Project description:Tissue fibrosis is a common pathway to organ injury and failure. It is characterized by an excessive deposition of extracellular matrix (ECM) in organs. Deciphering the fibrogenic processes is of utmost importance, as there are few effective therapies in fibrotic diseases 1. Systemic sclerosis (SSc) is a prototypical disease where fibroblasts (Fb) are key effector cells as they differentiate into myofibroblasts in response to chronic inflammation under the influence of transforming growth factor beta 1 (TGF-β1) pathway 2–4. In this study, we compared the proteome of primary Fb in different culture and stimulation conditions. Primary dermal normal human Fb were cultured at passage P3, P5 and P7 with and without Fetal Bovine Serum (FBS). At fifth passage, Fb were stimulated or not with different concentrations of recombinant human active TGF-β1 (0.04, 1 and 5 ng/mL) during 24, 48 and 72 hours.
Project description:Recent work has identified markers of fibroblast heterogeneity in human dermis. Transforming growth factor-β1 (TGF-β1) promotes fibroblast-to-myofibroblast differentiation, characterised by the expression of α-smooth muscle actin (α-SMA). Human dermal fibroblasts (hDF), treated with TGF-β1, were assayed for differentiation, proliferation and cell shape using the Operetta imaging system. One donor hDF, derived from female 64-year-old breast skin, expressed decreased levels of α-SMA protein. The gene expression profile of this donor hDF was determined using the Agilent microarray system. Four gene candidates (Asporin, ASPN; C-X-C motif chemokine ligand 1, CXCL1; Insulin-like growth factor 1, IGF1; and Wnt family member 4, WNT4) were chosen based on expression values and validated by TaqMan qPCR. Successful knockdown of IGF1 and WNT4 was achieved using MISSION shRNA-based lentiviral treatment. Fibroblast IGF1 knockdown (shIGF1) increased α-SMA mRNA and protein expression; no effect was seen with fibroblast WNT4 knockdown (shWNT4). Here I have characterised hDF phenotype and gene expression with regard to population heterogeneity. This work highlights the role of IGF-1 signalling on α-SMA expression and dermal fibroblast fate. Targeting IGF-1 signalling could provide therapeutic benefit for skin disorders involving aberrant wound healing and excessive fibrosis.
Project description:miRNA profiling of human H9-derived neural stem cells (H9-NSCs) comparing control human adult dermal fibroblasts (hDFs), SOX2-transduced human induced neural stem cells (hDF-iNSC (SOX2)), SOX2/HMGA2-transduced human induced neural stem cells (hDF-iNSC (SOX2/HMGA2)). Goal was to determine the global miRNA expression between the groups. H9-NSC vs hDF vs hDF-iNSC(SOX2) vs hDF-iNSC(SOX2/HMGA2)
Project description:Dermal fibroblasts deposit type I collagen, the dominant extracellular matrix molecule found in skin, during early postnatal development. Coincident with this biosynthetic program, fibroblasts proteolytically remodel pericellular collagen fibrils by mobilizing the membrane-anchored matrix metalloproteinase, Mmp14. Unexpectedly, dermal fibroblasts in Mmp14-/- mice commit to a large-scale apoptotic program that leaves skin tissues replete with dying cells. A requirement for Mmp14 in dermal fibroblast survival is recapitulated in vitro when cells are embedded within, but not cultured atop, 3-dimensional hydrogels of cross-linked type I collagen. In the absence of Mmp14-dependent pericellular proteolysis, dermal fibroblasts fail to trigger β1 integrin activation and instead actuate a TGF-β1/phospho-JNK stress response that leads to apoptotic cell death in vitro as well as in vivo. Taken together, these studies identify Mmp14 as a requisite cell survival factor that maintains dermal fibroblast viability in postnatal dermal tissues.
Project description:miRNA profiling of human H9-derived neural stem cells (H9-NSCs) comparing control human adult dermal fibroblasts (hDFs), SOX2-transduced human induced neural stem cells (hDF-iNSC (SOX2)), SOX2/HMGA2-transduced human induced neural stem cells (hDF-iNSC (SOX2/HMGA2)). Goal was to determine the global miRNA expression between the groups.
Project description:The effect at long term (15 days) of leukemia inhibitory factor (LIF), TGFβ or TGFβ + anti-Lif stimulation on the human primary dermal fibroblast (hDF) transcriptome was analyzed by whole genome microarray expression profiling.
Project description:The effect at short term (48 hours) of leukemia inhibitory factor (LIF), TGFβ or TGFβ + anti-Lif stimulation on the human primary dermal fibroblast (hDF) transcriptome was analyzed by whole genome microarray expression profiling.
Project description:Cell-cell communication between keratinocytes and fibroblasts is essential for skin homeostasis, regulation, and regeneration. How extracellular cytokine signaling, intracellular protein signaling and transcriptional regulation initiate and maintain this communication is still largely unknown. Here, we study in vitro the secretome and transcriptome dynamics that establish double paracrine cell-cell communication between normal human keratinocytes (NHK) and human dermal fibroblasts (HDF). To mimic double paracrine stimulation, we stimulate HDF with NHK-conditioned medium for 18 hours and use the then HDF-conditioned medium to stimulate the NHK. The NHK response is quantified at different timepoints for a duration of up to 24 hours. We find that double paracrine stimulation changes extracellular matrix composition and activates translation and enhances keratinocyte migration.
Project description:To investigate the contribution of fibroblast-derived extracellular matrices (ECMs) to the resistance to targeted therapies in BRAF-mutated melanoma cells, we generated native-like 3D ECMs from human primary fibroblasts obtained from healthy individuals or melanoma patients. Cell-derived matrices from human dermal fibroblasts (HDF), skin melanoma associated fibroblasts (MAF) and two different lymph node fibroblast reticular cells (FRC) were denuded of cells and their composition was analyzed by mass spectrometry.
Project description:Fibrotic diseases have significant health impact and have been associated with differentiation of the resident fibroblasts into myofibroblasts. In particular, stiffened extracellular matrix and TGF-β1 in fibrotic lesions have been shown to promote pathogenic myofibroblast activation and progression of fibrosis in various tissues. To better understand the roles of mechanical and chemical cues on myofibroblast differentiation and how they may crosstalk, we cultured primary valvular interstitial cells (VICs) isolated from porcine aortic valves and studied how traditional TCPS culture, which presents a non-physiologically stiff environment, and TGF-β1 affect native VIC phenotypes. We carried out gene expression profiling using porcine genome microarrays from Affymetrix and found that traditional TCPS culture induces major changes in gene expression of native VICs, rendering these cells more activated and similar to cells treated with TGF-β1. We also monitored time-dependent effects induced by TGF-β1 by examining gene expression changes induced by TGF-β1 at 8 hours and 24 hours. Porcine aortic VICs were isolated and cultured with or without TGF-β1 treatment for RNA extraction and hybridization on Affymetrix microarrays. We included 3 biological replicates for each condition. P0 VICs were freshly isolated cells which had not been cultured. P2 VICs were cells that had been passaged 2 times and cultured on plastic plates in low serum media. Some of the P2 VICs were treated with TGF-β1 at 5ng/ml for 8 hours or 24 hours. All the control and TGF-β1-treated conditions were collected at the same time on day 3 of culture.