Human Stromal Fibroblasts Research Articles

Progesterone Receptor Signaling Selectively Modulates Cytokine-Induced Global Gene Expression in Human Cervical Stromal Cells.

Preterm birth (PTB) is the leading cause of morbidity and mortality in infants <1 year of age. Intrauterine inflammation is a hallmark of preterm and term parturition; however, this alone cannot fully explain the pathobiology of PTB. For example, the cervix undergoes a prolonged series of biochemical and biomechanical events, including extracellular matrix (ECM) remodeling and mechanochemical changes, culminating in ripening. Vaginal progesterone (P4) prophylaxis demonstrates great promise in preventing PTB in women with a short cervix (<25 mm). We used a primary culture model of human cervical stromal fibroblasts to investigate gene expression signatures in cells treated with interleukin-1β (IL-1β) in the presence or absence of P4 following 17β-estradiol (17β-E2) priming for 7–10 days. Microarrays were used to measure global gene expression in cells treated with cytokine or P4 alone or in combination, followed by validation of select transcripts by semiquantitative polymerase chain reactions (qRT-PCR). Primary/precursor (MIR) and mature microRNAs (miR) were quantified by microarray and NanoString® platforms, respectively, and validated by qRT-PCR. Differential gene expression was computed after data normalization followed by pathway analysis using Kyoto Encyclopedia Genes and Genomes (KEGG), Panther, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) upstream regulator algorithm tools. Treatment of fibroblasts with IL-1β alone resulted in the differential expression of 1432 transcripts (protein coding and non-coding), while P4 alone led to the expression of only 43 transcripts compared to untreated controls. Cytokines, chemokines, and their cognate receptors and prostaglandin endoperoxide synthase-2 (PTGS-2) were among the most highly upregulated transcripts following either IL-1β or IL-1β + P4. Other prominent differentially expressed transcripts were those encoding ECM proteins, ECM-degrading enzymes, and enzymes involved in glycosaminoglycan (GAG) biosynthesis. We also detected differential expression of bradykinin receptor-1 and -2 transcripts, suggesting (prominent in tissue injury/remodeling) a role for the kallikrein–kinin system in cervical responses to cytokine and/or P4 challenge. Collectively, this global gene expression study provides a rich database to interrogate stromal fibroblasts in the setting of a proinflammatory and endocrine milieu that is relevant to cervical remodeling/ripening during preparation for parturition.

The Effect of the Geroprotectors Astragaloside IV, Cycloastragenol, and Timovial–Epivial Peptide Complex on Telomere Length and Telomerase Activity in Human Mesenchymal Stromal Cells and Senescent Fibroblasts

A search for geroprotectors that would affect the telomere length and/or telomerase activity and not exhibit tumorigenicity is under progress. The medical use of various extracts of plants from the Astragalus genus of the Fabaceae family was first described in 200 CE. However, scientific studies of the components of these extracts (cycloastragenol, astragaloside IV) have begun only recently. The goal of this work was to explore the effect of a composition consisting of astragaloside IV, cycloastragenol, and the Timovial–Epivial dipeptide and its components on telomere length and telomerase activity in human umbilical cord mesenchymal stromal cells and aging fibroblasts. Telomerase activity was determined by the telomeric repeat amplification protocol (TRAP). Telomere length was determined by measuring the intensity of fluorescence in situ hybridization signals using flow cytometry. None of the composition components caused a significant change in the determined parameters on its own. Only cycloastragenol had a negligible effect on the telomere length in fibroblasts. The combination of the key compound, astragaloside IV, aglycone of astragalosides—cycloastragenol (CAG), and biopeptide complex produced more significant changes in the telomere length and telomerase activity than did each component alone. The complex was shown to have a significant effect on telomere length and telomerase activity. Further studies are needed to determine the mechanism of the effect of the components combination on telomere length and telomerase activity.

The \u03b2-catenin/YAP signaling axis is a key regulator of melanoma-associated fibroblasts

β-catenin is a multifunctional protein that plays crucial roles in embryonic development, physiological homeostasis, and a wide variety of human cancers. Previously, we showed that in vivo targeted ablation of β-catenin in melanoma-associated fibroblasts after melanoma formation significantly suppressed tumor growth. However, when the expression of β-catenin was ablated in melanoma-associated fibroblasts before tumor initiation, melanoma development was surprisingly accelerated. How stromal β-catenin deficiency leads to opposite biological effects in melanoma progression is not completely understood. Here, we report that β-catenin is indispensable for the activation of primary human stromal fibroblasts and the mediation of fibroblast-melanoma cell interactions. Using coimmunoprecipitation and proximity ligation assays, we identified Yes-associated protein (YAP) as an important β-catenin-interacting partner in stromal fibroblasts. YAP is highly expressed in the nuclei of cancer-associated fibroblasts (CAFs) in both human and murine melanomas. Mechanistic investigation revealed that YAP nuclear translocation is significantly modulated by Wnt/β-catenin activity in fibroblasts. Blocking Wnt/β-catenin signaling in stromal fibroblasts inhibited YAP nuclear translocation. In the absence of YAP, the ability of stromal fibroblasts to remodel the extracellular matrix (ECM) was inhibited, which is consistent with the phenotype observed in cells with β-catenin deficiency. Further studies showed that the expression of ECM proteins and enzymes required for remodeling the ECM was suppressed in stromal fibroblasts after YAP ablation. Collectively, our data provide a new paradigm in which the β-catenin-YAP signaling axis regulates the activation and tumor-promoting function of stromal fibroblasts.

Bisphenol A impairs decidualization of human uterine stromal fibroblasts

This study examined the effect of bisphenol A (BPA) exposure on human uterine stromal fibroblast cells (HuF) undergoing decidualization. HuF cells were isolated and cultured for eight days in the presence of a decidualization-inducing cocktail, while concurrently exposed to physiological and supra-physiologic doses of BPA (1ng/mL, 10ng/mL, 0.5μg/mL, 10μg/mL and 20μg/mL). Decidualization markers, steroid hormone receptors and cell cycle gene expression were detected by qRT-PCR and cellular proliferation was assessed by KI-67 immunofluorescent staining and MTS assay. BPA impaired decidualization at 10μg/mL and 20μg/mL, but not at lower doses. Additionally, BPA at 20μg/mL decreased progesterone receptor and estrogen receptor-alpha compared to controls. The highest dose of BPA also reduced cellular proliferation and cyclin D2 expression compared to controls. These findings demonstrate that BPA disrupts in vitro decidualization of uterine stromal fibroblasts by altering steroid hormone receptor expression at higher concentrations but not at lower physiological doses.

Nerve regeneration by human corneal stromal keratocytes and stromal fibroblasts

Laser refractive surgeries reshape corneal stroma to correct refractive errors, but unavoidably affect corneal nerves. Slow nerve regeneration and atypical neurite morphology cause desensitization and neuro-epitheliopathy. Following injury, surviving corneal stromal keratocytes (CSKs) are activated to stromal fibroblasts (SFs). How these two different cell types influence nerve regeneration is elusive. Our study evaluated the neuro-regulatory effects of human SFs versus CSKs derived from the same corneal stroma using an in vitro chick dorsal root ganglion model. The neurite growth was assessed by a validated concentric circle intersection count method. Serum-free conditioned media (CM) from SFs promoted neurite growth dose-dependently, compared to that from CSKs. We detected neurotrophic and pro-inflammatory factors (interleukin-8, interleukin-15, monocyte chemoattractant protein-1, eotaxin, RANTES) in SFCM by Bio-Plex Human Cytokine assay. More than 130 proteins in SFCM and 49 in CSKCM were identified by nanoLC-MS/MS. Proteins uniquely present in SFCM had reported neuro-regulatory activities and were predicted to regulate neurogenesis, focal adhesion and wound healing. Conclusively, this was the first study showing a physiological relationship between nerve growth and the metabolically active SFs versus quiescent CSKs from the same cornea source. The dose-dependent effect on neurite growth indicated that nerve regeneration could be influenced by SF density.

Oxidized (non)-regenerated cellulose affects fundamental cellular processes of wound healing.

In this study we investigated how hemostats such as oxidized regenerated cellulose (ORC, TABOTAMP) and oxidized non-regenerated cellulose (ONRC, RESORBA CELL) influence local cellular behavior and contraction of the extracellular matrix (ECM). Human stromal fibroblasts were inoculated in vitro with ORC and ONRC. Cell proliferation was assayed over time, and migration was evaluated by Live Cell imaging microscopy. Fibroblasts grown in collagen-gels were treated with ORC or ONRC, and ECM contraction was measured utilizing a contraction assay. An absolute pH decline was observed with both ORC and ONRC after 1 hour. Mean daily cell proliferation, migration and matrix contraction were more strongly inhibited by ONRC when compared with ORC (p < 0.05). When control media was pH-lowered to match the lower pH values typically seen with ORC and ONRC, significant differences in cell proliferation and migration were still observed between ONRC and ORC (p < 0.05). However, in these pH conditions, inhibition of matrix contraction was only significant for ONRC (p < 0.05). We find that ORC and ONRC inhibit fibroblast proliferation, migration and matrix contraction, and stronger inhibition of these essential cellular processes of wound healing were observed for ONRC when compared with ORC. These results will require further validation in future in vivo experiments to clarify the clinical implications for hemostat use in post-surgical wound healing.

Abstract 1566: A cancer-stroma-cancer interacting loop promotes peritoneal metastasis of ovarian cancer through TNFα-TGFα-EGFR

Abstract Peritoneum is the commonest site for ovarian cancer metastasis. In peritoneum, stromal fibroblasts are the second most common cell types. In this study, we aimed to investigate the role of tumor-stromal interaction in peritoneal metastasis of ovarian cancer. Using three-dimensional (3D) organoid co-culture model, we found that the interaction between metastatic human ovarian cancer cells and normal human stromal fibroblasts promotes cancer colony formation. Using cytokine antibody array, we identified a group of cytokines are de novo produced in the 3D co-culture. Among them, transforming growth factor alpha (TGF-α) was produced anew from normal stromal fibroblasts in the 3D co-culture. In the co-culture, transcription of TGF-α in normal stromal fibroblasts is activated by tumor necrosis factor alpha (TNF-α) that derived by ovarian cancer cells. By epigenetic studies, we discovered that over-expression of TNF-α in human ovarian cancer cells is due to DNA hypomethylation and chromatin remodeling in TNF-α promoter. TGF-α is a ligand of epidermal growth factor receptor (EGFR), and we confirmed EGFR is over-expressed in a significant portion of human ovarian cancer. When applying EGFR-specific inhibitors (AG1478, Gefitini, and Erlotinib) in our 3D organoid models (3D mono-cultures and 3D co-cultures), we further found that the fibroblast-derived TGF-α promotes colony formation of metastatic ovarian cancer cells in vitro through EGFR activation. In fact, TGF-α activates Hippo/YAP signaling pathway in human ovarian cancer cells via EGFR. In addition, our in vivo experiments showed that normal stromal fibroblasts promote peritoneal metastasis of ovarian cancer through EGFR activation in a xenograft model. Finally, the TNFα-TGFα-EGFR interacting loop was detected in cancer-stroma-cancer compartments of omental metastases of ovarian cancer patients by immunohistochemistry. Based on our experimental results, we proposed a cancer-stroma-cancer interacting loop that promotes peritoneal metastasis of ovarian cancer through TNFα-TGFα-EGFR. The proposed mechanism involves: 1) TNF-α is over-expressed in human ovarian cancer cells by promoter DNA hypomethylation and chromatin remodeling; 2) the ovarian cancer cell-derived TNF-α induces de novo TGF-α production in normal human omental stromal fibroblasts; and 3) the fibroblast-derived TGF-α, in turn, promotes colony formation of metastatic ovarian cancer cells through EGFR-mediated Hippo/YAP signaling pathway. Our results thereby suggest that peritoneal metastasis in the ovarian cancer patients after cytoreductive surgery can be prevented by EGFR-targeted therapy. Citation Format: Tat-San Lau, Loucia Kit-Ying Chan, Chi-Hang Wong, Chi-Wai Man, Tak-Hong Cheung, So-Fan Yim, Jacqueline Ho-Sze Lee, Tony Kwok-Hung Chung, Joseph Kwong. A cancer-stroma-cancer interacting loop promotes peritoneal metastasis of ovarian cancer through TNFα-TGFα-EGFR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1566.

Abstract 630: Co-injection of human fibroblasts significantly enhances tumorigenicity of orthotopically implanted human non-small cell lung cancer cells in immunocompromised mice

Abstract Human tumor xenografts in immunodeficient mice have led to valuable insights into the biology of human cancer. The corresponding limitations and pitfalls of xenograft models have been described extensively. To tackle some limitations, we supplemented the murine tumor microenvironment (TME) with human stromal fibroblasts to mirror tumor-stroma cross-talk in vivo. Non-small cell lung cancer (NSCLC) cell lines Calu-1 and H1437 were co-injected with different types of fibroblasts into NOG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice in 3 independent experiments (n = 4-6/group; 106 in total). Human Dermal Fibroblasts (HDFs), lung cancer associated fibroblasts (CAFs), corresponding normal fibroblasts (NFs) and NSCLC cells were implanted into the lungs of 4-6 week old recipient mice. The influence of extracellular matrix components was evaluated by using either a matrigel/collagen mixture or alginate capsules. Tumor load was determined via overall survival (OS) and flow cytometry (FC). When mice had to be sacrificed, organs were collected and analyzed by FC and patho-histological examination. In a subsequent experiment, mice were sacrificed and tumors analyzed at defined time points and similar analyses performed. OS and metastatic pattern were similar in both cell lines when injected in mono-culture. OS of Calu-1 injected mice was not influenced when cells were co-injected with HDF but prolonged when co-injected with CAFs or NFs. In H1437 bearing mice, OS was significantly reduced when cells were co-injected with HDF or NF (Log-rank [Mantel-cox] test; p&amp;lt; 0.002 &amp; 0.005), whereas CAFs had no influence on OS. Co-injection of HDF and Calu-1 enhanced the number of tumor cells in bronchoalveolar lavage fluid, whereas CAFs had the opposite effect (1way ANOVA for all following statistics, p&amp;lt; 0.0001). Consistently, the number of circulating Calu-1 cells was highest in co-culture with HDF and significantly lower in mice bearing Calu-1 and CAF or NF (p&amp;lt;0.0021). Co-injection of fibroblasts enhanced the H1437 tumor load in all compartments. Of note, the effect was not statistically significant. The use of alginate capsules was favorable compared to matrigel/collagen, improving fibroblast engraftment in all investigated organs. Furthermore, alginate capsules enhanced the number of circulating H1437 cells when co-injected with HDF as compared to matrigel/collagen (p&amp;lt;0.0001). Our results demonstrate the major impact of fibroblasts on tumor cell behavior in a preclinical setting. With the successful co-cultivation of human fibroblast and NSCLC cells in vivo, it will be possible to study tumor-stroma interactions in a clinically relevant mouse model. Once validated by a compound screening approach, this model will help to reduce drug failure rates and contribute to a more efficient development of urgently needed novel anti-cancer treatments. Citation Format: Eva Oswald, Vitor E. Santo, Albin Rudisch, Catarina Brito, Wolfgang Sommergruber, Helmut Dolznig, Julia B. Schüler. Co-injection of human fibroblasts significantly enhances tumorigenicity of orthotopically implanted human non-small cell lung cancer cells in immunocompromised mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 630.

Ionizing radiation-mediated premature senescence and paracrine interactions with cancer cells enhance the expression of syndecan 1 in human breast stromal fibroblasts: the role of TGF-β.

The cell surface proteoglycan syndecan 1 (SDC1) is overexpressed in the malignant breast stromal fibroblasts, creating a favorable milieu for tumor cell growth. In the present study, we found that ionizing radiation, a well-established treatment in human breast cancer, provokes premature senescence of human breast stromal fibroblasts in vitro, as well as in the breast tissue in vivo. These senescent cells were found to overexpress SDC1 both in vitro and in vivo. By using a series of specific inhibitors and siRNA approaches, we showed that this SDC1 overexpression in senescent cells is the result of an autocrine action of Transforming Growth Factor-β (TGF-β) through the Smad pathway and the transcription factor Sp1, while the classical senescence pathways of p53 or p38 MAPK - NF-kB are not involved. In addition, the highly invasive human breast cancer cells MDA-MB-231 (in contrast to the low-invasive MCF-7) can also enhance SDC1 expression, both in early-passage and senescent fibroblasts via a paracrine action of TGF-β. The above suggest that radiation-mediated premature senescence and invasive tumor cells, alone or in combination, enhance SDC1 expression in breast stromal fibroblasts, a poor prognostic factor for cancer growth, and that TGF-β plays a crucial role in this process.

Uniform, fast, high concentration delivery of bone marrow stromal cells and gingival fibroblasts by gas-brushing

Delivering multipotent stem cells in adequate milieu directly to site of injury is important for successful tissue regeneration. Accelerated wound healing has been reported in recent publications when large area wounds were treated with sprayed cells. However, little is known about the basic physics of cell spraying in hydrogel free medium, causes of concomitant cell damage, and whether it can be applied for craniofacial tissue regeneration. To answer some of these questions, a simple co-axial CO2 gas operated brush (g-brush) was used to deliver human bone marrow stromal (hBMSC) and mouse gingival fibroblast (ESK-1) cells at low gas flow rates and in a small volume of medium. Live/dead, metabolic activity (MTS-1) and total DNA quantification assays were utilized to evaluate cell cultures after g-brush and typical pipette deposition. Viable hBMSC and ESK-1 cells were uniformly delivered at higher cell concentrations (≥60 000), in a short period of time (15 and 9 s), and in a small fluid volume (0.25 ml). Significantly, after the hBMSC were g-brushed and cultured up to 21 d, the cell multipotency was found to be preserved and comparable to the pipetted cells. Fluid dynamic calculations highlighted importance of low interfacial shear stress (<35 Pa) and gas velocity (<160 m s−1) in delivering concentrated, viable cells. Results of this research support applicability of the g-brushing technique for the quick, large number delivery of viable cells for tissue regeneration applications.

PH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants.

Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4th_FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4th_FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities.

TSLPR/STAT5 signaling modulates innate immunity in corneal fibroblasts triggered by Aspergillus fumigatus

To study the primary role of TSLPR/STAT5 signaling in inflammatory responses triggered by Aspergillus fumigatus(AF) in telomerase-immortalized human stromal fibroblasts (THSF). Experimental study. Baseline expression of TSLPR in THSF was assessed by immunofluorescence analysis. Human recombinant TSLP was added. At 5, 15, 30 and 60 min after incubation, cells were harvested for Western blot to assess the protein levels of p-STAT5 and STAT5. After stimulated with AF hyphae for 1, 3, 6, 12, 24 and 48 h, cells were collected for measurement of mRNA of TSLPR and IL-7Rα. After incubated with AF hyphae for 12, 24 and 48 h, cells were harvested for Western blot to assess the protein levels of TSLPR, p-STAT5 and STAT5. Incubation with anti-TSLPR antibody was performed for 4 h, and at 24 h after AF hyphae were added, cells were harvested to assess the protein levels of p-STAT5 and STAT5. Immunofluorescence staining evidenced that expression of TSLPR was visualized in THSF. Western blot assay showed that p-STAT5 protein was increased and peaked at 30 min after stimulation with hTSLP (AF group: 8.87±0.75; control group: 1.00±0.14; P<0.01). RT-PCR revealed that the expression levels of TSLPR mRNA were increased after incubation with AF hyphae for 3, 6, 12 and 24 h (AF group: 0.000 50±0.000 07, 0.001 20±0.000 11, 0.002 30±0.000 25 and 0.001 70±0.000 17; control group: 0.000 20±0.000 03, 0.000 20±0.000 05, 0.000 20±0.000 03 and 0.000 20±0.000 04; t=-9.955, -17.329, -16.735 and -18.214, P<0.01), but the expression levels of IL-7Rα mRNA were not increased significantly (t=-0.684,-0.029,-0.319,-1.034, P>0.05). In comparison with the control group, after being challenged with AF hyphae for 24 h, both TSLPR and p-STAT5 protein were increased significantly (p-STAT5: 9.46±2.08 vs. 1.00±0.06; TSLPR: 1.80±0.27 vs. 1.00±0.34; t=-7.055, -3.170, P<0.01). Western blot showed that the elevated p-STAT5 expression levels observed after AF hyphae stimulation can be inhibited by TSLPR antibody (anti-TSLPR-AF: 0.55 ± 0.20; CTR Ab-AF: 1.00 ± 0.08; t=3.506, P<0.05). TSLP/TSLPR/STAT5 signaling pathway plays an important role in inflammatory responses triggered by AF in THSF.

Human skin-derived fibroblasts used as a 'Trojan horse' for drug delivery.

Drug toxicity currently represents the main challenge of tumour chemotherapy. Our group recently developed a new method for drug delivery inspired by the 'Trojan Horse' concept. Human mesenchymal stem cells (hMSCs) have been shown to play the role of new 'horses' in delivering anti-tumour agents, without involving any genetic manipulation. As human stromal dermal fibroblasts (hSDFs) represent an interesting alternative to hMSCs, being easy to isolate, they could be an ideal candidate for this kind of procedure. To investigate whether hSDFs can take up and deliver paclitaxel (PTX) in sufficient concentrations to inhibit a very aggressive melanoma tumour (IgR39) invitro. hSDFs were primed with high doses of PTX, and then the effect of drug delivery on IgR39 melanoma proliferation invitro was evaluated using several assays (antiproliferation, transwell cocultures, rosette assays and colony growth assays). Furthermore, the cell cycle and PTX uptake/release mechanism of hSDFs were studied both under both normal and hypoxic conditions. hSDFs incorporated PTX and then released it with unaffected pharmacological activity, inhibiting human IgR39 melanoma growth invitro. The hypoxic conditions did not induce changes in cell cycle pattern and the uptake-release mechanism with PTX was not affected. hSDFs can be used as a Trojan horse, as the released drug was functionally active. These results indicated that these cells could be used for clinical treatment as the drug was released into the cellular environment and the primed cells underwent apoptosis.

ITF2357 transactivates Id3 and regulate TGFβ/BMP7 signaling pathways to attenuate corneal fibrosis.

Corneal fibrosis is often seen in patients with ocular trauma and infection that compromises corneal transparency resulting in vision loss. Treatment strategies including NSAIDs, steroids, MMC and corneal transplants have shown tremendous success but with several side effects and cellular toxicity. Histone deacetylase inhibitors (HDACi) have been shown to inhibit corneal fibrosis via TGFβ signaling pathway. In this study, we investigated safety, efficacy and mechanism of action of a HDACi, ITF2357 in TGFβ-stimulated in vitro primary human cornea stromal fibroblasts (pHCSFs) and in vivo in a photorefractive keratectomy-treated rabbit model of corneal fibrosis. We found that in vivo ITF2357 decreased collagen I, collagen IV, fibronectin, integrin αVβ3 expression with a reduction in corneal haze. In addition, ITF2357 reduced myofibroblast formation, suppressed phosphorylation of Smad proteins in TGFβ pathway and inhibited key responsive protein, P4HA1 involved in pro-collagen synthesis. Treatment of pHCSFs with ITF2357 activated BMP7 levels and expressed all the members of inhibitor of differentiation proteins (Id1-Id4), however, it failed to rescue TGFβ-driven transdifferentiation of fibroblasts to myofibroblasts in the presence of siRNA specific to Id3. We conclude that ITF2357 is a potential anti-fibrotic drug that exerts its action via activation of Id3, a downstream target of TGFβ/BMP7 signaling pathways.

Monocyte and Fibroblast Directed Generation of a Marrow Vascular Niche

Hematopoietic cells dynamically interact with their surrounding microenvironment during their residence, maturation and differentiation. Individual marrow components have been isolated and studied in 2D in vitro cultures, yet their functional contributions to a complete niche are not fully understood. In vivo studies in mouse models are complex, and the inaccessibility of marrow architecture has precluded systematic analysis of each component. Here, we employ an in vitro 3D microfluidic vascular system to study the effect of microenvironmental cues on cell trafficking in an engineered hematopoietic niche. Our system allows for control of 3D geometric cues, hydrodynamic flow, multi-cellular compositions, and cellular matrix remodeling by combining soft lithography and injection modeling in type I collagen gel (Zheng et al. PNAS 2012). Endothelial cells perfused through the embedded microfluidic network form a confluent, patent endothelium within the collagen. Incorporating hematopoietic cells and stromal cells into the extravascular collagen space allows us to visualize how cells interact with vasculature during culture.First, we developed a marrow stromal microenvironment to understand how stromal cells modify the marrow microvascular environment, by incorporating two different human marrow-derived stromal fibroblast cell lines (HS27a and HS5) in the extravascular space surrounding the vessels (Fig 1A). HS27a and HS5 cells created distinct vascular microenvironments by secreting divergent cytokine profiles: stem cell niche-associated and inflammatory cytokines, respectively. Both stromal lines modified the vascular phenotype by reducing endothelial expression of vWF and junctional proteins. In particular, co-culture with HS5 increased expression of inflammatory markers on the endothelium.Next, we evaluated the function of this modified vasculature on hematopoietic stem cell trafficking through the vessels. When CD34+ cells alone were perfused through the microvessels, they adhered on the vessel wall and migrated into the matrix. The HS27a and HS5-induced microenvironment did not significantly change trafficking behavior (Fig 1B, C). When monocytes were perfused through the microvessels, they also adhered on the vessel wall and migrated into the matrix. Monocytes are a critical part of the marrow space, and are known to modify the endothelium and stromal microenvironment. When CD34+ cells were perfused through the vessels 24 hours after monocytes, crosstalk shifted trafficking patterns of both cell types and led to increased adhesion and migration within the HS5- and HS27a-modified vessels (Fig 1D).To explore the competent marrow niche in vitro, we further examined hematopoietic cell trafficking from the extravascular space into the circulation by embedding cells from fresh human bone marrow screens within the collagen matrix. We found this cell fraction contained a mixed population of hematopoietic and stromal cells, and could be cultured in the microvascular niche for at least two weeks. Scanning electron microscopy showed various types of marrow cells residing in both the abluminal and luminal side of the endothelium (Fig 1E-G). Throughout culture with continuous vascular perfusion, we collected the media flow-through and identified different hematopoietic cell populations released from the matrix into the circulation over the course of two weeks. Specifically, we identified CD34+ hematopoietic progenitor cells along with megakaryocyte, erythroid, lymphoid, and myeloid lineage cells by flow cytometry analysis.In summary, we developed an in vitro 3D microvascular marrow niche and gained insight into hematopoietic cell trafficking between the stroma and the circulation. By guiding the interplay of heterogeneous cell populations, we have demonstrated the capacity to define distinct microenvironment spaces. This platform shows promise for long term culture of a whole marrow population and for the ex vivo generation of hematopoietic cells. Further development of this 3D marrow niche will allow us to better understand the complexities mediating stem cell trafficking, residence, proliferation, mobilization, and differentiation in both health and disease. [Display omitted] DisclosuresNo relevant conflicts of inteqerest to declare.

Proteomic identification of prognostic tumour biomarkers, using chemotherapy-induced cancer-associated fibroblasts.

Cancer cells grow in highly complex stromal microenvironments, which through metabolic remodelling, catabolism, autophagy and inflammation nurture them and are able to facilitate metastasis and resistance to therapy. However, these changes in the metabolic profile of stromal cancer-associated fibroblasts and their impact on cancer initiation, progression and metastasis are not well-known. This is the first study to provide a comprehensive proteomic portrait of the azathioprine and taxol-induced catabolic state on human stromal fibroblasts, which comprises changes in the expression of metabolic enzymes, myofibroblastic differentiation markers, antioxidants, proteins involved in autophagy, senescence, vesicle trafficking and protein degradation, and inducers of inflammation. Interestingly, many of these features are major contributors to the aging process. A catabolic stroma signature, generated with proteins found differentially up-regulated in taxol-treated fibroblasts, strikingly correlates with recurrence, metastasis and poor patient survival in several solid malignancies. We therefore suggest the inhibition of the catabolic state in healthy cells as a novel approach to improve current chemotherapy efficacies and possibly avoid future carcinogenic processes.

Application of Graphene as Candidate Biomaterial for Synthetic Keratoprosthesis Skirt.

Synthetic keratoprostheses are required for visual rehabilitation in patients with end-stage corneal blindness. This study aimed to assess the biocompatibility of graphene material and its potential as a novel synthetic keratoprosthesis skirt material for corneal tissue engineering. Human corneal stromal fibroblasts were cultured on material surfaces including pristine graphene film, graphene foam, pristine titanium (Ti) discs, and tissue culture plastic surface (TCPS). Cell attachment was assayed by immunostaining of paxillin and vinculin. Cell viability and proliferation were assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Click iT 5-ethynyl-2'-deoxyuridine (EdU) assays. The growth of fibroblasts on three-dimensional graphene foam was examined by scanning electron microscopy, and cytokine release was analyzed by enzyme-linked immunosorbent assay. Graphene films were implanted into rabbit corneal stromal pockets and examined by slit-lamp biomicroscopy, anterior segment optical coherence tomography, in vivo confocal microscopy, and histology. Pristine graphene demonstrated good biocompatibility with human stromal fibroblasts in terms of cell adhesion, viability, and proliferation. Cells on graphene films showed higher number than on TCPS control. Cells grown on graphene had 10% more proliferation than on Ti. The expression levels of IL-6 and IL-8 were reduced when cells were seeded on graphene foam as compared to Ti and graphene film. Implantation of graphene film into rabbit stroma (n = 6) did not show any signs of infection, neovascularization, or inflammation. Graphene displayed excellent short-term biocompatibility with corneal cells and tissue. This demonstrates that graphene can be developed as a tissue engineering material for use in cornea.

Self-Assembled Arginine-Capped Peptide Bolaamphiphile Nanosheets for Cell Culture and Controlled Wettability Surfaces.

The spontaneous assembly of a peptide bolaamphiphile in water, namely, RFL4FR (R, arginine; F, phenylalanine; L, leucine) is investigated, along with its novel properties in surface modification and usage as substrates for cell culture. RFL4FR self-assembles into nanosheets through lateral association of the peptide backbone. The L4 sequence is located within the core of the nanosheets, whereas the R moieties are exposed to the water at the surface of the nanosheets. Kinetic assays indicate that the self-assembly is driven by a remarkable two-step process, where a nucleation phase is followed by fast growth of nanosheets with an autocatalysis process. The internal structure of the nanosheets is formed from ultrathin bolaamphiphile monolayers with a crystalline orthorhombic symmetry with cross-β organization. We show that human corneal stromal fibroblast (hCSF) cells can grow on polystyrene films coated with films dried from RFL4FR solutions. For the first time, this type of amphiphilic peptide is used as a substrate to modulate the wettability of solid surfaces for cell culture applications.

Comparison of the Matrix Synthesizing Abilities of Human Adipose-Derived Stromal Vascular Fraction Cells and Fibroblasts.

For facial soft tissue augmentation or wound coverage using tissue-engineering technology, cultured fibroblasts have been most commonly used as key cells and their properties have been extensively studied. Clinical strategies based on human cultured fibroblasts, however, require Food and Drug Administration (FDA) approval for the facilities and the procedures used and time-consuming culture. Adipose tissue-derived stromal vascular fraction (SVF) cells may be a reliable alternative to fibroblasts because they are easily harvested by liposuction and do not require culture or FDA approval. No quantitative standard governing their use has, however, been issued. The purpose of this study was to quantitatively compare matrix-forming abilities of SVF cells and fibroblasts. Human dermal fibroblasts were obtained from the dermis of 10 healthy adults and cultured, and SVF cells were obtained from 10 patients who underwent liposuction. Monolayer and suspension cell cultures were performed using both cell types for 3 days. Cell proliferations, collagen synthesis levels, and glycosaminoglycan levels were compared using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, a collagen type I carboxy-terminal propeptide enzyme immunoassay, and the Blyscan Dye method, respectively. Cell proliferation ratios (fibroblasts versus SVF cells) in monolayer and suspension cultures were 1:0.75 and 1:0.99, respectively; collagen synthesis ratios in monolayer and suspension cultures were 1:0.50 and 1:0.52, respectively; and glycosaminoglycan production ratios were 1:0.70 and 1:0.74, respectively. The results of this in vitro study indicate that SVF cells have 50-74% of the matrix-forming ability of fibroblasts.

Sunitinib mesylate inhibits proliferation of human colonic stromal fibroblasts in vitro and in vivo.

Cancer stromal fibroblasts are important members of the cancer microenvironment. In this study, we determined the effect of sunitinib, a small molecule tyrosine kinase inhibitor, on the primary human colonic fibroblasts. Cell cycle analysis and cell proliferation assays were performed to evaluate the inhibitory effect of sunitinib in vitro. Western-blot analysis was performed to evaluate variations in the levels of phosphorylated platelet-derived growth factor receptor β (PDGFR-β), Akt, and ERK proteins. Co-injection of SW620 cells and colonic fibroblasts in nude mice was employed to test anti-growth efficacy in vivo. Sunitinib was found to effectively inhibit the growth of primary colonic fibroblasts. Low-dose sunitinib blocked the PDGF-BB-induced cell proliferation and PDGFR-β signaling. Co-injection of SW620 cells and colonic fibroblasts in nude mice generated greater tumor volumes than single injection of SW620 cells. Sunitinib treatment inhibited the SW620 cell+colonic fibroblast tumor growth more effectively than treatment of 5-fluorouracil. Sunitinib mesylate inhibited the proliferation of primary human colonic fibroblasts through target-inhibited PDGFR signaling in vitro and in vivo.