RESUMEN
Rationale: Stem cells self-organize to form organoids that generate mini-organs that resemble the physiologically-developed ones. The mechanism by which the stem cells acquire the initial potential for generating mini-organs remains elusive. Here we used skin organoids as an example to study how mechanical force drives initial epidermal-dermal interaction which potentiates skin organoids to regenerate hair follicles. Methods: Live imaging analysis, single-cell RNA-sequencing analysis, and immunofluorescence were used to analyze the contractile force of dermal cells in skin organoids. Bulk RNA-sequencing analysis, calcium probe detection, and functional perturbations were used to verify that calcium signaling pathways respond to the contractile force of dermal cells. In vitro mechanical loading experiment was used to prove that the stretching force triggers the epidermal Piezo1 expression which negatively regulates dermal cell attachment. Transplantation assay was used to test the regenerative ability of skin organoids. Results: We found that dermal cell-derived contraction force drives the movement of dermal cells surrounding the epidermal aggregates to trigger initial mesenchymal-epithelial interaction (MEI). In response to dermal cell contraction force, the arrangement of the dermal cytoskeleton was negatively regulated by the calcium signaling pathway which further influences dermal-epidermal attachment. The native contraction force generated from the dermal cell movement exerts a stretching force on the adjacent epidermal cells, activating the stretching force sensor Piezo1 in the epidermal basal cells during organoid culture. Epidermal Piezo1 in turn drives strong MEI to negatively regulate dermal cell attachment. Proper initial MEI by mechanical-chemical coupling during organoid culture is required for hair regeneration upon transplantation of the skin organoids into the back of the nude mice. Conclusion: Our study demonstrated that mechanical-chemical cascade drives the initial event of MEI during skin organoid development, which is fundamental to the organoid, developmental, and regenerative biology fields.
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Folículo Piloso , Piel , Ratones , Animales , Ratones Desnudos , Organoides , ARN , Canales IónicosRESUMEN
INTRODUCTION: Although it has been reported that the antidiabetic drug metformin has multiple extra-hypoglycemic activities, such as anti-oxidation, antiaging, and even antitumor, topical metformin also can induce hair regeneration, but the precise mechanism involved in that process is still unclear. OBJECTIVES: The aim of this study was to assess the effect of metformin on hair growth in a mouse hair-follicle reconstitution model generated by in vitro self-assembled three-dimensional aggregates of epidermal and dermal cells (DCs) (3D aggregates). METHODS: Epidermal cells and DCs were isolated and cultured from the mouse skin of 50 C57BL/6 mouse pups (1-day-old). For tracing the distribution of DCs during the self-assembly process of 3D aggregates, the DCs were labeled with Vybrant Dil Cell-Labeling Solution and mixed with epidermal cells at a 1:1 ratio. Formed 3D aggregates were treated with 10 mM metformin and then were grafted into recipient BALB/c nude mice. The biomarkers (hepatocyte growth factor [HGF], prominin-1 [CD133], alkaline phosphatase [ALP], ß-catenin, and SRY-box transcription factor 2 [SOX2]) associated with the hair-inductive activity of DCs were detected in the grafted skin tissues and in cultured 3D aggregates treated with metformin using immunofluorescent staining, quantitative real-time RT-PCR (qRT-PCR), and Western blotting. Furthermore, the expression levels of CD133 were also examined in DCs with different passage numbers using qRT-PCR and Western blotting. RESULTS: Metformin directly stimulates the activity of ALP of cultured 3D aggregates, upregulates both the protein and mRNA expression levels of molecular markers (HGF, CD133, ALP, ß-catenin, and SOX2), and improves the survival rate of reconstituted hair follicles. Moreover, we also found that metformin increases the expression of CD133 in DCs thus maintaining their trichogenic capacity that would normally be lost by serial subculture. CONCLUSIONS: These results suggest that metformin can promote hair follicle regeneration in vitro through upregulation of the hair-inductive capability of DCs, warranting further evaluation in the clinical treatment of male or female pattern hair loss.
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Metformina , beta Catenina , Animales , Células Cultivadas , Femenino , Cabello , Folículo Piloso , Masculino , Metformina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , beta Catenina/genéticaRESUMEN
BACKGROUND: Laminitis, an inflammation of the claw laminae, is one of the major causes of bovine lameness, which can lead to enormous economic losses and animal welfare problems in dairy farms. Angelica polysaccharide (AP) is proved to possess anti-inflammatory properties. But the role of AP on inflammatory response of the claw dermal cells has not been reported. The aim of this study was to investigate the anti-inflammatory effects of AP on lipopolysaccharide (LPS)-induced primary claw dermal cells of dairy cow and clarify the potential mechanisms. In the current research, the primary claw dermal cells were exposed to gradient concentrations of AP (10, 50, 100 µg/mL) in the presence of 10 µg/mL LPS. The levels of cytokines and nitric oxide (NO) were detected with ELISA and Griess colorimetric method. The mRNA expressions of TLR4, MyD88 and chemokines were measured with qPCR. The activation of NF-κB and MAPK signaling pathways was detected with western blotting. RESULTS: The results indicated that AP reduced the production of inflammatory mediators (TNF-α, IL-1ß, IL-6 and NO), downregulated the mRNA expression of TLR4, MyD88 and some pro-inflammatory chemokines (CCL2, CCL20, CXCL2, CXCL8, CXCL10), and suppressed the NF-κB and MAPK signaling pathways evidenced by inhibition of the phosphorylation of IκBα, p65 and ERK, JNK, p38. CONCLUSIONS: Our results demonstrated that AP may exert its anti-inflammatory effects on claw dermal cells of dairy cow by regulating the NF-κB and MAPK signaling pathways.
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Angelica/química , Pezuñas y Garras/citología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/metabolismo , Animales , Bovinos , Enfermedades de los Bovinos/tratamiento farmacológico , Células Cultivadas , Dermis/citología , Dermis/efectos de los fármacos , Femenino , Pezuñas y Garras/efectos de los fármacos , Inflamación/inducido químicamente , Lipopolisacáridos/toxicidad , Extractos Vegetales/farmacologíaRESUMEN
In the present study, the influence of the infection with equine herpesvirus type 1 (non-neuro-pathogenic and neuropathogenic strains of EHV-1) on the morphology and distribution of mitochondrial network in equine dermal cell line was investigated. Our results indicate that EHV-1-infection caused changes in the mitochondrial morphology manifested mostly by fission and reactive oxygen species generation.
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Dermis/citología , Herpesvirus Équido 1/fisiología , Caballos , Mitocondrias/virología , Animales , Línea Celular , Replicación ViralRESUMEN
BACKGROUND: Laminitis is considered as one of the most important causes of hoof lameness in dairy cows, which can lead to enormous economic losses. However, the etiology and pathogenesis of laminitis have not been clarified yet. Besides, it is of great significant to find alternative herbs for the prevention and treatment of dairy hooves to avoid the antibiotic abuse. In this study, the primary hoof dermal cells of dairy cows were isolated, the inflammatory model was induced by LPS, and treated with silymarin to find whether silymarin has protective effect on the inflammatory dermal cells. The viability of dermal cells, the levels of IL-1ß and TNF-α, the degree of p65 NF-κB and p38 MAPK phosphorylation, the expressions of CYP3A4 and CYP1A1 were measured. RESULTS: Hoof dermal cells of dairy cows were successfully isolated and cultured by tissue adherent culture method. Certain concentrations of LPS can increase the levels of IL-1ß and TNF-α, promote the phosphorylation of p65 NF-κB and p38 MAPK, and inhibit the mRNA expressions of CYP3A4 and CYP1A1. The optimal concentration for LPS to establish a hoof dermal cells inflammatory model was 10 µg/mL. Certain concentrations of silymarin can markedly decrease the secretions of IL-1ß and TNF-α, inhibit the phosphorylation of p65 NF-κB and p38 MAPK, and promote the mRNA expressions of CYP3A4 and CYP1A1 in LPS-induced dermal inflammatory model. CONCLUSIONS: LPS can be used for inducing the hoof dermal cells inflammatory model of dairy cows. Silymarin has protective effects on the LPS-induced inflammatory model.
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Sistema Enzimático del Citocromo P-450/metabolismo , Pezuñas y Garras/citología , Silimarina/farmacología , Factor de Transcripción ReIA/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Bovinos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Dermis/citología , Dermis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Pezuñas y Garras/efectos de los fármacos , Inflamación/inducido químicamente , Interleucina-1beta/metabolismo , Lipopolisacáridos/toxicidad , Fosforilación , Factor de Transcripción ReIA/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
BACKGROUND: Available options for correction of facial volume loss, such as synthetic fillers, autologous fat and cultured fibroblasts, have limitations viz. temporary effect and high cost. AIM: To assess the use of a novel technique, autologous non-cultured dermal cell suspension transplantation, for correction of localized facial volume loss due to inflammatory pathologies. METHODS: It was a pilot study conducted in the Dermatology Outpatient Department, All India Institute of Medical Sciences (AIIMS), New Delhi, India. Autologous non-cultured dermal cell suspension was transplanted in a total of 10 patients, out of which 5 had predominantly dermal loss and the rest had predominantly lipoatrophy. The donor tissue from the gluteal region was digested into a single cell suspension using collagenase-1 and injected into the recipient area. The outcome was assessed subjectively by patients and investigators and objectively using ultrasonography. Cell count, viability testing and measurement of mesenchymal stem cells were also done. RESULTS: On assessment of patients, the median improvement in the predominantly dermal atrophy group at 3 and 6 months was 70% (range: 10-90%) and 80% (range: 0-90%), respectively, and in the predominantly lipoatrophy group, 0% (range: 0-40) and 0% (range: 0-50), respectively. Mean thickness of dermis + subcutis at the baseline was 1.835 mm (range: 0.89-6.04 mm), which increased to 2.912 mm (range: 0.88-7.07 mm, P = 0.03) at 6 months. LIMITATIONS: Our pilot study has some limitations such as small sample size and heterogeneity of the recruited patients. CONCLUSIONS: Autologous non-cultured dermal cell suspension transplantation appears to be safe and effective in localized facial dermal defects because of inflammatory pathologies, but not effective in deeper defects.