Human Fetal Foreskin Fibroblasts Research Articles

Technetium-99m radiolabeling through conjugation with l,l-ethylene dicysteine chelator of a trimethoxylated flavone and its bioevaluation in rat with induced C6 glioma tumor as a new cancer diagnostic agent

Abstract Xanthomicrol (4′,5-dihydroxy-6,7,8-trimethoxyflavone) is the main active component of Dracocephalum kotschyi Boiss leaf extract. It has showed selective cytotoxic activity against some cancer cell lines and little effect on human fetal foreskin fibroblast cells used as nonmalignant control. This study aimed to develop 99mTc-labeled xanthomicrol and to evaluate its efficiency as a new tumor imaging agent. l,l-Ethylene dicysteine (EC) chelator was conjugated to xanthomicrol. EC-Xanthomicrol was labeled with technetium-99m by using tin chloride as a reducing agent and incubating at room temperature. Radiochemical purity and in vitro stability were analyzed by thin layer chromatography and high-performance liquid chromatography. In vitro cellular uptake and binding profile of radio-conjugate was determined on C6 glioma cells. In vivo bioevaluation and imaging studies of [99mTc]Tc-EC-Xanthomicrol were performed in C6 glioma tumor induced rat at different time points after injection of radio-conjugate. The high radiochemical yield (>95 %) was achieved for [99mTc]Tc-EC-Xanthomicrol which was stable up to 6 h. The radio-conjugate indicated high cell uptake (35.12 % at 2 h) which demonstrated to be specific. Tumor uptake was seen for [99mTc]Tc-EC-Xanthomicrol (1.23 ± 0.14 %ID/g) at 1 h post injection. Scintigraphy confirmed that tumors could be visualized clearly with [99mTc]Tc-EC-Xanthomicrol. The results indicated that [99mTc]Tc-EC-Xanthomicrol has potential to be considered as a new radiotracer in glioma tumor imaging.

Polyurethane-based nanofibrous mat containing porphyrin with photosensitivity and bactericidal properties can promote cutaneous tissue healing in rats

The regeneration of cutaneous tissue is one of the most challenging issues in human regenerative medicine. To date, several studies have been done to promote cutaneous tissue healing with minimum side effects. The healing potential of polyurethane (PU)/Poly (caprolactone)–poly (ethylene glycol)–poly (caprolactone) (PCEC)/chitosan (CS) (PCS) nanofibrous mat with cationic photosensitizer meso tetrakis (N-methyl pyridinium-4-yl) porphyrin tetratosylate salt (TMP) was examined. The CS tripolyphosphate nanoparticles (CSNPs) were prepared and loaded by TMP to provide an efficient drug release system (TMPNPs) for delivery of TMP to promote wound healing. In in vitro setting, parameters such as bactericidal effects, cytocompatibility, and hemolytic effects were examined. The healing potential of prepared nanofibrous mats was investigated in a rat model of full-thickness cutaneous injury. PCS/TMP/TMPNPs nanofibers can efficiently release porphyrin in the aqueous phase. The addition of TMPNPs and CS to the PU backbone increased the hydrophilicity, degradation, and reduced mechanical properties. The culture of human fetal foreskin fibroblasts (HFFF2) on PCS/TMP/TMPNPs scaffold led to an increased survival rate and morphological adaptation analyzed by MTT and SEM images. Irradiation with a red laser (635 nm, 3 J/cm2) for the 30 s reduced viability of S. aureus and E. Coli bacteria plated on PCS/TMP and PCS/TMP/TMPNPs nanofibrous mats compared to PU/PCEC (PC) and PU/PCEC/CS (PCS) groups, indicating prominent antibacterial effects of PCS/TMP and PCS/TMP/TMPNPs nanofibrous (p < 0.05). Data indicated that PCS/TMP/TMPNPs mat enhanced healing of the full-thickness excisional wound in a rat model by the reduction of inflammatory response and fibrotic changes compared to the PC, and PCS groups (p < 0.05). Immunofluorescence imaging indicated that levels of Desmoglein were increased in rats that received PCS/TMP/TMPNPs compared to the other groups. It is found that a PU-based nanofibrous mat is an appropriate scaffold to accelerate the healing of injured skin.Graphical abstract

Chitosan-Based Membranes for Skin Wound Repair in a Dorsal Fold Chamber Rat Model.

Frequently, deep partial and full-thickness skin wounds do not spontaneously regenerate. To restore the normal function of skin, epidermal and dermal components have to be supplied to the wound bed by grafting various substrates. Available options are limited and frequently costly. Herein, authors present a possible approach using 3D skin scaffolds capable of mimicking structure and biological functions of the extracellular matrix, providing, in parallel, a good environment for cell attachment, proliferation and differentiation. Low-molecular weight chitosan-based membranes were prepared by freeze-drying and ionizing radiation techniques to be used as skin scaffolds. Poly (vinyl alcohol), PVA, vinyl pyrrolidone, VP, and gelatin from cold water fish were incorporated. Information regarding membranes' physical-chemical properties from SEM analysis, swelling and weight loss, together with biological response through in vitro assays (using Human Caucasian Fetal Foreskin Fibroblast) allowed the selection of an optimized batch of membranes that was used as skin scaffold in a dorsal rat model wound. The in vivo implantation assays (in Wistar rats) resulted in very promising results: (i) healing process faster than control; (ii) good vascularization; (iii) viable new tissues morphologically functional.

A Biodegradable Flexible Micro/Nano-Structured Porous Hemostatic Dental Sponge.

A biodegradable micro/nano-structured porous hemostatic gelatin-based sponge as a dentistry surgery foam was prepared using a freeze-drying method. In vitro function evaluation tests were performed to ensure its hemostatic effect. Biocompatibility tests were also performed to show the compatibility of the sponge on human fetal foreskin fibroblasts (HFFF2) cells and red blood cells (RBCs). Then, 10 patients who required the extraction of two teeth were selected, and after teeth extraction, for dressing, the produced sponge was placed in one of the extracavities while a commercial sponge was placed in the cavity in the other tooth as a control. The total weight of the absorbed blood in each group was compared. The results showed a porous structure with micrometric and nanometric pores, flexibility, a two-week range for degradation, and an ability to absorb blood 35 times its weight in vitro. The prepared sponge showed lower blood clotting times (BCTs) (243.33 ± 2.35 s) and a lower blood clotting index (BCI) (10.67 ± 0.004%) compared to two commercial sponges that displayed its ability for faster coagulation and good hemostatic function. It also had no toxic effects on the HFFF2 cells and RBCs. The clinical assessment showed a better ability of blood absorption for the produced sponge (p-value = 0.0015). The sponge is recommended for use in dental surgeries because of its outstanding abilities.

The Association of Cell Surface Fibromodulin Expression and Bladder Carcinoma.

Fibromodulin (FMOD) is a secretory protein which is considered a major component of extracellular matrix. Its dysregulation in different types of cancer implies it as a promising target for cancer therapy. Within the scope of its rather wide expression in different tumors, we studied expression of FMOD and effect of anti-FMOD antibody in bladder cancer cells in order to identify new target for diagnostic and therapeutic interventions. We report here for the first time the expression of FMOD in bladder cancer cell lines in comparison to the normal cell line and tissues. A peptide-based produced anti-FMOD murine monoclonal antibody (mAb) (clone 2C2-A1) was applied for evaluation of FMOD expression in bladder cancer and normal tissues by immunohistochemistry (IHC) staining. Furthermore, the expression of FMOD was examined in human bladder cell lines, 5637 and EJ138, as well as a non-cancerous human cell line, human fetal foreskin fibroblast (HFFF), by immunocytochemistry (ICC) and flow cytometry. The apoptosis induction of anti-FMOD mAb was also evaluated in bladder cancer cells. IHC and ICC analyses revealed that the qualitative expression of FMOD in bladder cancer tissues and cell lines is higher than in normal tissues and cell lines. Flow cytometry analyses revealed that 2C2-A1 mAb could recognize FMOD expression in 84.05 ± 1.85%, 46.1 ± .4% , and 2.56 ± 1.26% of 5637, EJ138, and HFFF cells, respectively. An effective apoptosis induction was detected in 5637 and EJ138 cells with no significant effect on HFFF cell. To our knowledge, this is for the first time reporting surface expression of FMOD in bladder cancer. This significant surface expression of FMOD in bladder cancer with no expression in normal bladder tissues and the capacity of inducing apoptosis through directed targeting of FMOD with specific monoclonal antibody might candidates FMOD as a diagnostic marker as well as a potential immunotargeting with monoclonal antibody.

Sodium metabisulfite as a cytotoxic food additive induces apoptosis in HFFF2 cells

Sodium metabisulfite (SMB), an antioxidant agent, is extensively used as a preservative in food industry. The current study was aimed to clarify its potential toxic effects on human fetal foreskin fibroblasts (HFFF2) cells, in vitro. Subsequently, MTT results illustrated that exposure to SMB significantly (p < 0.0001) decreased HFFF2 cell viability in a dose-dependent manner, and the concentration of 25 μM reduced cell survival rates to 50% as the half-maximal inhibitory concentration of SMB. It was further shown that SMB exerted this cytotoxic effect on HFFF2 cells through apoptosis induction. qRT-PCR and western blotting results showed that treatment of HFFF2 cells with this food additive led to significant upregulation of Bax, caspase 8, and caspase 9 pro-apoptotic genes and downregulation of Bcl-2 expression as a pro-survival agent. Furthermore, SMB remarkably increased caspase 3 levels and promoted its activation through cleavage in treated cells. Besides, exposure to SMB increased ROS levels and activated autophagy in treated cells, which are considered as the other indicators for cell damage. Taken together, our findings suggested that SMB could exert remarkable toxic effects on human normal cells through multiple mechanisms, including apoptosis activation, and its widespread usage in food safety should be reconsidered.

In vitro antifungal activity of Thymus vulgaris essential oil nanoemulsion

BackgroundAn essential oil derived from the evergreen plant Thymus vulgaris acts as an antifungal agent with strong antimicrobial activity, but it has low water solubility. The current study therefore aimed to enhance the solubility of thyme essential oil by formulating it into a nanoemulsion and investigating its effectiveness on a broad range of fungi. MethodThe identification of the compositions of essential oils was performed using GC–MS. Thyme essential oil nanoemulsion (TV EO-NE) was prepared by using a high-pressure homogenization method. Both viability and cytotoxicity outcomes on Human fetal foreskin fibroblast cells were gauged by different concentrations of Thymus vulgaris essential oils and TV EO-NE after 24 and 48 h. Antifungal susceptibility tests were carried out based on the modified CLSI M60 document for yeasts and CLSI M28-A3 documents for filamentous fungi. ResultsThyme essential oil was mainly composed of thymol (22.10 %), p-cymene (21.31 %), carvacrol (13.02 %), carvacrol acetate (6.72 %), and linalool (5.58 %). The TV EO-NEs were spherical with a mean diameter of 127.6 ± 62.52 nm and zeta potential of −9.82 ± 6.07 mV. The MIC90 for C. albicans and C. glabrata isolates was 0.031 μg/mL and 0.0625 μg/mL for C. parapsilosis. Also, MIC90 for both dermatophytes and Aspergillus fumigatus isolates was determined to be 0.016 μg/mL. For yeasts and filamentous fungi, a significant reduction in MIC values was observed using TV EO-NE (p < 0.05). ConclusionThe study highlighted T. vulgaris and thymol to be promising alternatives for the treatment of cutaneous mycoses especially when the etiological agents are resistant to conventional antifungal drugs.

Purification of Soluble Membrane-Bound Ambystoma mexicanum Epidermal Lipoxygenase from E. coli and Its Growth Effect on Human Fetal Foreskin Fibroblast.

Lipoxygenases are non-heme iron-containing lipid dioxygenases enzymes that catalyze the hydroperoxidation of lipids. The Mexican axolotl (Ambystoma mexicanum) is a prominent source of the enzyme with a regeneration capacity in limbs. It has been shown that transfected human osteosarcoma and keratinocyte cells with epidermal lipoxygenase (LOXe) have an increased rate of cell migration. In the present study, LOXe, a peripheral membrane protein, was produced in Escherichia coli. The enzyme was purified using different detergents, anionic solutions, and gel filtration chromatography. Kinetic assay of the enzyme activity was carried out by the spectroscopy method using arachidonic acid as a substrate. Finally, the enzyme was characterized and its growth effect on human fibroblast cells was examined by MTT viability assay. Enzyme kinetic parameters including Km of 90.4µM and Vmax of 2.63IU were determined for LOXe. The enzyme with 0.1nM end concentration promoted the growth of 5000 cells/well human fibroblast cells up to 11% (P < 0.01). In the present study, we introduce an E. coli expression system to produce an excessive amount of soluble LOXe and the efficient purification method to provide a soluble and active form of LOXe that is effective in stimulating human fibroblast cell proliferation.

Fibrillar Self-Assembly of a Chimeric Elastin-Resilin Inspired Engineered Polypeptide.

In the field of tissue engineering, recombinant protein-based biomaterials made up of block polypeptides with tunable properties arising from the functionalities of the individual domains are appealing candidates for the construction of medical devices. In this work, we focused our attention on the preparation and structural characterization of nanofibers from a chimeric-polypeptide-containing resilin and elastin domain, designed on purpose to enhance its cell-binding ability by introducing a specific fibronectin-derived Arg-Gly-Asp (RGD) sequence. The polypeptide ability to self-assemble was investigated. The molecular and supramolecular structure was characterized by Scanning Electronic Microscopy (SEM) and Atomic Force Microscopy (AFM), circular dichroism, state-of-the-art synchrotron radiation-induced techniques X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The attained complementary results allow us to assess as H-bonds influence the morphology of the aggregates obtained after the self-assembling of the chimeric polypeptide. Finally, a preliminary investigation of the potential cytotoxicity of the polypeptide was performed by culturing human fetal foreskin fibroblast (HFFF2) for its use as biomedical device.

Human iPSC-derived iMSCs improve bone regeneration in mini-pigs

Autologous bone marrow concentrate (BMC) and mesenchymal stem cells (MSCs) have beneficial effects on the healing of bone defects. To address the shortcomings associated with the use of primary MSCs, induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) have been proposed as an alternative. The aim of this study was to investigate the bone regeneration potential of human iMSCs combined with calcium phosphate granules (CPG) in critical-size defects in the proximal tibias of mini-pigs in the early phase of bone healing compared to that of a previously reported autograft treatment and treatment with a composite made of either a combination of autologous BMC and CPG or CPG alone. iMSCs were derived from iPSCs originating from human fetal foreskin fibroblasts (HFFs). They were able to differentiate into osteoblasts in vitro, express a plethora of bone morphogenic proteins (BMPs) and secrete paracrine signaling-associated cytokines such as PDGF-AA and osteopontin. Radiologically and histomorphometrically, HFF-iMSC + CPG transplantation resulted in significantly better osseous consolidation than the transplantation of CPG alone and produced no significantly different outcomes compared to the transplantation of autologous BMC + CPG after 6 weeks. The results of this translational study imply that iMSCs represent a valuable future treatment option for load-bearing bone defects in humans.

Human pluripotent stem cell derived HLC transcriptome data enables molecular dissection of hepatogenesis

Induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) differentiated into hepatocyte-like cells (HLCs) provide a defined and renewable source of cells for drug screening, toxicology and regenerative medicine. We previously reprogrammed human fetal foreskin fibroblast cells (HFF1) into iPSCs employing an episomal plasmid-based integration-free approach, this iPSC-line and the hESC lines H1 and H9 were used to model hepatogenesis in vitro. Biochemical characterisation confirmed glycogen storage, ICG uptake and release, urea and bile acid production, as well as CYP3A4 activity. Microarray-based transcriptome analyses was carried out using RNA isolated from the undifferentiated pluripotent stem cells and subsequent differentiation stages- definitive endoderm (DE) hepatic endoderm (HE) and HLCs. K-means identified 100 distinct clusters, for example, POU5F1/OCT4 marking the undifferentiated stage, SOX17 the DE stage, HNF4α the HE stage, and ALB specific to HLCs, fetal liver and primary human hepatocytes (PHH). This data descriptor describes these datasets which should be useful for gaining new insights into the molecular basis of hepatogenesis and associated gene regulatory networks.

Elusive Role of the CD94/NKG2C NK Cell Receptor in the Response to Cytomegalovirus: Novel Experimental Observations in a Reporter Cell System.

Human cytomegalovirus (HCMV) infection promotes the differentiation and persistent expansion of a mature NK cell subset, which displays high surface levels of the activating CD94/NKG2C NK cell receptor, together with additional distinctive phenotypic and functional features. The mechanisms underlying the development of adaptive NK cells remain uncertain but some observations support the involvement of a cognate interaction of CD94/NKG2C with ligand(s) displayed by HCMV-infected cells. To approach this issue, the heterodimer and its adaptor (DAP12) were expressed in the human Jurkat leukemia T cell line; signaling was detected by transfection of a reporter plasmid encoding for Luciferase (Luc) under NFAT/AP1-dependent control. Engagement of the receptor by solid-phase bound CD94- or NKG2C-specific monoclonal antibodies (mAbs) triggered Luc expression. Moreover, reporter activation was detectable upon interaction with HLA-E+ 721.221 (.221-AEH) cells, as well as with 721.221 cells incubated with synthetic peptides, which stabilized surface expression of endogenous HLA-E; the response was specifically antagonized by soluble NKG2C- and HLA-E-specific mAbs. By contrast, activation of Jurkat-NKG2C+ was undetectable upon interaction with Human Fetal Foreskin Fibroblasts (HFFF) infected with HCMV laboratory strains (i.e., AD169, Towne), regardless of their differential ability to preserve surface HLA-E expression. On the other hand, infection with two clinical isolates or with the endotheliotropic TB40/E strain triggered Jurkat-NKG2C+ activation; yet, this response was not inhibited by blocking mAbs and was independent of CD94/NKG2C expression. The results are discussed in the framework of previous observations supporting the hypothetical existence of specific ligand(s) for CD94/NKG2C in HCMV-infected cells.

Footprint-free human fetal foreskin derived iPSCs: A tool for modeling hepatogenesis associated gene regulatory networks

Induced pluripotent stem cells (iPSCs) are similar to embryonic stem cells and can be generated from somatic cells. We have generated episomal plasmid-based and integration-free iPSCs (E-iPSCs) from human fetal foreskin fibroblast cells (HFF1). We used an E-iPSC-line to model hepatogenesis in vitro. The HLCs were characterized biochemically, i.e. glycogen storage, ICG uptake and release, UREA and bile acid production, as well as CYP3A4 activity. Ultra-structure analysis by electron microscopy revealed the presence of lipid and glycogen storage, tight junctions and bile canaliculi- all typical features of hepatocytes. Furthermore, the transcriptome of undifferentiated E-iPSC, DE, HE and HLCs were compared to that of fetal liver and primary human hepatocytes (PHH). K-means clustering identified 100 clusters which include developmental stage-specific groups of genes, e.g. OCT4 expression at the undifferentiated stage, SOX17 marking the DE stage, DLK and HNF6 the HE stage, HNF4α and Albumin is specific to HLCs, fetal liver and adult liver (PHH) stage. We use E-iPSCs for modeling gene regulatory networks associated with human hepatogenesis and gastrulation in general.

Modulation of Human Leukocyte Antigen-C by Human Cytomegalovirus Stimulates KIR2DS1 Recognition by Natural Killer Cells.

The interaction of inhibitory killer cell Ig-like receptors (KIRs) with human leukocyte antigen (HLA) class I molecules has been characterized in detail. By contrast, activating members of the KIR family, although closely related to inhibitory KIRs, appear to interact weakly, if at all, with HLA class I. KIR2DS1 is the best studied activating KIR and it interacts with C2 group HLA-C (C2-HLA-C) in some assays, but not as strongly as KIR2DL1. We used a mouse 2B4 cell reporter system, which carries NFAT-green fluorescent protein with KIR2DS1 and a modified DAP12 adaptor protein. KIR2DS1 reporter cells were not activated upon coculture with 721.221 cells transfected with different HLA-C molecules, or with interferon-γ stimulated primary dermal fibroblasts. However, KIR2DS1 reporter cells and KIR2DS1+ primary natural killer (NK) cells were activated by C2-HLA-C homozygous human fetal foreskin fibroblasts (HFFFs) but only after infection with specific clones of a clinical strain of human cytomegalovirus (HCMV). Active viral gene expression was required for activation of both cell types. Primary NKG2A−KIR2DS1+ NK cell subsets degranulated after coculture with HCMV-infected HFFFs. The W6/32 antibody to HLA class I blocked the KIR2DS1 reporter cell interaction with its ligand on HCMV-infected HFFFs but did not block interaction with KIR2DL1. This implies a differential recognition of HLA-C by KIR2DL1 and KIR2DS1. The data suggest that modulation of HLA-C by HCMV is required for a potent KIR2DS1-mediated NK cell activation.

In vitro Cytotoxicity Comparison of MTA Fillapex, AH-26 and Apatite Root Canal Sealer at Different Setting Times.

Introduction:This study aimed to compare the cytotoxicity of MTA Fillapex, AH-26 and Apatite root canal sealers at different times after mixing. Methods and Materials:In this in vitro study, MTA Fillapex, AH-26 and Apatite root canal sealer were spilled uniformly by 40 µm mesh in a 96-well plate. Then, human fetal foreskin fibroblast cell line (HFFF2) were added to each sealer cell culture medium. Cytotoxicity was measured using MTT assay after 24, 48 and 72 h and seven days. Multiple comparisons were done using analysis of variances (ANOVA) and Scheffe’s post hoc test. Results:All studied sealers exhibited severe cytotoxicity (more than 70%) except for Apatite sealer (95%) at 48 h after mixing. Cytotoxicity of MTA Fillapex and AH-26 were similar (P>0.05) at 24, 48 and 72 h and 7 days after mixing of sealers. Cytotoxicity of MTA Fillapex and Apatite root canal sealer, at 24 and 48 h, were significantly different (P=0.003 and P=0.000, respectively); MTA Fillapex was more cytotoxic. However in 72 h and 7 days after mixing, the difference was not significant (P>0.05). At 24 and 48 h after mixing, AH-26 was more cytotoxic (P=0.002 and P=0.000, respectively). Same as above at 72 h and 7 days after mixing, their cytotoxicity were similar (P>0.05). Conclusion:Overall cytotoxicity of all studied materials were severe. However, it was observed that the cytotoxicity of MTA Fillapex, AH-26 and Apatite root canal sealer decreased over time. Apatite root canal sealer exhibited the least cytotoxicity. Cytotoxicity of MTA Fillapex and AH-26 were similar at different time intervals.

Dose-dependent and cell type-specific cell death and proliferation following in vitro exposure to radial extracorporeal shock waves.

Radial extracorporeal shock wave (rESW) therapy is widely used in musculoskeletal disorders and wound repair. However, the mechanisms of action are still largely unknown. The current study compared the effects of rESWs on two cell types. Human fetal foreskin fibroblasts (HFFF2) and human placental choriocarcinoma cell line JEG-3 were exposed to 0, 100, 200, 500 or 5000 rESWs generated with a Swiss DolorClast device (2.5 bar, 1 Hz). FACS analysis immediately after rESW exposure showed that initially, rESWs rather induced mechanical cell destruction than regulated or programmed cell death. Cell damage was nearly negated by reducing cavitation. Furthermore, cell viability decreased progressively with higher numbers of rESWs. Exposure to rESWs had no impact on growth potential of JEG-3 cells, but dose-dependently increased growth potential of HFFF2 cells. Cultivation of cells that were initially exposed to sham-rESWs in conditioned media increased the growth potential of HFFF2 cells, nevertheless, an even stronger effect was achieved by direct exposure to rESWs. Additionally, cell cycle distribution analysis demonstrated a shift in proportion from G0/G1 to G2/M phase in HFFF2 cells, but not in JEG-3 cells. These data demonstrate that rESWs leads to initial and subsequent dose-dependent and cell type-specific effects in vitro.

Abstract B15: Targeting NOX4 in the tumor microenvironment inhibits myofibroblast differentiation and suppresses tumor progression

Abstract Purpose: Smooth muscle actin (SMA)-positive myofibroblasts (cancer-associated fibroblasts), within the tumor microenvironment, promote many of the hallmarks of malignancy and correlate with poor prognosis in multiple cancer types. The purpose of this study was to investigate the mechanisms regulating myofibroblast differentiation in solid tumors in order to therapeutically target this cell population. Experimental procedures: Clinical Samples: Human tumor samples were analyzed by immunohistochemistry and RNA sequencing followed by bioinformatics analyses. In vitro: Myofibroblast differentiation was analyzed in Human fetal foreskin fibroblasts (HFFF2), adult primary fibroblasts and cancer associated fibroblasts (CAFs) by Western blotting for α-SMA; immunofluorescence for α-SMA stress fiber formation; collagen gel contraction assays and analysis of gene expression by RNA sequencing/PCR. Intracellular Reactive Oxygen Species (ROS) generation was measured using FACS analysis of DCFH-DA fluorescence. In vivo: To monitor tumor growth in vivo, a xenograft model of Head &amp; Neck squamous cell carcinoma (HNSCC; 5PT) co-injected with HFFF2s in B6.129S7-Rag1tm1Mom/J mice; and an isograft model (K2) in C57BL/6-Tg(TRAMP)8247Ng/J mice was used. Lymph node (LN) metastases were assayed in the 5PT model using the LI-COR IRDye® 800CW 2-DG Optical Probe. Myofibroblast accumulation was assessed by immunohistochemistry for α-SMA and collagen deposition by Masson's trichrome staining. Results: Using TGF-β1 treatment to model fibroblast-to-myofibroblast differentiation in vitro, we found that a delayed phase of intracellular ROS generation, beginning 24/48 hours post-treatment was required to induce differentiation in HFFF2s and adult primary fibroblasts. RNA-Sequencing of fibroblasts treated with TGF-β1 showed that NOX4 was the most significantly up-regulated ROS-producing enzyme. NOX4 was also found to be up-regulated in the stroma of multiple solid tumors, co-localized with α-SMA, and correlating with a myofibroblast gene signature. Genetic (si/shRNA) and pharmacological (GKT137831) inhibition of NOX4 abrogated multiple processes associated with myofibroblast differentiation, down-stream of TGF-β1: suppressing ROS generation; α-SMA expression; collagen gel contraction and expression of genes associated with ECM deposition (COL1A1, COL3A1, FN1 and CTGF). Furthermore, NOX4 inhibition reversed the myofibroblast phenotype in Cancer Associated Fibroblasts (CAFs) isolated from HNSCC and esophageal adenocarcinoma. Genetic and pharmacological inhibition of NOX4 in fibroblasts significantly slowed tumor growth and myofibroblast accumulation in the 5PT and K2, in vivo, model systems. Notably, LN metastases in the 5PT model were significantly reduced following shRNA mediated NOX4 knockdown in HFFF2s. Conclusions: NOX4 is a critical regulator of myofibroblast accumulation in multiple tumor types. Fibroblast-specific inhibition of this enzyme suppresses tumor growth and metastatic spread, suggesting that NOX4 is a promising therapeutic target for preventing myofibroblast differentiation within the tumor microenvironment. Citation Format: Christopher J. Hanley, Massimiliano Mellone, Gareth J. Thomas. Targeting NOX4 in the tumor microenvironment inhibits myofibroblast differentiation and suppresses tumor progression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B15.

Episomal-based generation of an iPS cell line from human fetal foreskin fibroblasts

Human fetal foreskin fibroblasts (HFF1) were used to generate the iPSC line epiHFF1-B1 employing a combination of three episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC, and KLF4. Pluripotency was confirmed both in vivo and in vitro. The transcriptome profile of epiHFF1-B1 and the human embryonic stem cell line—H1 have a pearson correlation of 0.936.

Human cytomegalovirus transcriptome activity differs during replication in human fibroblast, epithelial and astrocyte cell lines

Broad cell tropism contributes to the pathogenesis of human cytomegalovirus (HCMV), but the extent to which cell type influences HCMV gene expression is unclear. A bespoke HCMV DNA microarray was used to monitor the transcriptome activity of the low passage Merlin strain of HCMV at 12, 24, 48 and 72 h post-infection, during a single round of replication in human fetal foreskin fibroblast cells (HFFF-2s), human retinal pigmented epithelial cells (RPE-1s) and human astrocytoma cells (U373MGs). In order to correlate transcriptome activity with concurrent biological responses, viral cytopathic effect, growth kinetics and genomic loads were examined in the three cell types. The temporal expression pattern of viral genes was broadly similar in HFFF-2s and RPE-1s, but dramatically different in U373MGs. Of the 165 known HCMV protein-coding genes, 41 and 48 were differentially regulated in RPE-1s and U373MGs, respectively, compared with HFFF-2s, and 22 of these were differentially regulated in both RPE-1s and U373MGs. In RPE-1s, all differentially regulated genes were downregulated, but, in U373MGs, some were down- and others upregulated. Differentially regulated genes were identified among the immediate-early, early, early late and true-late viral gene classes. Grouping of downregulated genes according to function at landmark stages of the replication cycle led to the identification of potential bottleneck stages (genome replication, virion assembly, and virion maturation and release) that may account for cell type-dependent viral growth kinetics. The possibility that cell type-specific differences in expressed cellular factors are responsible for modulation of viral gene expression is discussed.

Early Induction of Autophagy in Human Fibroblasts after Infection with Human Cytomegalovirus or Herpes Simplex Virus 1

The infection of human fetal foreskin fibroblasts (HFFF2) with human cytomegalovirus (HCMV) resulted in the induction of autophagy. This was demonstrated by the increased lipidation of microtubule-associated protein 1 light chain 3 (LC3), a hallmark of autophagy, and by the visualization of characteristic vesicles within infected cells. The response was detected first at 2 h postinfection and persisted for at least 3 days. De novo protein synthesis was not required for the effect, since HCMV that was irradiated with UV light also elicited the response, and furthermore the continuous presence of cycloheximide did not prevent induction. Infection with herpes simplex virus type 1 (HSV-1) under conditions that inhibited viral gene expression provoked autophagy, whereas UV-irradiated respiratory syncytial virus did not. The induction of autophagy occurred when cells were infected with HCMV or HSV-1 that was gradient purified, but HCMV dense bodies and HSV-1 light particles, each of which lack nucleocapsids and genomes, were inactive. The depletion of regulatory proteins Atg5 and Atg7, which are required for autophagy, reduced LC3 modification in response to infection but did not result in any detectable difference in viral or cellular gene expression at early times after infection. The electroporation of DNA into HFFF2 cultures induced the lipidation of LC3 but double-stranded RNA did not, even though both agents stimulated an innate immune response. The results show a novel, early cellular response to the presence of the incoming virion and additionally demonstrate that autophagy can be induced by the presence of foreign DNA within cells.