Unknown,Transcriptomics,Genomics,Proteomics

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MRNA differential expression analysis in a human ex vivo model of chronic wounds

ABSTRACT: To study the fibroblast to myofibroblast differentiation in a normal or pathological situation, NHDF (Normal human dermal fibroblast) obtained from two different healthy donors (donors A and B) were either left untreated (T-E-) either treated with TGF-beta alone (T+E-), or with exudate from chronic wounds (T-E+) or both (T+E+). For each different treatment we performed mRNA deep sequencing 3 times : twice with cells from donor A and once with cells from donor B.We focused our study on gene expression profile as a representation of cell fate. We performed mRNA deep sequencing analysis of the 4 different conditions. For each condition, mRNA deep sequencing was performed 3 times : twice with cells from donor A and once with cells from donor B.Comparing the mRNA abundance between the different treatments, we identified 3 lists of genes characterizing the different gene expression states: 171 genes in List I representing the genes differentially expressed during normal fibroblast to myofibroblast differentiation, 409 genes in List II representing the genes differentially expressed upon exudate-only treatment and 1006 genes in List III representing the genes differentially expressed upon the combination of exudate and TGF-beta treatments. 4 different treatments are compared : (T-E-) NHFD left untreated, (T+E-) NHDF treated with TGFbeta for 4 days, (T-E+) NHDF treated with exudate for 4 days and (T+E+) NHDF treated with both TGFbeta and exudate. NHDF : Normal Human Dermal Fibroblast

ORGANISM(S): Homo sapiens

SUBMITTER: Claire Dugast-Darzacq

PROVIDER: E-GEOD-49159 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Pericryptal myofibroblasts in the colon and rectum play an important role in regulating the normal colorectal stem cell niche and facilitating tumour progression. Myofibroblasts have previously mostly been distinguished from normal fibroblasts only by the expression of Î± smooth muscle actin (Î±SMA). We now identify AOC3, a surface monoamine oxidase, as a new marker of myofibroblasts by showing that it is the target protein of the myofibroblast reacting monoclonal antibody (mAb), PR2D3. The normal and tumour tissue distribution and the cell line reactivity of AOC3 match that expected for myofibroblasts. We have shown that the surface expression of AOC3 is sensitive to digestion by trypsin and collagenase and that anti-AOC3 antibodies can be used for FACS sorting of myofibroblasts obtained by non-enzymatic procedures. Whole genome microarray mRNA expression profiles of myofibroblasts and skin fibroblasts revealed four additional genes that are significantly expressed differentially between these two cell types; NKX2-3 and LRRC17 are expressed in myofibroblasts and SHOX2 and TBX5 in skin fibroblasts. Transforming Growth Factor Î² (TGFÎ²) substantially down-regulated AOC3 expression in myofibroblasts but not in skin fibroblasts, in which it dramatically increased the expression of Î±SMA. A knockdown of NKX2-3 in myofibroblasts caused a decrease of myofibroblast-related gene expression and an increased expression of the fibroblast associated gene, SHOX2, suggesting that NKX2-3 is a key mediator for maintaining myofibroblast characteristics. Our results show that colorectal myofibroblasts, as defined by the expression of AOC3, NKX2-3 and other markers, are a distinctly different cell type from TGFÎ² activated fibroblasts. colorectal myofibroblast specific markers and expression profiles were sought by comparing four primary myofibroblast cultures to a panel of four dermal and foreskin fibroblast cell lines Four primary myofibroblast cultures established from adult human colon compared to four skin fibroblast cell lines to identify intestinal myofibroblast specific markers

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MRNA differential expression analysis in a human ex vivo model of chronic wounds

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