In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line.
Ontology highlight
ABSTRACT:
SUBMITTER: Deyrieux AF
PROVIDER: S-EPMC2267500 | biostudies-other | 2007 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:ObjectiveOligodendrocytes (OL) are the glial cell type in the CNS that are responsible for myelin formation. The ability to culture OLs in vitro has provided critical insights into the mechanisms underlying their function. However, primary OL cultures are tedious to obtain, difficult to propagate and are not easily conducive to genetic manipulation. To overcome these obstacles, researchers have generated immortalized OL like cell lines derived from various species. One such cell line is the mouse Oli-neu line which is thought to recapitulate characteristics of OLs in early stages of maturity. They have been extensively utilized in multiple studies as surrogates for OLs, especially in analyzing epigenetic modifications and regulatory pathways in the OL lineage.ResultsIn this report we present the development of optimized culture media and growth conditions that greatly facilitate the differentiation of Oli-neu cells. Oli-neu cells differentiated using these new protocols exhibit a higher expression of myelin related genes and increased branching, both of which are defining characteristics of mature OLs, when compared to previous culture protocols. We envision that these new culture conditions will greatly facilitate the use of Oli-neu cells and enhance their ability to recapitulate the salient features of primary OLs.
| S-EPMC10401818 | biostudies-literature
Project description:Radioactive elements, such as tritium, have been released into the ocean in large quantities as a result of the reactor leakage accident. In this study, an MTT assay demonstrated that the viability of HacaT cells decreased after tritiated water treatment. Bioinformatics analysis was used to analyze gene changes in the HacaT cells. The sequencing results showed 267 significantly differentially expressed genes (DEGs), and GO enrichment analysis showed that the DEGs were mainly divided into three parts. The KEGG pathway analysis showed that the up-regulated DEGs were involved in Wnt and other pathways, while the down-regulated DEGs were involved in Jak-STAT and others. A Western blot assay was used to verify the parts of the sequencing results. This study was the first to explore the mechanism of tritiated water on HacaT cells using Transcriptome analysis. The results will provide a theoretical basis for the study of tritiated water hazard mechanisms.
| S-EPMC10045445 | biostudies-literature
Project description:Vitamin C plays an important role in neutralizing toxic free radicals formed during oxidative metabolism or UV exposure of human skin. This study was performed to investigate the mechanisms that regulate the homoeostasis of vitamin C in HaCaT cells by identifying the events involved in the transport and in the reduction of dehydroascorbic acid. Dehydroascorbic acid accumulated to a greater extent and faster compared with ascorbic acid; its transport appeared to be mediated by hexose transporters and was entirely distinct from ascorbic acid transport. Dehydroascorbate reductase activity was unaffected by glutathione depletion, although it was sensitive to thiol protein reagents. These observations, as well as the subcellular distribution of this enzymic activity and the cofactor specificity, indicate that thioredoxin reductase and lipoamide dehydrogenase play an important role in this reduction process. HaCaT cells were able to enhance their dehydroascorbic acid reductase activity in response to oxidative stress.
| S-EPMC1220802 | biostudies-other
Project description:BackgroundRecent studies have revealed that particulate matter induces inflammation, oxidative stress, and several skin diseases. Experimental results have also shown that negative air ions are highly effective in removing particulate matter-induced inflammation.ObjectiveThe present study aimed to investigate whether negative air ions can inhibit inflammatory responses and reduce oxidative stress in HaCaT cells exposed to particulate matters.MethodsHaCaT cells were treated with particulate matter in the presence or absence of negative air ions and the viability was evaluated by the MTT assay. Reactive oxygen species (ROS) generation was quantified by the dichlorodihydrofluorescein diacetate assay. The expression of genes and proteins was analyzed by real-time polymerase chain reaction and Western blot. Levels of inflammatory cytokines were quantified by enzyme-linked immunosorbent assay.ResultsNegative air ions were observed to downregulate the mRNA and protein levels of particulate matter-induced pro-inflammatory cytokines in HaCaT cells. In addition, negative air ion treatment suppressed particulate matter-induced intracellular ROS generation, p38 mitogen-activated protein kinase activation, and activator protein 1 (c-Fos and c-Jun) activation.ConclusionOur findings indicate that negative air ions exert anti-inflammatory and antioxidant effects in HaCaT cells exposed to particulate matter. Therefore, negative air ions can be used for the prevention and treatment of particulate matter-related inflammatory skin diseases.
| S-EPMC8082002 | biostudies-literature
Project description:Aim of the study was to characterize at a molecular level (changes in transcriptomes) the effect of monosodium urate crystal (MSU) on HaCaT keratinocyte cell line. This was adressed by using a culture model. The HaCaT cell line (human keratinocytes) was stimulated by MSU (1mg/mL) vs control for 12 hrs. By using genome-wide expression profiling, we identified deregulation of functionally relevant gene networks.
2015-10-29 | GSE65931 | GEO
Project description:Aim of the study was to characterize at a molecular level (changes in transcriptomes) the effect of monosodium urate crystal (MSU) on HaCaT keratinocyte cell line. This was adressed by using a culture model. The HaCaT cell line (human keratinocytes) was stimulated by MSU (1mg/mL) vs control for 12 hrs. By using genome-wide expression profiling, we identified deregulation of functionally relevant gene networks. HaCaT were obtained from Cell Lines Service (Eppelheim, Germany) and grown in DMEM medium (PAN biotech, Aidenbach, Germany) supplemented with 10% FBS (Life Technology, Grand Island, NY, USA), L-glutamine and non-essential amino acid. Before the treatment HaCaT cells were cultured in serum-free medium for 12hrs. HaCaT were treated with MSU (1mg/ml) vs DMEM control for 12hrs then submitted to RNA extration and gene expression profiling. Triplicate experiments were performed: HaCaT control (n=3), MSU-treated (n=3).
2015-10-29 | E-GEOD-65931 | biostudies-arrayexpress
Project description:Histamines suppress epidermal keratinocyte differentiation. Previously, we reported that konjac ceramide (kCer) suppresses histamine-stimulated cell migration of HaCaT keratinocytes. kCer specifically binds to Nrp1 and does not interact with histamine receptors. The signaling mechanism of kCer in HaCaT cells is also controlled by an intracellular signaling cascade activated by the Sema3A-Nrp1 pathway. In the present study, we demonstrated that kCer treatment induced HaCaT keratinocyte differentiation after migration of immature cells. kCer-induced HaCaT cell differentiation was accompanied by some features of keratinocyte differentiation markers. kCer induced activating phosphorylation of p38MAPK and c-Fos, which increased the protein levels of involucrin that was the latter differentiation marker. In addition, we demonstrated that the effects of both kCer and histamines are regulated by an intracellular mechanism of Rac1 activation/RhoA inhibition downstream of the Sema3A/Nrp1 receptor and histamine/GPCR pathways. In summary, the effects of kCer on cell migration and cell differentiation are regulated by cascade crosstalk between downstream Nrp1 and histamine-GPCR pathways in HaCaT cells.
| S-EPMC8772740 | biostudies-literature
Project description:BackgroundChronic arsenic exposure via drinking water is associated with an increased risk of developing cancer and noncancer chronic diseases. Pre-mRNAs are often subject to alternative splicing, generating mRNA isoforms encoding functionally distinct protein isoforms. The resulting imbalance in isoform species can result in pathogenic changes in critical signaling pathways. Alternative splicing as a mechanism of arsenic-induced toxicity and carcinogenicity is understudied.ObjectiveThis study aimed to accurately profile differential alternative splicing events in human keratinocytes induced by chronic arsenic exposure that might play a role in carcinogenesis.MethodsIndependent quadruplicate cultures of immortalized human keratinocytes (HaCaT) were maintained continuously for 28 wk with 0 or 100 nM sodium arsenite. RNA-sequencing (RNA-Seq) was performed with poly(A) RNA isolated from cells harvested at 7, 19, and 28 wk with subsequent replicate multivariate analysis of transcript splicing (rMATS) analysis to detect and quantify differential alternative splicing events. Reverse transcriptase-polymerase chain reaction (RT-PCR) for selected alternative splicing events was performed to validate RNA-Seq predictions. Functional enrichment was performed by gene ontology (GO) analysis of the differential alternative splicing event data set at each time point.ResultsAt least 600 differential alternative splicing events were detected at each time point tested, comprising all the five main types of alternative splicing and occurring in both open reading frames (ORFs) and untranslated regions (UTRs). Based on functional relevance ELK4, SHC1, and XRRA1 were selected for validation of predicted alternative splicing events at 7 wk by RT-PCR. Densitometric analysis of RT-PCR data corroborated the rMATS predicted alternative splicing for all three events. Protein expression validation of the selected alternative splicing events was challenging given that very few isoform-specific antibodies are available. GO analysis demonstrated that the enriched terms in differential alternatively spliced mRNAs changed dynamically with the time of exposure. Notably, RNA metabolism and splicing regulation pathways were enriched at the 7-wk time point, when the greatest number of differentially alternatively spliced mRNAs are detected. Our preliminary proteomic analysis demonstrated that the expression of the canonical isoforms of the splice regulators DDX42, RMB25, and SRRM2 were induced upon chronic arsenic exposure, corroborating the splicing predictions.DiscussionThese results using cultures of HaCaT cells suggest that arsenic exposure disrupted an alternative splice factor network and induced time-dependent genome-wide differential alternative splicing that likely contributed to the changing proteomic landscape in arsenic-induced carcinogenesis. However, significant challenges remain in corroborating alternative splicing data at the proteomic level. https://doi.org/10.1289/EHP9676.
| S-EPMC8785870 | biostudies-literature
Project description:In vitro differentiation or expansion of stem and progenitor cells under chemical stimulation or genetic manipulation is used for understanding the molecular mechanisms of cell differentiation and self-renewal. However, concerns around the cell identity of in vitro-cultured cells exist. Bioinformatics methods, which rely heavily on signatures of cell types, have been developed to estimate cell types in bulk samples. The Tabula Muris Senis project provides an important basis for the comprehensive identification of signatures for different cell types. Here, we identified 46 cell type-specific (CTS) gene clusters for 83 mouse cell types. We conducted Gene Ontology term enrichment analysis on the gene clusters and revealed the specific functions of the relevant cell types. Next, we proposed a simple method, named CTSFinder, to identify different cell types between bulk RNA-Seq samples using the 46 CTS gene clusters. We applied CTSFinder on bulk RNA-Seq data from 17 organs and from developing mouse liver over different stages. We successfully identified the specific cell types between organs and captured the dynamics of different cell types during liver development. We applied CTSFinder with bulk RNA-Seq data from a growth factor-induced neural progenitor cell culture system and identified the dynamics of brain immune cells and nonimmune cells during the long-time cell culture. We also applied CTSFinder with bulk RNA-Seq data from reprogramming induced pluripotent stem cells and identified the stage when those cells were massively induced. Finally, we applied CTSFinder with bulk RNA-Seq data from in vivo and in vitro developing mouse retina and captured the dynamics of different cell types in the two development systems. The CTS gene clusters and CTSFinder method could thus serve as promising toolkits for assessing the cell identity of in vitro culture systems.
| S-EPMC8267371 | biostudies-literature
Project description:The dermatophyte Trichophyton rubrum is the major fungal pathogen of skin, hair, and nails that uses keratinized substrates as the primary nutrients during infection. Few strategies are available that permit a better understanding of the molecular mechanisms involved in the interaction of T. rubrum with the host because of the limitations of models mimicking this interaction. Dual RNA-seq is a powerful tool to unravel this complex interaction since it enables simultaneous evaluation of the transcriptome of two organisms. Using this technology in an in vitro model of co-culture, this study evaluated the transcriptional profile of genes involved in fungus-host interactions in 24 h. Our data demonstrated the induction of glyoxylate cycle genes, ERG6 and TERG_00916, which encodes a carboxylic acid transporter that may improve the assimilation of nutrients and fungal survival in the host. Furthermore, genes encoding keratinolytic proteases were also induced. In human keratinocytes (HaCat) cells, the SLC11A1, RNASE7, and CSF2 genes were induced and the products of these genes are known to have antimicrobial activity. In addition, the FLG and KRT1 genes involved in the epithelial barrier integrity were inhibited. This analysis showed the modulation of important genes involved in T. rubrum⁻host interaction, which could represent potential antifungal targets for the treatment of dermatophytoses.
| S-EPMC6070946 | biostudies-literature