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Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632

ABSTRACT: Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632

PROVIDER: PRJNA488732 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7773470.fastq.gz	Fastqsanger.gz
	SRR7773480.fastq.gz	Fastqsanger.gz
	SRR7773490.fastq.gz	Fastqsanger.gz
	SRR7773500.fastq.gz	Fastqsanger.gz
	SRR7773471.fastq.gz	Fastqsanger.gz

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Gene regulation in human epidermal keratinocytes by a Rho kinase inhibitor (Y-27632)

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

Gene regulation in human epidermal keratinocytes by a Rho kinase inhibitor (Y-27632)

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.

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Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632

Project description:Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense may be hindered by invasive isolation methods and/or improper culture conditions to support stem cell maintenance and other potential mechanisms contributing to long-term subcultivation in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases.

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RAS induced senescence of skin keratinocytes is mediated through Rho kinase

Project description:RAS induced senescence of skin keratinocytes is mediated through Rho kinase

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Short-Term Treatment with Rho-Associated Kinase Inhibitor Preserves Keratinocyte Stem Cell Characteristics In Vitro

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.

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Immortalization and characterization of rat lingual keratinocytes in a high-calcium and feeder-free culture system using ROCK inhibitor Y-27632

Project description:The study tried the identify some differentially expressed proteins in rat lingual keratinocytes treated with or without the ROCK inhibitor Y-27632. In brief, rat lingual keratinocytes were treated with or without Y-27632 for 36 h and were subjected to proteomic analysis.

2022-06-23 | PXD025812 | JPOST Repository

Transcription profiling of mouse primary keratinocytes expressing Rho/CRIK to study the impact of Rho signaling pathway on gene expression.

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

TRANSCRIPTOMIC PROFILING OF ASTROCYTES TREATED WITH THE RHO KINASE INHIBITOR FASUDIL REVEALS CYTOSKELETAL AND PRO-SURVIVAL RESPONSESTRANSFORMATION INDUCED BY THE RHO KINASE INHIBITOR FASUDIL.

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

Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632.

| S-EPMC6135349 | biostudies-literature

TRANSCRIPTOMIC PROFILING OF ASTROCYTES TREATED WITH THE RHO KINASE INHIBITOR FASUDIL REVEALS CYTOSKELETAL AND PRO-SURVIVAL RESPONSESTRANSFORMATION INDUCED BY THE RHO KINASE INHIBITOR FASUDIL.

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. Total RNA was extracted from primary astrocyte cultures prepared from mouse forebrain from 1.5 day old mice and treated with Fasudil or vehicle control for 2, 6, 12 or 24 hours.

2011-05-10 | E-GEOD-25829 | biostudies-arrayexpress

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