Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro
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ABSTRACT:
PROVIDER: PRJNA853776 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19887957.fastq.gz | Fastqsanger.gz | |||
| SRR19887958.fastq.gz | Fastqsanger.gz | |||
| SRR19887959.fastq.gz | Fastqsanger.gz | |||
| SRR19887960.fastq.gz | Fastqsanger.gz | |||
| SRR19887961.fastq.gz | Fastqsanger.gz |
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Similar Datasets
Project description:Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications.
2023-01-17 | GSE207130 | GEO
Project description:Rho GTPases integrate control of cell structure and adhesion with downstream signaling events. In keratinocytes, RhoA is activated at early times of differentiation and plays an essential function in establishment of cell-cell adhesion. We report here that, surprisingly, Rho signaling suppresses downstream gene expression events associated with differentiation. Similar inhibitory effects are exerted by a specific Rho effector, CRIK (Citron kinase), which is selectively down-modulated with differentiation, thereby allowing the normal process to occur. The suppressing function of Rho/CRIK on differentiation is associated with induction of KyoT1/2, a LIM domain protein gene implicated in integrin-mediated processes and/or Notch signaling. Like activated Rho and CRIK, elevated KyoT1/2 expression suppresses differentiation. Thus, Rho signaling exerts an unexpectedly complex role in keratinocyte differentiation, which is coupled with induction of KyoT1/2, a LIM domain protein gene with a potentially important role in control of cell self renewal. Keywords: epithelial cell differentiation | gene expression | Rho signaling | stem cell
2005-08-18 | GSE3162 | GEO
Project description:We previously demonstrated that the lifespan of primary human keratinocytes could be extended indefinitely by culture in the presence of the Rho kinase (ROCK) inhibitor Y-27632. Here, gene expression analysis of primary human epidermal keratinocytes cells grown in the presence of Y-27632 demonstrates that ROCK inhibition primarily inhibits keratinocyte differentiation.
2014-03-31 | GSE52515 | GEO
Project description:Rho GTPases integrate control of cell structure and adhesion with downstream signaling events. In keratinocytes, RhoA is activated at early times of differentiation and plays an essential function in establishment of cellcell adhesion. We report here that, surprisingly, Rho signaling suppresses downstream gene expression events associated with differentiation. Similar inhibitory effects are exerted by a specific Rho effector, CRIK (Citron kinase), which is selectively down-modulated with differentiation, thereby allowing the normal process to occur. The suppressing function of Rho/CRIK on differentiation is associated with induction of KyoT1/2, a LIM domain protein gene implicated in integrin-mediated processes and/or Notch signaling. Like activated Rho and CRIK, elevated KyoT1/2 expression suppresses differentiation. Thus, Rho signaling exerts an unexpectedly complex role in keratinocyte differentiation, which is coupled with induction of KyoT1/2, a LIM domain protein gene with a potentially important role in control of cell self renewal. Experiment Overall Design: Total RNA from primary mouse keratinocytes infected with adeno-GFP, adeno-RhoV14, or adeno-CRIK-SK for 48 h was used for biotin-labeled cRNA probe preparation and hybridization to Affymetrix U74A gene chips. cRNA probes from each condition were tested in duplicate.
2007-09-07 | E-GEOD-3162 | biostudies-arrayexpress
Project description:We quantified protein changes associated with long-term Rho-kinase inhibition, using SILAC proteomics in conjugation with long-termH1152 treatment of mouse embryonic fibroblast cells.
2016-01-18 | PXD002521 | Pride
Project description:IntroductionWe previously demonstrated that the lifespan of primary human keratinocytes could be extended indefinitely by culture in the presence of the Rho kinase (ROCK) inhibitor Y-27632. This technique has proven to be very useful in diverse areas of basic and clinical research.MethodsIn this follow-up study we determine whether the continual presence of Y-27632 is required for sustained proliferation. We also test whether different ROCK inhibitors can be used for this technique and whether it can also promote indefinite proliferation of animal keratinocytes. We measure keratinocyte gene expression, proliferation, behaviour and lifespan in the presence and absence of Y-27632.ResultsWe demonstrate that the extension of lifespan observed by culture of keratinocytes in the presence of fibroblast feeders and a ROCK inhibitor is reversible and that cells senesce gradually when the inhibitor is removed from the medium. Conversely, keratinocytes that are close to the end of their replicative life span can be revived by ROCK inhibition. We demonstrate that different inhibitors of ROCK can also efficiently extend the lifespan of human keratinocytes and that ROCK inhibition extends the lifespan of animal keratinocytes derived from mouse and bovine epithelia. Gene expression analysis of human epidermal keratinocytes cells grown in the presence of Y-27632 demonstrates that ROCK inhibition primarily inhibits keratinocyte differentiation. Live-imaging of keratinocytes cultured with ROCK inhibitors show that the effect of ROCK inhibition on cellular proliferation is immediate and ROCK inhibited cells proliferate rapidly without differentiation or stratification.ConclusionsROCK inhibition rapidly and conditionally induces indefinite proliferation of keratinocytes. This method has far-reaching applications for basic research, as well as for regenerative and personalized medicine.
| S-EPMC4055106 | biostudies-literature
Project description:In this study, we use the epidermis as a model system to elucidate the cellular effects and signaling feedback sequelae of mTORC1 loss-of-function in epithelial tissue. In mice with conditional epidermal loss of mTORC1 components Rheb or Rptor, we find that mTORC1 loss-of-function unexpectedly results in a profound skin barrier defect with epidermal abrasions, blistering and early postnatal lethality, due to a thinned epidermis with decreased desmosome expression and incomplete biochemical differentiation. Impaired cell-cell adhesion in the context of mTORC1 loss-of-function is caused by constitutive activation of Rho kinase (ROCK) signaling with increased cytoskeletal tension, and inhibition or silencing of ROCK1 is sufficient to rescue keratinocyte adhesion and biochemical differentiation. mTORC1 loss-of-function results in marked feedback up-regulation of upstream TGF-β signaling, triggering ROCK activity and its downstream effects on desmosomal gene expression. These findings elucidate a novel role for mTORC1 in the regulation of epithelial barrier formation, cytoskeletal tension and cell adhesion and underscore the complexity of signaling feedback after mTORC1 inhibition
2020-04-14 | GSE102026 | GEO
Project description:Corneal epithelial stem cells are located in the limbus, the junction between the cornea and the conjunctiva. A limbal epithelium model in vitro would be useful for the study of epithelial stem cells, as well as improving the quality of cultivated epithelial sheets for the treatment of limbal stem cell deficiency. In this study, we succeeded in constructing a limbal epithelium-like structure that could be maintained for at least 5 months in vitro. We modified conventional medium by replacing epidermal growth factor with keratinocyte growth factor (KGF) and adding Y-27632, a rho kinase inhibitor. Using this medium, epithelial cells freshly isolated from human limbus were cocultured with human mesenchymal stem cell-derived feeder cells. Cells formed a stratified layer without air exposure, and both basal and suprabasal layers maintained their unique morphologies for up to 5 months. Basal layers expressed the progenitor marker p63 uniformly and K15 heterogeneously. Expressions of PAX6, K3, and K12 indicated that cell sheets underwent normal differentiation in the corneal epithelium lineage. Although medium was changed daily after day 7, cell debris was observed every day, suggesting that cell sheets underwent turnover. Furthermore, secondary colonies were observed from cells dissociated from 1-month and 3-month cultured sheets. In conclusion, human limbal epithelial cell sheet cultures with KGF and Y-27632 maintained stratification, high expression of both stem/progenitor markers and differentiation markers, and colony-forming cells long-term. This protocol may be useful as an in vitro limbal epithelial model for basic studies.
| S-EPMC3785260 | biostudies-literature
Project description:Analysis of genome wide gene expression changes over a time course of 24 hours induced by the Rho kinase inhibitor Fasudil in primary mouse astrocyte cultures.
2011-05-10 | GSE25829 | GEO
Project description:Keratinocytes are a targeted cells for infection of human papillomaviruses (HPV). But, there are very limited information for gene regulation of human karatinocytes by HPV infection. Y-27632, a rho-kinase inhibitor, stimulates proparation of keratinocytes. We identified genes, which are regulated by a drug for karatinocyte proparation (Y-27632). mRNA profiles of primary HEKs regulated by a rho kinase inhibitor (Y-27632, Y-drug) were generated by microarray analysis using Illumina BeadChip.
2014-03-31 | E-GEOD-52515 | biostudies-arrayexpress