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1.
Nat Commun ; 12(1): 5127, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34493721

RESUMO

Intricate color patterns are a defining aspect of morphological diversity in the Felidae. We applied morphological and single-cell gene expression analysis to fetal skin of domestic cats to identify when, where, and how, during fetal development, felid color patterns are established. Early in development, we identify stripe-like alterations in epidermal thickness preceded by a gene expression pre-pattern. The secreted Wnt inhibitor encoded by Dickkopf 4 plays a central role in this process, and is mutated in cats with the Ticked pattern type. Our results bring molecular understanding to how the leopard got its spots, suggest that similar mechanisms underlie periodic color pattern and periodic hair follicle spacing, and identify targets for diverse pattern variation in other mammals.


Assuntos
Gatos/genética , Regulação da Expressão Gênica no Desenvolvimento , Pigmentação/genética , Animais , Animais Domésticos , Gatos/crescimento & desenvolvimento , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Genótipo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratinócitos/metabolismo , Mutação , Fenótipo , Análise de Célula Única , Pele/anatomia & histologia , Pele/crescimento & desenvolvimento , Pele/metabolismo , Via de Sinalização Wnt
2.
Methods Mol Biol ; 2319: 61-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331243

RESUMO

The blood vascular system is a tree-like hierarchical branching structure and needs to function even before fully established. Abnormal formation of blood vessels results in embryonic lethality and also contributes to the pathogenesis of a number of human diseases, including cancer metastasis. To understand the molecular events associated with blood vessel formation, we established a fluorescence staining-based protocol on mouse embryonic skin. We harvested mouse embryonic skin and performed whole-mount staining. The reconstructed three-dimensional vascular structure provided detailed information on angiogenesis.


Assuntos
Células Endoteliais/citologia , Imuno-Histoquímica/métodos , Neovascularização Fisiológica , Pele/irrigação sanguínea , Pele/citologia , Coloração e Rotulagem/métodos , Animais , Células Endoteliais/metabolismo , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Pele/crescimento & desenvolvimento , Pele/metabolismo
3.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199374

RESUMO

BACKGROUND: Skinboosters represent the latest category of hyaluronan (HA) hydrogels released for aesthetic purposes. Different from originally developed gels, they are intended for more superficial injections, claiming a skin rejuvenation effect through hydration and possibly prompting biochemical effects in place of the conventional volumetric action. Here, three commercial skinboosters were characterized to unravel the scientific basis for such indication and to compare their performances. METHODS: Gels were evaluated for water-soluble/insoluble-HA composition, rheology, hydration, cohesivity, stability and effect, in vitro, on human dermal fibroblasts towards the production of extracellular matrix components. RESULTS: Marked differences in the insoluble-hydrogel amount and in the hydrodynamic parameters for water-soluble-HA chains were evidenced among the gels. Hydration, rigidity and cohesivity also varied over a wide range. Sensitivity to hyaluronidases and Reactive Oxygen Species was demonstrated allowing a stability ranking. Slight differences were found in gels' ability to prompt elastin expression and in ColIV/ColI ratio. CONCLUSIONS: A wide panel of biophysical and biochemical parameters for skinboosters was provided, supporting clinicians in the conscious tuning of their use. Data revealed great variability in gels' behavior notwithstanding the same clinical indication and unexpected similarities to the volumetric formulations. Data may be useful to improve customization of gel design toward specific uses.


Assuntos
Ácido Hialurônico/química , Hialuronoglucosaminidase/genética , Hidrogéis/química , Pele/efeitos dos fármacos , Elastina/química , Fibroblastos/efeitos dos fármacos , Humanos , Hialuronoglucosaminidase/química , Injeções , Espécies Reativas de Oxigênio/química , Rejuvenescimento/fisiologia , Reologia , Pele/crescimento & desenvolvimento , Pele/patologia , Envelhecimento da Pele/genética , Viscosidade
4.
Biomater Sci ; 9(15): 5227-5236, 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34190240

RESUMO

Scarless skin regeneration remains a challenge due to the complicated microenvironment involved in wound healing. Here, the hydrophobic drug, asiaticoside (AC), was loaded inside silk nanofiber hydrogels to achieve bioactive and injectable matrices for skin regeneration. AC was dispersed in aqueous silk nanofiber hydrogels with retention of biological functions that regulated inflammatory reactions and vascularization in vitro. After implantation in full-thickness wound defects, these AC-laden hydrogel matrices achieved scarless wound repair. Inflammatory reactions and angiogenesis were regulated during inflammation and remodeling, which was responsible for wound regeneration similar to normal skin. Both in vitro and in vivo studies demonstrated promising applications of these AC-laden silk hydrogels towards scarless tissue regeneration.


Assuntos
Nanofibras , Regeneração , Seda , Pele/crescimento & desenvolvimento , Animais , Cicatriz/prevenção & controle , Humanos , Hidrogéis , Inflamação/tratamento farmacológico , Masculino , Camundongos , Células RAW 264.7 , Ratos Sprague-Dawley , Triterpenos
5.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071405

RESUMO

In vitro skin tissue engineering is challenging due to the manifold differences between the in vivo and in vitro conditions. Yet, three-dimensional (3D) human skin equivalents (HSEs) are able to mimic native human skin in many fundamental aspects. However, the epidermal lipid barrier formation, which is essential for the functionality of the skin barrier, remains compromised. Recently, HSEs with an improved lipid barrier formation were generated by (i) incorporating chitosan in the dermal collagen matrix, (ii) reducing the external oxygen level to 3%, and (iii) inhibiting the liver X receptor (LXR). In this study, we aimed to determine the synergic effects in full-thickness models (FTMs) with combinations of these factors as single-, double-, and triple-targeted optimization approaches. The collagen-chitosan FTM supplemented with the LXR inhibitor showed improved epidermal morphogenesis, an enhanced lipid composition, and a better lipid organization. Importantly, barrier functionality was improved in the corresponding approach. In conclusion, our leading optimization approach substantially improved the epidermal morphogenesis, barrier formation, and functionality in the FTM, which therefore better resembled native human skin.


Assuntos
Células Epidérmicas/metabolismo , Epiderme/metabolismo , Morfogênese , Pele/metabolismo , Engenharia Tecidual/métodos , Células Cultivadas , Quitosana/metabolismo , Cromatografia Líquida , Colágeno/metabolismo , Epiderme/crescimento & desenvolvimento , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Metabolismo dos Lipídeos , Lipídeos/análise , Receptores X do Fígado/metabolismo , Espectrometria de Massas , Espalhamento a Baixo Ângulo , Pele/citologia , Pele/crescimento & desenvolvimento , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
6.
J Exp Med ; 218(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33561194

RESUMO

T cells in human skin play an important role in the immune defense against pathogens and tumors. T cells are present already in fetal skin, where little is known about their cellular phenotype and biological function. Using single-cell analyses, we identified a naive T cell population expressing αß and γδ T cell receptors (TCRs) that was enriched in fetal skin and intestine but not detected in other fetal organs and peripheral blood. TCR sequencing data revealed that double-positive (DP) αßγδ T cells displayed little overlap of CDR3 sequences with single-positive αß T cells. Gene signatures, cytokine profiles and in silico receptor-ligand interaction studies indicate their contribution to early skin development. DP αßγδ T cells were phosphoantigen responsive, suggesting their participation in the protection of the fetus against pathogens in intrauterine infections. Together, our analyses unveil a unique cutaneous T cell type within the native skin microenvironment and point to fundamental differences in the immune surveillance between fetal and adult human skin.


Assuntos
Feto/imunologia , Vigilância Imunológica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Pele/embriologia , Pele/imunologia , Linfócitos T/imunologia , Adulto , Células Cultivadas , Citocinas/metabolismo , Voluntários Saudáveis , Humanos , Intestinos/embriologia , Intestinos/imunologia , Pessoa de Meia-Idade , RNA-Seq/métodos , Análise de Célula Única/métodos , Pele/crescimento & desenvolvimento , Transcriptoma
7.
Methods Mol Biol ; 2273: 151-158, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604851

RESUMO

The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is the progenitor of the outer epithelial component of the placenta and supports the fetus during intrauterine life. Our understanding of these events is limited, particularly in human, because of ethical and legal restrictions and availability of adequate in vitro models would be very advantageous. Here we describe a method that converts human fibroblasts into trophoblast-like cells, combining the use of 5-azacytidine-CR (5-aza-CR) to erase the original cell phenotype and a cocktail containing bone morphogenetic protein 4 (BMP4) with inhibitors of the Activin/Nodal/ERK signaling pathways, to drive erased fibroblasts into the trophoblastic differentiation. This innovative method uses very easily accessible cells to derive trophoblast-like cells and it can be useful to study embryo implantation disorders related to aging.


Assuntos
Técnicas de Cultura de Células/métodos , Fibroblastos/citologia , Trofoblastos/citologia , Ativinas/antagonistas & inibidores , Animais , Azacitidina/farmacologia , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Implantação do Embrião , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteína Nodal/antagonistas & inibidores , Placenta/citologia , Gravidez , Transdução de Sinais , Pele/citologia , Pele/crescimento & desenvolvimento
8.
Sci Rep ; 11(1): 1779, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469169

RESUMO

Pannexin 3 (Panx3), a member of the gap junction pannexin family is required for the development of hard tissues including bone, cartilage and teeth. However, the role of Panx3 in skin development remains unclear. Here, we demonstrate that Panx3 regulates skin development by modulating the transcription factor, Epiprofin (Epfn). Panx3-/- mice have impaired skin development and delayed hair follicle regeneration. Loss of Panx3 in knockout mice and suppression by shRNA both elicited a reduction of Epfn expression in the epidermis. In cell culture, Panx3 overexpression promoted HaCaT cell differentiation, cell cycle exit and enhanced Epfn expression. Epfn-/- mice and inhibition of Epfn by siRNA showed no obvious differences of Panx3 expression. Furthermore, Panx3 promotes Akt/NFAT signaling pathway in keratinocyte differentiation by both Panx3 ATP releasing channel and ER Ca2+ channel functions. Our results reveal that Panx3 has a key role factor for the skin development by regulating Epfn.


Assuntos
Conexinas/metabolismo , Folículo Piloso/crescimento & desenvolvimento , Fatores de Transcrição Kruppel-Like/metabolismo , Organogênese/genética , Pele/crescimento & desenvolvimento , Animais , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Conexinas/genética , Regulação da Expressão Gênica/genética , Células HaCaT , Humanos , Queratinócitos/citologia , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Knockout , Organogênese/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/genética
9.
Int J Mol Sci ; 22(2)2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33430241

RESUMO

Several types of 3-dimensional (3D) biological matrices are employed for clinical and surgical applications, but few indications are available to guide surgeons in the choice among these materials. Here we compare the in vitro growth of human primary fibroblasts on different biological matrices commonly used for clinical and surgical applications and the activation of specific molecular pathways over 30 days of growth. Morphological analyses by Scanning Electron Microscopy and proliferation curves showed that fibroblasts have different ability to attach and proliferate on the different biological matrices. They activated similar gene expression programs, reducing the expression of collagen genes and myofibroblast differentiation markers compared to fibroblasts grown in 2D. However, differences among 3D matrices were observed in the expression of specific metalloproteinases and interleukin-6. Indeed, cell proliferation and expression of matrix degrading enzymes occur in the initial steps of interaction between fibroblast and the investigated meshes, whereas collagen and interleukin-6 expression appear to start later. The data reported here highlight features of fibroblasts grown on different 3D biological matrices and warrant further studies to understand how these findings may be used to help the clinicians choose the correct material for specific applications.


Assuntos
Diferenciação Celular/genética , Colágeno Tipo I/genética , Dermatopatias/cirurgia , Pele/crescimento & desenvolvimento , Movimento Celular/genética , Proliferação de Células/genética , Matriz Extracelular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Interleucina-6/genética , Metaloproteases/genética , Microscopia Eletrônica de Varredura , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Cultura Primária de Células , Pele/metabolismo , Dermatopatias/metabolismo
10.
Methods Mol Biol ; 2193: 1-11, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32808253

RESUMO

Cutaneous wound healing is an intricate and multifaceted process. Despite these complexities, the distinct phases of wound healing provide a unique opportunity to evaluate the roles of different targets in these coordinated responses. This protocol details an in vivo wound healing assay to study the intersection of cellular, molecular, and systemic effector pathways. The role of certain proteins in the wound healing process can be efficiently explored in vivo through the generation of tissue-specific deficient mice. This approach, although optimized for use with animal models displaying epithelial deficiencies, can be used for other tissue-specific deficiencies, and utilizes simple and cost-effective methods, allowing investigators to precisely devise their experimental design. The coordination of immunological, epithelial, vascular, and microenvironmental factors in wound healing makes this technique a valuable tool for investigators across fields.


Assuntos
Bioensaio/métodos , Microambiente Celular/fisiologia , Pele/crescimento & desenvolvimento , Cicatrização/fisiologia , Animais , Modelos Animais de Doenças , Epiderme/crescimento & desenvolvimento , Camundongos
11.
Dokl Biochem Biophys ; 494(1): 252-255, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33119828

RESUMO

Expression of cell death regulators RIPK-1 and RIPK-3 in mouse and human hair follicle structures was studied by immunohistochemistry. At anagen and catagen stages of mouse hair follicle, RIPK-1+ cells were located in the inner root sheath, whereas RIPK-3+ cells were found in the inner and outer root sheath, dermal papilla, and interfollicular epidermis. RIPK-1 expression intensity was low in the early anagen and increased as mature anagen and catagen approached. RIPK-1+ and RIPK-3+ cells were also found in human hair follicle. It is assumed that the role of necroptosis markers in hair follicle life activity is independent of programmed cell death and that they may have yet unknown functions and take part in noncanonical signal cascades.


Assuntos
Folículo Piloso/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Pele/metabolismo , Animais , Apoptose/fisiologia , Folículo Piloso/crescimento & desenvolvimento , Humanos , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pele/crescimento & desenvolvimento
12.
J S Afr Vet Assoc ; 91(0): e1-e8, 2020 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-33054248

RESUMO

Accurate diet formulations are required to fulfil the nutrient requirements of birds in order to achieve optimal production. Knowing how the skin, nodule and feather production characteristics vary with diets of different nutrient densities will help in least-cost modelling. Feather growth and nodule development are factors that were previously neglected in ostrich diet formulation, both of which are essential for the development of a predictive production model. In this trial, 120 birds were placed in 15 pens. Varying energy regimes (high, medium and low) and accompanying protein and amino acid profile levels (level 1-5) were assigned ad libitum to each pen. A randomly selected bird from each pen was slaughtered at 1, 35, 63, 103, 159, 168 and 244 days of age. During the slaughter, each bird was weighed, stunned, exsanguinated, defeathered and eviscerated. Feathers from four regions of the skin were plucked and weighed. The shaft diameter of the wing feathers was measured. The nodule size of the tanned skin was measured for each slaughter age. The data were transformed to natural logarithms and regressed against the total feather weight and the total featherless empty body protein weight to set up allometric growth equations. A prediction equation to determine nodule size of the live bird was proposed. Feed cost optimisation is paramount, and results from this study will aid in setting up least-cost optimisation (simulation) formulation models.


Assuntos
Proteínas na Dieta/metabolismo , Ingestão de Energia , Plumas/crescimento & desenvolvimento , Struthioniformes/crescimento & desenvolvimento , Animais , Proteínas na Dieta/administração & dosagem , Relação Dose-Resposta a Droga , Distribuição Aleatória , Pele/crescimento & desenvolvimento , Struthioniformes/metabolismo
13.
Plast Reconstr Surg ; 146(4): 792-798, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32970001

RESUMO

BACKGROUND: Tissue expansion relies on the ability of skin to grow in response to sustained mechanical strain. This study focuses on correlation of cellular and histologic changes with skin growth and deformation during tissue expansion. METHODS: Tissue expanders were placed underneath the skin of five Yucatan minipigs and inflated with one fill of 60 cc of saline 1 hour, 24 hours, 3 days, and 7 days before the animals were killed, or two fills of either 30 cc or 60 cc at 10 and 3 days or 14 and 7 days before the animals were killed. Skin biopsy specimens and three-dimensional photographs were used to calculate skin growth and stretch according to the authors' novel finite element analysis model. RESULTS: The mitotic index of keratinocytes in the basal layer increased 1 hour after stimulus was applied (4 percent) (p = 0.022), peaked at approximately day 3 (26 percent) (p < 0.0001), and tapered by day 7 (12.5 percent) (p = 0.012) after tissue expansion. The authors demonstrated that it is the volume per fill rather than the total volume in the expander that scales the magnitude of response. Lastly, the authors demonstrated that the ratio of deformation attributable to growth versus stretch (Fgrowth/Fstretch) after 60 cc of tissue expansion fill was 1.03 at 1 hour, 0.82 at 1 day, 0.85 at day 3, and 0.95 at 7 days. CONCLUSIONS: Peak cell proliferation occurred 3 days after tissue expansion fill and is scaled in response to stimulus magnitude. The growth component of deformation equilibrates to the stretch component at day 7, as cell proliferation has started to translate to skin growth.


Assuntos
Modelos Estatísticos , Pele/crescimento & desenvolvimento , Expansão de Tecido/métodos , Animais , Feminino , Modelos Animais , Tamanho do Órgão , Pele/anatomia & histologia , Suínos , Porco Miniatura , Fatores de Tempo
14.
Sci Rep ; 10(1): 15991, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994433

RESUMO

Tissue expansion procedures (TE) utilize mechanical forces to induce skin growth and regeneration. While the impact of quick mechanical stimulation on molecular changes in cells has been studied extensively, there is a clear gap in knowledge about sequential biological processes activated during long-term stimulation of skin in vivo. Here, we present the first genome-wide study of transcriptional changes in skin during TE, starting from 1 h to 7 days of expansion. Our results indicate that mechanical forces from a tissue expander induce broad molecular changes in gene expression, and that these changes are time-dependent. We revealed hierarchical changes in skin cell biology, including activation of an immune response, a switch in cell metabolism and processes related to muscle contraction and cytoskeleton organization. In addition to known mechanoresponsive genes (TNC, MMPs), we have identified novel candidate genes (SFRP2, SPP1, CCR1, C2, MSR1, C4A, PLA2G2F, HBB), which might play crucial roles in stretched-induced skin growth. Understanding which biological processes are affected by mechanical forces in TE is important for the development of skin treatments to maximize the efficacy and minimize the risk of complications during expansion procedures.


Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Pele/crescimento & desenvolvimento , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Modelos Biológicos , Análise de Sequência de RNA , Pele/química , Suínos , Fatores de Tempo , Expansão de Tecido , Sequenciamento Completo do Exoma
15.
Biomolecules ; 10(10)2020 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-32992554

RESUMO

Acute and chronic skin wounds due to burns, pressure injuries, and trauma represent a substantial challenge to healthcare delivery with particular impacts on geriatric, paraplegic, and quadriplegic demographics worldwide. Nevertheless, the current standard of care relies extensively on preventive measures to mitigate pressure injury, surgical debridement, skin flap procedures, and negative pressure wound vacuum measures. This article highlights the potential of adipose-, blood-, and cellulose-derived products (cells, decellularized matrices and scaffolds, and exosome and secretome factors) as a means to address this unmet medical need. The current status of this research area is evaluated and discussed in the context of promising avenues for future discovery.


Assuntos
Queimaduras/terapia , Exossomos/transplante , Hidrogéis/uso terapêutico , Cicatrização/genética , Queimaduras/patologia , Terapia Baseada em Transplante de Células e Tecidos/tendências , Celulose/uso terapêutico , Exossomos/genética , Humanos , Hidrogéis/química , Transplante de Células-Tronco Mesenquimais/tendências , Células-Tronco Mesenquimais/citologia , Pele/crescimento & desenvolvimento , Pele/lesões , Pele/metabolismo
16.
Sci Rep ; 10(1): 13920, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811876

RESUMO

The expression of hair features is an evolutionary adaptation resulting from interactions between many organisms and their environment. Elucidation of the mechanisms that underlie the expression of such traits is a topic in evolutionary biology research. Therefore, we assessed the de novo transcriptome of Atelerix albiventris at three developmental stages and compared gene expression profiles between abdomen hair and dorsal spine tissues. We identified 328,576 unigenes in our transcriptome, among which 4,435 were differentially expressed between hair- and spine-type tissues. Dorsal and abdomen skin tissues 5 days after birth were compared and the resulting DEGs were mainly enriched in keratin filament, epithelium cell differentiation, and epidermis development based on GO enrichment analysis, and tight junction, p53, and cell cycle signaling pathways based on KEGG enrichment analysis. MBP8, SFN, Wnt1 and KRT1 gene may involve in the development of hedgehog skin and its appendages. Strikingly, DEGs in hair-type tissues were also significantly enriched in immune-related terms and pathways with hair-type tissues exhibiting more upregulated immune genes than spine-type tissues. Our study provided a list of potential genes involved in skin appendage development and differentiation in A. albiventris, and the candidate genes provided valuable information for further studies of skin appendages.


Assuntos
Ouriços-Cacheiros/genética , Ouriços-Cacheiros/imunologia , Pele/metabolismo , Animais , China , Biologia Computacional/métodos , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Ontologia Genética , Cabelo/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anotação de Sequência Molecular/métodos , Transdução de Sinais/genética , Pele/crescimento & desenvolvimento , Transcriptoma/genética
17.
Immunity ; 53(2): 371-383.e5, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32673566

RESUMO

Cutaneous wound healing is associated with the unpleasant sensation of itching. Here we investigated the mechanisms underlying this type of itch, focusing on the contribution of soluble factors released during healing. We found high amounts of interleukin 31 (IL-31) in skin wound tissue during the peak of itch responses. Il31-/- mice lacked wound-induced itch responses. IL-31 was released by dermal conventional type 2 dendritic cells (cDC2s) recruited to wounds and increased itch sensory neuron sensitivity. Transfer of cDC2s isolated from late-stage wounds into healthy skin was sufficient to induce itching in a manner dependent on IL-31 expression. Addition of the cytokine TGF-ß1, which promotes wound healing, to dermal DCs in vitro was sufficient to induce Il31 expression, and Tgfbr1f/f CD11c-Cre mice exhibited reduced scratching and decreased Il31 expression in wounds in vivo. Thus, cDC2s promote itching during skin would healing via a TGF-ß-IL-31 axis with implications for treatment of wound itching.


Assuntos
Interleucinas/metabolismo , Células de Langerhans/fisiologia , Prurido/patologia , Células Receptoras Sensoriais/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Feminino , Humanos , Interleucinas/genética , Células de Langerhans/transplante , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Receptores de Interleucina/metabolismo , Pele/citologia , Pele/crescimento & desenvolvimento , Pele/lesões , Canais de Cátion TRPV/metabolismo , Cicatrização/fisiologia
18.
Int J Mol Sci ; 21(13)2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32629914

RESUMO

An important problem for researchers working in the field of dermatology is the preparation of the human skin equivalent (HSE). Here, we describe a simple and reliable protocol for preparing a skin model from the commercially available cell lines: keratinocytes, fibroblasts, and melanocytes. Importantly, in our 3D model, the keratinocytes are diverse that brings this model closer to the natural skin. For the production of HSE, we used available primary PCS-200-010, PCS-201-010, PCS-200-013, and immortalized CRL-4048 and CRL-4001 cell lines. We used genipin, which is necessary for collagen cross-linking and studied its cytotoxicity for keratinocytes and fibroblasts. The addition of 20 µM genipin reduced the shrinkage of the collagen in the constructs from 59% to 24% on day 12 of the culture of the construct. A higher concentration (80-200 µM) of genipin reduced shrinkage by 14% on average. Genipin in concentration 10 µM and below was not cytotoxic to the keratinocytes, and 150 µM and below to the fibroblasts. Hematoxylin and eosin staining showed that the morphology of HSEs was identical to that of native human skin. The immunohistochemical staining of the constructs showed the presence of vimentin-positive fibroblasts in the skin layer, while the melanocytes were in the epidermis and in the basal layer. We observed that the longer differentiation of constructs led to the higher secretion of GM-CSF, IL-10, IL-15, IL-1α, IL-6, IL-7, IL-8, and MCP-1. We also observed that the longer time of differentiation led to a more stable secretion of all analytes, which was reflected in the coefficient of variation. We described here a simple, reliable, and cost-effective production of the full-thickness human skin equivalents that can be used in the research and industry. With the global trend to decrease animal use for the research and testing, our HSE could be a useful testing tool and an alternative research model.


Assuntos
Técnicas de Cultura de Células/métodos , Pele/crescimento & desenvolvimento , Pele/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Colágeno/metabolismo , Derme/citologia , Células Epidérmicas/citologia , Epiderme/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Iridoides/farmacologia , Queratinócitos/metabolismo , Melanócitos/metabolismo , Modelos Biológicos , Pele Artificial
19.
Nature ; 584(7820): 268-273, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32728211

RESUMO

The ability of the skin to grow in response to stretching has been exploited in reconstructive surgery1. Although the response of epidermal cells to stretching has been studied in vitro2,3, it remains unclear how mechanical forces affect their behaviour in vivo. Here we develop a mouse model in which the consequences of stretching on skin epidermis can be studied at single-cell resolution. Using a multidisciplinary approach that combines clonal analysis with quantitative modelling and single-cell RNA sequencing, we show that stretching induces skin expansion by creating a transient bias in the renewal activity of epidermal stem cells, while a second subpopulation of basal progenitors remains committed to differentiation. Transcriptional and chromatin profiling identifies how cell states and gene-regulatory networks are modulated by stretching. Using pharmacological inhibitors and mouse mutants, we define the step-by-step mechanisms that control stretch-mediated tissue expansion at single-cell resolution in vivo.


Assuntos
Mecanotransdução Celular/fisiologia , Análise de Célula Única , Pele/citologia , Pele/crescimento & desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Redes Reguladoras de Genes/efeitos dos fármacos , Hidrogéis/administração & dosagem , Hidrogéis/farmacologia , Mecanotransdução Celular/efeitos dos fármacos , Mecanotransdução Celular/genética , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , RNA Mensageiro/genética , RNA-Seq , Pele/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Transativadores/antagonistas & inibidores , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição Genética/efeitos dos fármacos
20.
Nature ; 582(7812): 399-404, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494013

RESUMO

The skin is a multilayered organ, equipped with appendages (that is, follicles and glands), that is critical for regulating body temperature and the retention of bodily fluids, guarding against external stresses and mediating the sensation of touch and pain1,2. Reconstructing appendage-bearing skin in cultures and in bioengineered grafts is a biomedical challenge that has yet to be met3-9. Here we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use stepwise modulation of the transforming growth factor ß (TGFß) and fibroblast growth factor (FGF) signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During an incubation period of 4-5 months, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis and pigmented hair follicles that are equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking the neural circuitry associated with human touch. Single-cell RNA sequencing and direct comparison to fetal specimens suggest that the skin organoids are equivalent to the facial skin of human fetuses in the second trimester of development. Moreover, we show that skin organoids form planar hair-bearing skin when grafted onto nude mice. Together, our results demonstrate that nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo. We anticipate that our skin organoids will provide a foundation for future studies of human skin development, disease modelling and reconstructive surgery.


Assuntos
Cabelo/citologia , Cabelo/crescimento & desenvolvimento , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Pele/citologia , Animais , Ectoderma/citologia , Feminino , Cabelo/transplante , Cor de Cabelo , Folículo Piloso/citologia , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/inervação , Folículo Piloso/transplante , Cabeça , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Organoides/crescimento & desenvolvimento , Organoides/inervação , Organoides/transplante , RNA-Seq , Análise de Célula Única , Pele/crescimento & desenvolvimento , Pele/inervação , Transplante de Pele
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