Fibroblast growth factor (fgf)

Manufactured by Lonza

Sourced in Switzerland, United States

Fibroblast growth factor is a type of lab equipment used in cell culture and tissue engineering applications. It is a protein that stimulates the growth and proliferation of fibroblasts, which are important cells in the extracellular matrix. The core function of fibroblast growth factor is to support and promote the growth of fibroblasts in laboratory settings.

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12 protocols using fibroblast growth factor (fgf)

Culturing Vascular Endothelial Cells

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HPMECs (Lonza, Basel, Switzerland) were maintained in EBM-2MV media supplemented with 5% FBS and proprietary concentrations of human epidermal growth factor, fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor, hydrocortisone and gentamicin, all provided by Lonza. HBCMECs were obtained from Sciencell (Carlsbad, CA) and were maintained in EBM-2 containing 5% FBS, 1.4 μM hydrocortisone, 5 μg ml⁻¹ ascorbic acid, 1 ng ml⁻¹ basic fibroblast growth factor (Sigma, St Louis, MO), 1X chemically defined lipid concentrate, 10 mM HEPES and Penicillin–Streptomycin (Life Technologies, Carlsbad, CA). Media were changed every 2–3 days and cells were used up to passage 7.

Satterfield B.A., Cross R.W., Fenton K.A., Agans K.N., Basler C.F., Geisbert T.W, & Mire C.E. (2015). The immunomodulating V and W proteins of Nipah virus determine disease course. Nature Communications, 6, 7483.

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Cardiac Fibroblasts: TGF-β and S-NO-HSA Effects

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Human ventricular cardiac fibroblasts (Lonza, Basel, Switzerland) were cultured in fibroblast basal medium supplemented with 0.1% insulin, 0.1% fibroblast growth factor, 0.1% GA-1000, and 10% FBS (all Lonza, Basel, Switzerland) as described previously (19 (link)). Cultures were washed once with DPBS (Thermo Fisher Scientific, CA, United States) when indicated, and split at a confluency level of 70%. Cells were treated for 24 h follows: 1) No treatment—control; 2) 20 ng/ml TGF-β (Abcam, Cambridge, United Kingdom); 3) 25 μmol/L HSA; 4) 25 μmol/L S-NO-HSA; 5) 20 ng/ml TGF-β + 25 μmol/L HSA and 6) 20 ng/ml TGF-β + 25 μmol/L S-NO-HSA. Total RNA was extracted, and expression of target genes (Supplementary Table S5) were assessed by RT-qPCR (Supplementary Appendix S1).

Schaefer A.K., Kiss A., Oszwald A., Nagel F., Acar E., Aliabadi-Zuckermann A., Hackl M., Zuckermann A., Kain R., Jakubowski A., Ferdinandy P., Hallström S, & Podesser B.K. (2022). Single Donor Infusion of S-Nitroso-Human-Serum-Albumin Attenuates Cardiac Isograft Fibrosis and Preserves Myocardial Micro-RNA-126-3p in a Murine Heterotopic Heart Transplant Model. Transplant International, 35, 10057.

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Cardiac Fibroblast Culture and Stimulation

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Human ventricular cardiac fibroblasts (Lonza, Basel, Switzerland) were cultured in fibroblast basal medium supplemented with 0.1% insulin, 0.1% fibroblast growth factor, 0.1% GA-1000, and 10% FBS (all Lonza, Basel, Switzerland). Cultures were washed with HEPES buffered saline (Lonza, Basel, Switzerland) when indicated, and split at a confluency level of 70%. Cells were serum starved for 24 h, with subsequent treatment with one of the following: (1) No treatment—control; (2) 20 ng/mL TGFβ (Abcam, Cambridge, UK); or (3) recombinant human tenascin C (1 µg/mL rh TN-C, Fisher Scientific, Waltham, MA, USA) for an additional 24 h. Then, the total RNA was extracted as described previously [19 (link)].

Perera-Gonzalez M., Kiss A., Kaiser P., Holzweber M., Nagel F., Watzinger S., Acar E., Szabo P.L., Gonçalves I.F., Weber L., Pilz P.M., Budinsky L., Helbich T, & Podesser B.K. (2021). The Role of Tenascin C in Cardiac Reverse Remodeling Following Banding–Debanding of the Ascending Aorta. International Journal of Molecular Sciences, 22(4), 2023.

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Cardiac Fibroblast Culture and Transcription Analysis

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Human ventricular cardiac fibroblasts (HVCFs, cryopreserved ampules of normal human ventricular cardiac fibroblasts containing ≥ 500,000 cells, #CC-2904, Lonza, Basel, Switzerland, https://bioscience.lonza.com/lonza_bs/CH/en/Primary-and-Stem-Cells/p/000000000000197234/NHCF-V-%E2%80%93-Human-Ventricular-Cardiac-Fibroblasts) were cultured in a fibroblast basal medium supplemented with 0.1% insulin, 0.1% fibroblast growth factor, 0.1% GA-1000, 1% pen-strep, and 10% FBS (all Lonza, Basel, Switzerland) as previously described^{40 (link),57 (link)}. Cultures were washed with HEPES buffered saline (Lonza, Basel, Switzerland) when indicated and split at a confluency level of 70%^{40 (link),57 (link)}. Cells were treated as follows: (i) without treatment—control; (ii) 20 ng/mL transforming growth factor-beta (TGF-β, R&D systems, Minneapolis, Minnesota, USA); (iii) 10 nmol/L P234; and (iv) 20 ng/mL TGF-β with 10 nmol/L P234 for 24 h. Then total RNA was extracted from HVCFs, and gene expressions of Col1a1, Mmp9, and Acta2 were calculated relative to glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and hypoxanthine–guanine phosphoribosyltransferase (Hgprt) expressions (the specific primer sequences and the description of the RT-qPCR method from HVCFs are detailed in the Supplementary Material).

Dinh H., Kovács Z.Z., Márványkövi F., Kis M., Kupecz K., Szűcs G., Freiwan M., Lauber G.Y., Acar E., Siska A., Ibos K.E., Bodnár É., Kriston A., Kovács F., Horváth P., Földesi I., Cserni G., Podesser B.K., Pokreisz P., Kiss A., Dux L., Csabafi K, & Sárközy M. (2023). The kisspeptin-1 receptor antagonist peptide-234 aggravates uremic cardiomyopathy in a rat model. Scientific Reports, 13, 14046.

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Cultivation of Human Microvascular Endothelial Cells

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Human pulmonary microvascular endothelial cells (HPMEC; Lonza, Basel, Switzerland) were maintained in EBM-2MV media supplemented with 5% FBS and proprietary concentrations of human epidermal growth factor, fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor, hydrocortisone, and gentamicin all provided by Lonza. Human brain cerebrum microvascular endothelial cells (HBCMEC) were obtained from Sciencell (Carlsbad, CA) and maintained in EBM-2 containing 5% FBS, 1.4 μM hydrocortisone, 5 μg/ml ascorbic acid, 1 ng/ml basic fibroblast growth factor (Sigma, St. Louis, MO), 1X chemically defined lipid concentrate, 10 mM HEPES, and Penicillin-Streptomycin (Life Technologies, Carlsbad, CA). Media was changed every 2-3 days and cells were used up to passage 7.

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Isolation and Characterization of Endothelial Outgrowth Cells

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Mouse EOCs were cultured as previously described [19 (link)]. Briefly, macrophage-depleted BMMNCs were seeded in fibronectin-coated plates and maintained in endothelial cell basal medium-2 supplemented with 5% FBS, VEGF-A, fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, ascorbic acid and antibiotics (Lonza, Walkersville, MD, USA). Non-adherent cells were removed after 4 days of culture and new medium was applied. EOCs, recognised as an attached cluster of spindle-shaped cells [20 (link)], were characterised on day 7 by immunostaining using primary antibodies against CD34, CD31, VEGFR2 and VE-cadherin (ESM Table 1). After incubation with Alexa Flour 488- or 594-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA), the cells were visualised under an Olympus BX53 microscope (Olympus, Tokyo, Japan).

Bhatta M., Ma J.H., Wang J.J., Sakowski J, & Zhang S.X. (2015). Enhanced endoplasmic reticulum stress in bone marrow angiogenic progenitor cells in a mouse model of long-term experimental type 2 diabetes. Diabetologia, 58(9), 2181-2190.

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Culturing Cell Lines for Research

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HUVECs and hMSCs used in the study were obtained from Lonza, and were each used until passage 10. HUVECs were cultured in EGM-2 (Lonza), and hMSCs were cultured in mesenchymal stem cell growth medium (MSCGM) (Lonza). MDA-MB-231 cells were obtained from ATCC and were cultured in DMEM supplemented with 10% FBS and 2 mM L-Glutamine. HEK293T cells were obtained from ATCC and were cultured in DMEM supplemented with 10% FBS. The Med411-FH medulloblastoma patient-derived xenograft line [77 (link),81 (link)] was maintained in the Wechsler-Reya lab. Tumors were harvested and dissociated into single cells the day of culture, and were subsequently encapsulated in their respective growth environments. For Med411-FH, media was composed of NeuroCult basal medium supplemented with Proliferation Kit (StemCell Technologies), 2 μg/mL heparin (Sigma), 20 ng/mL epidermal growth factor (Sigma), and 20 ng/mL fibroblast growth factor (Lonza).

Hu M., Lei X.Y., Larson J.D., McAlonis M., Ford K., McDonald D., Mach K., Rusert J.M., Wechsler-Reya R.J, & Mali P. (2021). Integrated genome and tissue engineering enables screening of cancer vulnerabilities in physiologically relevant perfusable ex vivo cultures. Biomaterials, 280, 121276.

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Epigallocatechin-3-Gallate and DNA Methylation

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Human umbilical vein endothelial cells (HUVEC) (Lonza, Walkersville, MD, USA) were cultured in a phenol-free endothelial growth medium (EGM) supplemented with 2% fetal bovine serum (FBS), 0.4% fibroblast growth factor, 0.1% vascular endothelial growth factor, 0.1% heparin, 0.1% insulin-like growth factor, 0.1% ascorbic acid, 0.1% epidermal growth factor and 0.04% hydrocortisone (all from Lonza). HUVECs, at passage 4, were seeded into the 24-well plates (BD-Falcon, Le Pont-De-Claix, France) and grown to reach 60-70% confluence. The medium was then replaced to expose the cells for 3h to the culture medium containing either solvent alone (ethanol 0.5‰, control wells, vehicle) or different metabolites: 4′-O-methyl(-)-epicatechin, 4′-O-methyl(-)epicatechin-7-β-D-glucuronide or (-)-epicatechin-4′-sulfate at 1µM. At the end of the incubation period, the medium was replaced with flavanol free medium and left for further 18h until confluence. The confluent monolayer was then stimulated for 4 hours with TNFα (R&D Systems Lille, France) at 0.1 ng/mL or only incubated with PBS/BSA (0,001%, negative control).
Following gDNA isolation, genomewide DNA methylation profiles were generated by Illumina 450k BeadChip (Illumina, San Diego, CA, USA) arrays covering >450,000 CpG dinucleotides.

Milenkovic D., Declerck K., Guttman Y., Kerem Z., Claude S., Weseler A.R., Bast A., Schroeter H., Morand C, & Vanden Berghe W. (2020). (-)-Epicatechin metabolites promote vascular health through epigenetic reprogramming of endothelial-immune cell signaling and reversing systemic low-grade inflammation. Biochemical pharmacology, 173.

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Co-culture of Hematopoietic Progenitors

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Sort-purified CD133⁺CD34⁺ cells (200/cm²) were seeded on stromal cells and co-cultured for 14 days (Fig. 1D). Co-culture was performed in IMDM (Lonza, Basel, Switzerland) supplemented with 20% FBS (Biochrom, Berlin, Germany), 100 U/ml penicillin, 100 U/ml streptomycin (Life Technologies, Karlsruhe, Germany) and SCF, TPO, FLT3-L (Peprotech, Rocky Hill, USA) each at 10 ng/ml final concentration with 50% of exchange of culture medium on day 7. For the co-culture experiments with HUVECs and ECFCs, the basic culture medium was supplemented with additional cytokines (FGF, EGF) from the endothelial growth medium (EGM, Lonza) to support survival and proliferation of endothelial cells throughout the entire 14 days of co-culture.

Radtke S., Görgens A., da Conceição Castro S.V., Kordelas L., Köninger A., Dürig J., Möllmann M., Horn P.A, & Giebel B. (2019). Human multipotent hematopoietic progenitor cell expansion is neither supported in endothelial and endothelial/mesenchymal co-cultures nor in NSG mice. Scientific Reports, 9, 12914.

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Culturing Endothelial Cell Lines

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Human microvascular endothelial cell line-1 (HMEC-1) was grown in MCDB131 medium (Gibco) supplemented with EGF (1 ng/mL; Promega) and hydrocortisone (1 ng/mL; Sigma). On the other hand, human umbilical vein endothelial cells (HUVECs) were grown on EBM-2 medium (Lonza), supplemented with SingleQuots (Lonza) endothelial growth medium-2 (EGM-2). All culture media were supplemented with 10% of FBS (Gibco), 2 mM of L-Glutamine and 100 units/mL of penicillin/estreptomicine (Gibco). Blood outgrowth endothelial cells (BOECs) from healthy and HHT donor primary cultures were grown in EGM-2 (Lonza), supplemented with 20% FBS, following the reported protocol.
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Treatments in the absence or presence of FGF were performed in medium EGM with 2% FBS. BOECs from passages 4 and 5, three control groups, and three different HHT donors were used in the experiments.

Albiñana V., Giménez-Gallego G., García-Mato A., Palacios P., Recio-Poveda L., Cuesta A.M., Patier J.L, & Botella L.M. (2019). Topically Applied Etamsylate: A New Orphan Drug for HHT-Derived Epistaxis (Antiangiogenesis through FGF Pathway Inhibition). TH Open: Companion Journal to Thrombosis and Haemostasis, 3(3), e230-e243.

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