RESUMO
PURPOSE: While meibomian gland dysfunction (MGD) is widely recognized as a major cause of evaporative dry eye disease, little is known about normal gland differentiation and lipid synthesis or the mechanism underlying gland atrophy and abnormal lipid secretion. The purpose of this study was to use single-cell and spatial transcriptomics to probe changes in cell composition, differentiation, and gene expression associated with two murine models of MGD: age-related gland atrophy in wild-type mice and altered meibum quality in acyl-CoA wax alcohol acyltransferase 2 (Awat2) knockout (KO) mice. METHODS: Young (6 month) and old (22 month) wild type, C57Bl/6 mice and young (3 month) and old (13 month) Awat2 KO mice were used in these studies. For single-cell analysis, the tarsal plate was dissected from the upper and lower eyelids, and single cells isolated and submitted to the UCI Genomic Core, while for the spatial analysis frozen tissue sections were shipped to Resolve Biosciences on dry ice and sections probed in duplicate using a meibomian gland specific, 100 gene Molecular Chartography panel. RESULTS: Analysis of gene expression patterns identified the stratified expression of lipogenic genes during meibocyte differentiation, which may control the progressive synthesis of meibum lipids; an age-related decrease in meibocytes; and increased immune cell infiltration. Additionally, we detected unique immune cell populations in the Awat2 KO mouse suggesting activation of psoriasis-like, inflammatory pathways perhaps caused by ductal dilation and hyperplasia. CONCLUSION: Together these findings support novel mechanism controlling gland function and dysfunction.
RESUMO
Early wound intervention and closure is critical for reducing infection and improving aesthetic and functional outcomes for patients with acute burn wounds and nonthermal full-thickness skin defects. Treatment of partial-thickness burns or full-thickness injuries with autologous skin cell suspension (ASCS) achieves robust wound closure while limiting the amount of donor skin compared with standard autografting. A Next Generation Autologous Cell Harvesting Device (NG-ACHD) was developed to standardize the preparation process for ASCS to ensure biological attributes are obtained known to correlate with well-established safety and performance data. This study compared ASCS prepared using the NG-ACHD and ACHD following the manufacturer's guidance, evaluating cellular yields, viability, apoptotic activity, aggregates, phenotypes and functional capacity. Non-inferiority was established for all biological attributes tested and comparable healing trajectories were demonstrated using an in vitro skin regeneration model. In addition to standardization, the NG-ACHD also provides workflow efficiencies with the potential to decrease training requirements and increase the ease of incorporation and utilization of ASCS in clinical practice.
Assuntos
Transplante Autólogo , Cicatrização , Humanos , Transplante Autólogo/métodos , Cicatrização/fisiologia , Queimaduras/terapia , Regeneração/fisiologia , Transplante de Pele/métodos , Coleta de Tecidos e Órgãos/métodos , Coleta de Tecidos e Órgãos/instrumentação , Células Epidérmicas , Epiderme/fisiologia , Masculino , FemininoRESUMO
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.
Assuntos
Queratinócitos , Pele , Humanos , Diferenciação Celular , Mãos , Células Cultivadas , EpidermeRESUMO
The migrating keratinocyte wound front is required for skin wound closure. Despite significant advances in wound healing research, we do not fully understand the molecular mechanisms that orchestrate collective keratinocyte migration. Here, we show that, in the wound front, the epidermal transcription factor Grainyhead like-3 (GRHL3) mediates decreased expression of the adherens junction protein E-cadherin; this results in relaxed adhesions between suprabasal keratinocytes, thus promoting collective cell migration and wound closure. Wound fronts from mice lacking GRHL3 in epithelial cells (Grhl3-cKO) have lower expression of Fascin-1 (FSCN1), a known negative regulator of E-cadherin. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on wounded keratinocytes shows decreased wound-induced chromatin accessibility near the Fscn1 gene in Grhl3-cKO mice, a region enriched for GRHL3 motifs. These data reveal a wound-induced GRHL3/FSCN1/E-cadherin pathway that regulates keratinocyte-keratinocyte adhesion during wound-front migration; this pathway is activated in acute human wounds and is altered in diabetic wounds in mice, suggesting translational relevance.
Assuntos
Proteínas de Transporte/genética , Adesão Celular/genética , Proteínas de Ligação a DNA/genética , Epiderme/lesões , Regulação da Expressão Gênica , Proteínas dos Microfilamentos/genética , RNA/genética , Fatores de Transcrição/genética , Cicatrização , Animais , Proteínas de Transporte/biossíntese , Linhagem Celular , Movimento Celular/genética , Proteínas de Ligação a DNA/biossíntese , Modelos Animais de Doenças , Epiderme/metabolismo , Epiderme/patologia , Queratinócitos/metabolismo , Camundongos , Proteínas dos Microfilamentos/biossíntese , Fatores de Transcrição/biossínteseRESUMO
Regulatory regions of the genome harbor most genetic variance associated with skin diseases. Because gene-regulatory networks are cell-type and cell-state specific and are subject to variation from the genetic background, current cell models that link genetic variation to gene expression are imperfect. Emerging single-cell genomics approaches may provide a new approach to understand the genetics of common skin diseases.
Assuntos
Psoríase , Dermatopatias , Redes Reguladoras de Genes , Genômica , HumanosRESUMO
The fusion of digits or toes, syndactyly, can be part of complex syndromes, including van der Woude syndrome. A subset of van der Woude cases is caused by dominant-negative mutations in the epithelial transcription factor Grainyhead like-3 (GRHL3), and Grhl3-/-mice have soft-tissue syndactyly. Although impaired interdigital cell death of mesenchymal cells causes syndactyly in multiple genetic mutants, Grhl3-/- embryos had normal interdigital cell death, suggesting alternative mechanisms for syndactyly. We found that in digit separation, the overlying epidermis forms a migrating interdigital epithelial tongue (IET) when the epithelium invaginates to separate the digits. Normally, the non-adhesive surface periderm allows the IET to bifurcate as the digits separate. In contrast, in Grhl3-/- embryos, the IET moves normally between the digits but fails to bifurcate because of abnormal adhesion of the periderm. Our study identifies epidermal developmental processes required for digit separation.
Assuntos
Movimento Celular , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Membro Anterior/embriologia , Sindactilia/genética , Dedos do Pé/embriologia , Fatores de Transcrição/genética , Animais , Células Epiteliais/fisiologia , Membro Anterior/anormalidades , Membro Anterior/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Dedos do Pé/anormalidadesRESUMO
During tissue development, transcription factors bind regulatory DNA regions called enhancers, often located at great distances from the genes they regulate, to control gene expression. The enhancer landscape during embryonic stem cell differentiation has been well characterized. By contrast, little is known about the shared and unique enhancer regulatory mechanisms in different ectodermally derived epithelial cells. Here we use ChIP sequencing (ChIP-seq) to identify domains enriched for the histone marks histone H3 lysine 4 trimethylation, histone H3 lysine 4 monomethylation, and histone H3 lysine 27 acetylation (H3K4me3, H3K4me1, and H3K27ac) and define, for the first time, the super enhancers and typical enhancers active in primary human corneal epithelial cells. We show that regulatory regions are often shared between cell types of the ectodermal lineage and that corneal epithelial super enhancers are already marked as potential regulatory domains in embryonic stem cells. Kruppel-like factor (KLF) motifs were enriched in corneal epithelial enhancers, consistent with the important roles of KLF4 and KLF5 in promoting corneal epithelial differentiation. We now show that the Kruppel family member KLF7 promotes the corneal progenitor cell state; on many genes, KLF7 antagonized the corneal differentiation-promoting KLF4. Furthermore, we found that two SNPs linked previously to corneal diseases, astigmatism, and Stevens-Johnson syndrome fall within corneal epithelial enhancers and alter their activity by disrupting transcription factor motifs that overlap these SNPs. Taken together, our work defines regulatory enhancers in corneal epithelial cells, highlights global gene-regulatory relationships shared among different epithelial cells, identifies a role for KLF7 as a KLF4 antagonist in corneal epithelial cell differentiation, and explains how two SNPs may contribute to corneal diseases.