Células Epidérmicas/citología , Células Epidérmicas/metabolismo , Técnicas de Cultivo Tridimensional de Células/métodos , Perfilación de la Expresión Génica , Técnicas de Cultivo de Célula/métodosRESUMEN
Autologous skin grafting is a standard treatment for skin defects such as burns. No artificial skin substitutes are functionally equivalent to autologous skin grafts. The cultured epidermis lacks the dermis and does not engraft deep wounds. Although reconstituted skin, which consists of cultured epidermal cells on a synthetic dermal substitute, can engraft deep wounds, it requires the wound bed to be well-vascularized and lacks skin appendages. In this study, we successfully generate complete skin grafts with pluripotent stem cell-derived epidermis with appendages on p63 knockout embryos' dermis. Donor pluripotent stem cell-derived keratinocytes encroach the embryos' dermis by eliminating p63 knockout keratinocytes based on cell-extracellular matrix adhesion mediated cell competition. Although the chimeric skin contains allogenic dermis, it is engraftable as long as autologous grafts. Furthermore, we could generate semi-humanized skin segments by human keratinocytes injection into the amnionic cavity of p63 knockout mice embryos. Niche encroachment opens the possibility of human skin graft production in livestock animals.
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Dermis , Queratinocitos , Ratones Noqueados , Trasplante de Piel , Animales , Trasplante de Piel/métodos , Queratinocitos/citología , Queratinocitos/trasplante , Humanos , Dermis/citología , Dermis/trasplante , Ratones , Epidermis/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/trasplante , Piel Artificial , Células Epidérmicas/trasplante , Células Epidérmicas/citología , Matriz Extracelular/metabolismo , Piel/citologíaRESUMEN
Numerous long non-coding RNAs (lncRNAs) were shown to have a functional impact on cellular processes such as human epidermal homeostasis. However, the mechanism of action for many lncRNAs remains unclear to date. Here, we report that lncRNA LINC00941 regulates keratinocyte differentiation on an epigenetic level through association with the NuRD complex, one of the major chromatin remodelers in cells. We find that LINC00941 interacts with NuRD-associated MTA2 and CHD4 in human primary keratinocytes. LINC00941 perturbation changes MTA2/NuRD occupancy at bivalent chromatin domains in close proximity to transcriptional regulator genes, including the EGR3 gene coding for a transcription factor regulating epidermal differentiation. Notably, LINC00941 depletion resulted in reduced NuRD occupancy at the EGR3 gene locus, increased EGR3 expression in human primary keratinocytes, and increased abundance of EGR3-regulated epidermal differentiation genes in cells and human organotypic epidermal tissues. Our results therefore indicate a role of LINC00941/NuRD in repressing EGR3 expression in non-differentiated keratinocytes, consequentially preventing premature differentiation of human epidermal tissues.
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Diferenciación Celular , Epidermis , Histona Desacetilasas , Queratinocitos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , ARN Largo no Codificante , Proteínas Represoras , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Diferenciación Celular/genética , Queratinocitos/metabolismo , Queratinocitos/citología , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Epidermis/metabolismo , Histona Desacetilasas/metabolismo , Histona Desacetilasas/genética , Proteína 3 de la Respuesta de Crecimiento Precoz/genética , Proteína 3 de la Respuesta de Crecimiento Precoz/metabolismo , Epigénesis Genética , Células Epidérmicas/metabolismo , Células Epidérmicas/citología , Cromatina/metabolismo , Cromatina/genética , Regulación de la Expresión Génica , Células CultivadasRESUMEN
Intricate signaling systems are required to maintain homeostasis and promote differentiation in the epidermis. Receptor tyrosine kinases are central in orchestrating these systems in epidermal keratinocytes. In particular, EPHA2 and EGFR transduce distinct signals to dictate keratinocyte fate, yet how these cell communication networks are integrated has not been investigated. Our work shows that loss of EPHA2 impairs keratinocyte stratification, differentiation, and barrier function. To determine the mechanism of this dysfunction, we drew from our proteomics data of potential EPHA2 interacting proteins. We identified EGFR as a high-ranking EPHA2 interactor and subsequently validated this interaction. We found that when EPHA2 is reduced, EGFR activation and downstream signaling are intensified and sustained. Evidence indicates that prolonged SRC association contributes to the increase in EGFR signaling. We show that hyperactive EGFR signaling underlies the differentiation defect caused by EPHA2 knockdown because EGFR inhibition restores differentiation in EPHA2-deficient 3-dimensional skin organoids. Our data implicate a mechanism whereby EPHA2 restrains EGFR signaling, allowing for fine tuning in the processes of terminal differentiation and barrier formation. Taken together, we purport that crosstalk between receptor tyrosine kinases EPHA2 and EGFR is critical for epidermal differentiation.
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Diferenciación Celular , Epidermis , Receptores ErbB , Queratinocitos , Receptor EphA2 , Transducción de Señal , Receptor EphA2/metabolismo , Receptor EphA2/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Humanos , Queratinocitos/metabolismo , Queratinocitos/citología , Epidermis/metabolismo , Células Cultivadas , Ratones , Animales , Células Epidérmicas/metabolismo , Células Epidérmicas/citología , Proteómica/métodosRESUMEN
Diabetic foot ulcers (DFUs) pose a significant threat to the health and well-being of individuals with diabetes, often leading to lower limb amputations. Fortunately, epidermal stem cell therapy offers hope for improving the treatment of DFUs. By leveraging 3D culture techniques, the scalability of stem cell manufacturing can be greatly enhanced. In particular, using bioactive materials and scaffolds can promote the healing potential of cells, enhance their proliferation, and facilitate their survival. Furthermore, 3D tissue-mimicking cultures can accurately replicate the complex interactions between cells and extracellular matrix, thereby ensuring that the stem cells are primed for therapeutic application. To ensure the safety and quality of these stem cells, it is essential to adhere to good manufacturing practice (GMP) principles during cultivation. This chapter provides a comprehensive overview of the step-by-step process for GMP-based 3D epidermal stem cell cultivation, thus laying the groundwork for developing reliable regenerative medicine therapies.
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Pie Diabético , Células Madre , Pie Diabético/terapia , Pie Diabético/patología , Humanos , Células Madre/citología , Células Epidérmicas/citología , Técnicas de Cultivo Tridimensional de Células/métodos , Técnicas de Cultivo de Célula/métodos , Andamios del Tejido/química , Medicina Regenerativa/métodos , Trasplante de Células Madre/métodos , Proliferación Celular , Cicatrización de Heridas , Células Cultivadas , Diferenciación CelularRESUMEN
The mammalian integumentary system, including skin and its appendages, serves as a protective barrier for the body. During development, skin epidermis undergoes rapid cell division and differentiation to form multiple stratified layers of keratinocytes. Concurrently the epidermis also gives rise to hair follicles that invaginate into the dermis. In adult skin, the hair follicle undergoes cyclic regeneration fueled by hair follicle stem cells located in the bulge. Three-dimensional and high-resolution imaging of these structures using whole-mount immunofluorescent staining allows to better characterize epidermal progenitors and stem cells.
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Epidermis , Técnica del Anticuerpo Fluorescente , Folículo Piloso , Animales , Folículo Piloso/citología , Folículo Piloso/metabolismo , Ratones , Epidermis/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Animales Recién Nacidos , Coloración y Etiquetado/métodos , Piel/citología , Piel/metabolismo , Células Epidérmicas/citología , Células Epidérmicas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismoRESUMEN
To investigate the effect of human adipose tissue-derived multilineage-differentiating stress-enduring (Muse) cells on the oxidative stress injury of human epidermal melanocytes (HEMs) in vitro. HEMs were treated with H2O2 to establish an oxidative stress injury model and then were co-cultured with adipose tissue-derived Muse cells. Immunohistochemistry, flow cytometry and Western blotting were used to assess changes in autophagy flux, apoptosis, expression of melanin synthesis related proteins and proliferation of melanocytes. Our findings demonstrate that co-culture with Muse cells significantly increased the tolerance of HEMs to oxidative stress, enhanced autophagy flux and reduced apoptosis. The expression of proteins related to the formation of melanin increased as did cell proliferation. Treatment with the autophagy inhibitor, 3-methyladenine (3MA), partially counteracted the improvement of oxidative stress tolerance in melanocytes elicited by co-culture with Muse cells. Muse cells promote autophagy and oxidative stress tolerance of melanocytes.
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Tejido Adiposo , Autofagia , Melanocitos , Células Madre Mesenquimatosas , Tejido Adiposo/citología , Humanos , Femenino , Células Epidérmicas/citología , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Melanocitos/patología , Estrés Oxidativo , Apoptosis , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Técnicas de Cocultivo , Exosomas/metabolismo , Peróxido de Hidrógeno/farmacología , Proliferación Celular , AdultoRESUMEN
BACKGROUND: The biological activity and regenerative medicine of bone marrow mesenchymal stem cells (BMSCs) have been focal topics in the broad fields of diabetic wound repair. However, the molecular mechanisms are still largely elusive for other cellular processes that are regulated during BMSC treatment. Our previous studies have shown that hypoxia is not only a typical pathological phenomenon of wounds but also exerts a vital regulatory effect on cellular bioactivity. In this study, the beneficial effects of hypoxic BMSCs on the cellular behaviors of epidermal cells and diabetic wound healing were investigated. METHOD: The viability and secretion ability of hypoxic BMSCs were detected. The autophagy, proliferation and migration of HaCaT cells cultured with hypoxic BMSCs-derived conditioned medium were assessed by estimating the expression of autophagy-related proteins, MTS, EdU proliferation and scratch assays. And the role of the SMAD signaling pathway during hypoxic BMSC-evoked HaCaT cell autophagy was explored through a series of in vitro gain- and loss-of-function experiments. Finally, the therapeutic effects of hypoxic BMSCs were evaluated using full-thickness cutaneous diabetic wound model. RESULTS: First, we demonstrated that hypoxic conditions intensify HIF-1α-mediated TGF-ß1 secretion by BMSCs. Then, the further data revealed that BMSC-derived TGF-ß1 was responsible for the activation of epidermal cell autophagy, which contributed to the induction of epidermal cell proliferation and migration. Here, the SMAD signaling pathway was identified as downstream of BMSC-derived TGF-ß1 to regulate HaCaT cell autophagy. Moreover, the administration of BMSCs to diabetic wounds increased epidermal autophagy and the rate of re-epithelialization, leading to accelerated healing, and these effects were significantly attenuated, accompanied by the downregulation of Smad2 phosphorylation levels due to TGF-ß1 interference in BMSCs. CONCLUSION: In this report, we present evidence that uncovers a previously unidentified role of hypoxic BMSCs in regulating epidermal cell autophagy. The findings demonstrate that BMSC-based treatment by restoring epidermal cell autophagy could be an attractive therapeutic strategy for diabetic wounds and that the process is mediated by the HIF-1α/TGF-ß1/SMAD pathway.
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Diabetes Mellitus , Subunidad alfa del Factor 1 Inducible por Hipoxia , Células Madre Mesenquimatosas , Proteínas Smad , Factor de Crecimiento Transformador alfa , Cicatrización de Heridas , Autofagia , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Diabetes Mellitus/terapia , Células Epidérmicas/citología , Células Epidérmicas/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate the number, functional status, and responsiveness of hair follicle bulge stem cells. This is linked to gene expression programs related to the reinforcement of the identity and the quiescent state of normal SSCs. By contrast, in the case of cancer stem cells, they promote transcriptomal programs associated with the identity, activation state, and cytoskeletal remodeling. These results underscore the role of these Rho exchange factors in the regulation of normal and tumor epidermal stem cells.