Basic fibroblast growth factor (bfgf)

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BFGF is a recombinant human protein that functions as a basic fibroblast growth factor. It is a potent mitogen for a variety of cell types, including fibroblasts, endothelial cells, and smooth muscle cells.

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657 protocols using basic fibroblast growth factor (bfgf)

Optimizing Cell Differentiation with Growth Factors

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The isolated cells were divided into eight groups as follows: control group included cells without any treatment, FSH group included cells treated with 0.5 IU/ml human urinary FSH (Utrofol, Kuanart Pharmaceuticals, India), bFGFG group included cells treated with 100 ng/ml basic FGF (R&D Systems Inc, USA), NT3 group included cells treated with 100 ng/ml NT3 (R&D Systems Inc, USA), and other groups included cells treated with one of the combination of mentioned growth factors including FSH+NT3, FSH+ bFGF, NT3+bFGF, and FSH+NT3+bFGF. Each group included 3 same members (10000 cells). They were kept at 37°C in 5% CO
₂followed by monitoring by inverted microscope using Hoffman modulation contrast (Eclipse TE2000-S, Nikon, Japan). Cortical tissue sections were sampled on day three and RNA extraction was done after processing.

Tanbakooei S., Haramshahi S.M., Vahabzadeh G., Barati M., Katebi M., Golab F., Shetabi Q., Niknam N., Roudbari L., Rajabi Fomeshi M, & Amini Moghadam S. (2021). Ovarian Stem Cells Differentiation into Primary Oocytes Using Follicle Stimulating Hormone, Basic Fibroblast Growth Factor, and Neurotrophin 3. Journal of Reproduction & Infertility, 22(4), 241-250.

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Culturing Human Osteosarcoma and Mesenchymal Stem Cells

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The human osteosarcoma cell line MG-63 purchased from ATCC was cultured in a 5% CO₂ atmosphere at 37 °C in DMEM supplemented with 5% of fetal bovine serum (Hyclone Perbio, France). Three days before infection, cells were seeded in 24-well culture plates to obtain confluent monolayers. To confirm the results obtained with MG-63 cells, the same method was applied with two representative isolates (ATCC6919 and BL strains) on human mesenchymal stem cells (hMSC). hMSC were obtained from healthy donors. Blood draws were performed at the “Etablissement Français du Sang” (Nantes, France) after obtaining the informed consent of all healthy donors. The experimental protocol was approved by the ethics committee of the Medical School of Nantes. All experiments were performed in accordance with the Good Scientific Practice guidelines of the Medical School of Nantes and all relevant guidelines and regulations. hMSC were cultured in DMEM supplemented with 10% of fetal bovine serum, 1 ng/mL of basic Fibroblast Growth Factor (bFGF; R&D systems, UK), 100 U/mL of penicillin/streptomycin, and 2 mM L-glutamine. Adherent cells were frozen at passage 2 after characterization by flow cytometry (CD45−, CD34−, CD105+, CD73+, and CD90+, purity ≥99%) prior to further experiments27 (link).

Aubin G.G., Baud’huin M., Lavigne J.P., Brion R., Gouin F., Lepelletier D., Jacqueline C., Heymann D., Asehnoune K, & Corvec S. (2017). Interaction of Cutibacterium ( formerly Propionibacterium) acnes with bone cells: a step toward understanding bone and joint infection development. Scientific Reports, 7, 42918.

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Culturing Astrocytes and Glioma Cells

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Normal human astrocytes (NHA) were maintained in astrocyte media (Welgene) supplemented with 10% fetal bovine serum (FBS; HyClone, Thermo Fisher Scientific), and 1% penicillin‐streptomycin (Welgene). Glioma cells (A172, A1207, and U87MG) were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Welgene) supplemented with 10% fetal bovine serum (FBS; HyClone, Thermo Fisher Scientific) and 1% penicillin‐streptomycin (Welgene). GBM patient‐derived glioma stem cells (GSC11, GSC20, GSC23, and GSC267) were provided by The University of Texas MD Anderson Cancer Center. GSCs were cultured under stem cell culture conditions (NBE, serum‐free neurobasal media supplemented with growth factors). The NBE comprised DMEM/F12 (Welgene) supplemented with B27 (Gibco), 1% penicillin/streptomycin (Welgene), epidermal growth factor (EGF; 20 ng/mL; R&D Systems), and basic fibroblast growth factor (bFGF; 20 ng/mL; R&D Systems).¹⁴ Growth factors (bFGF and EGF) were added twice a week. To induce differentiation, GSC11 and GSC23 cells were cultured for 10 days in DMEM/F12 containing 10% FBS.

Jeong H.Y., Park S., Kim H., Moon S., Lee S., Lee S.H, & Kim S. (2020). B3GNT5 is a novel marker correlated with stem‐like phenotype and poor clinical outcome in human gliomas. CNS Neuroscience & Therapeutics, 26(11), 1147-1154.

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Human ESC Endothelial Differentiation and Glutamine

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H1 human ESCs (WiCell) were cultured with conditioned medium containing fresh 10ng/ml basic fibroblast growth factor (bFGF, R&D Systems) and 100ng/ml heparin (Sigma Aldrich). For spontaneous differentiation, medium was not conditioned and bFGF/heparin were left out. Endothelial differentiation was started by adding 20ng/ml bFGF, 25ng/ml bone morphogenetic protein 4 (BMP4), and 50ng/ml vascular endothelial growth factor (VEGF) (all from R&D Systems). To measure the effect of glutamine withdrawal on endothelial differentiation, glutamine was removed from the medium for the final 2 days of a 4 day differentiation.

Marsboom G., Zhang G.F., Pohl-Avila N., Zhang Y., Yuan Y., Kang H., Hao B., Brunengraber H., Malik A.B, & Rehman J. (2016). Glutamine Metabolism Regulates the Pluripotency Transcription Factor OCT4. Cell reports, 16(2), 323-332.

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Mesendodermal and Ectodermal Differentiation

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Differentiation was induced at 70% cell confluency. For mesendodermal differentiation, the cells were subjected to a medium composed of DMEM-F12, L-glutamine, ITS, non-essential amino acids, B27, and β-mercaptoethanol supplemented with the following growth factors: day 1: 25 ng/μL Wnt3a (R&D Systems), 10 μg/μL Activin-A (PeproTech); day 2: 25 ng/μL Wnt3a, 10 μg/mL Activin-A, 4 ng/mL bFGF (R&D Systems); and day 3: 25 ng/μL Wnt3a, 10 μg/μL Activin-A, 4 ng/μL bFGF, 50 ng/μL BMP4 (R&D Systems) (Maldonado et al., 2016a (link)). The medium was exchanged daily in the QCM-EIS device, as well as for the positive controls.
For ectodermal differentiation, the cells were maintained in neurobasal medium, supplemented with B27, N2, L-glutamine, and non-essential amino acids. The medium was exchanged every 36 hr with the growth factors, 0.1 μM retinoic acid (Sigma-Aldrich), and 2 μM dorsomorphin (Sigma-Aldrich), according to an established protocol for ectodermal differentiation (Maldonado et al., 2016a (link)). The positive controls were treated on the same schedule.

Low K., Wong L.Y., Maldonado M., Manjunath C., Horner C.B., Perez M., Myung N.V, & Nam J. (2017). Physico-electrochemical Characterization of Pluripotent Stem Cells during Self-Renewal or Differentiation by a Multi-modal Monitoring System. Stem Cell Reports, 8(5), 1329-1339.

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Mineral-Coated Matrix Loading with bFGF

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MCMs were spread onto a flat surface and sterilized via UV irradiation for 30 min, then aliquotted into sterile tubes and stored until use. For MCM loading, sterilized MCMs were suspended in sterile PBS to make a 1 mg/mL stock solution, and appropriate volumes of recombinant human basic fibroblast growth factor (bFGF, carrier-free; R&D Systems) and MCM stock solutions were combined to achieve the desired final concentration of each in the loading solution (e.g., 1.0 μg/mL bFGF, 1.0 mg/mL MCMs for non-optimized loading; 0.456 μg/mL bFGF, 0.375 mg/mL MCMs for optimized loading). The MCM-bFGF solution was incubated at 37°C under constant rotation for 1 h and protein binding was mediated through ionic interactions between the mineral coating and protein as previously described¹⁶. The suspension was then centrifuged at 2000g for 3 min to pellet the MCMs. For cell culture experiments, the MCMs were resuspended in E7 media and used immediately unless otherwise stated. bFGF-loaded MCMs were prepared fresh every three passages for Transwell culture studies (unless otherwise specified) and prepared fresh at each passage in the direct culture studies. Protein binding efficiency was determined indirectly by Quantikine bFGF ELISA, by measuring the amount of bFGF in original loading solution and subtracting the amount of bFGF in the supernatant after MCM loading.

Khalil A.S., Xie A.W., Johnson H.J, & Murphy W.L. (2020). Sustained release and protein stabilization reduce the growth factor dosage required for human pluripotent stem cell expansion. Biomaterials, 248, 120007.

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Optimizing Chondrocyte Culture Conditions

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Human or rabbit chondrocytes were seeded in 25 cm² cultured flasks in a humidified Thermo Scientific Forma Series II Water Jacket CO₂ incubator in chondrocyte culture medium: Ham's F12 culture medium with l-glutamine, 25 μg mL⁻¹ of l-ascorbic acid, 100 units per mL of penicillin, 100 μg mL⁻¹ of streptomycin, 0.25 μg mL⁻¹ of Fungizone® Antimycotic (AM) and 10% FBS. The culture medium was changed twice a week. To optimize the culture, an MTS assay was performed with different chondrocyte medium compositions: Ham's F12, 1% AM (Invitrogen, Life Technologies, Carlsbad, CA, USA), 25 μg mL⁻¹ ascorbic acid (Sigma-Aldrich, USA), different concentrations of fetal bovine serum (FBS, Lonza, Walkersville, MD, USA) and the presence or absence of basic fibroblast growth factor (b-FGF at 10 ng mL⁻¹, R&D Systems Inc, Minneapolis, Minn, USA) to find the best option for chondrocytes culture (10% FBS + b-FGF). MSC were cultured with DMEM media supplemented with 100 units per mL of penicillin, 100 μg mL⁻¹ of streptomycin, 0.25 μg mL⁻¹ of AM and 10% FBS.

Melgar-Lesmes P., Bosch O., Zubajlo R., Molins G., Comfort S., Luque-Saavedra A., López-Moya M., García-Polite F., Parri Ferrandis F.J., Rogers C., Gelabertó A., Martorell J., Edelman E.R, & Balcells M. (2023). Optimization of 3D autologous chondrocyte-seeded polyglycolic acid scaffolds to mimic human ear cartilage. Biomaterials Science, 11(10), 3695-3708.

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Corneal Endothelial Cell Differentiation from hPSCs

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The hPSCs (2.5 × 10⁵ cells) were seeded on 1% Matrigel-coated dishes and grew into 30% confluence after 4 days’ culture, then cultured in neural crest differentiation medium (NDM) consisting of DMEM/F12 (Gibco), 20% knockout serum replacement (KSR, Gibco), L-GlutaMAX (2 mM, Gibco), MEM nonessential amino acids (0.1 mM, Gibco), β-mercaptoethanol (0.1 mM, Gibco), basic fibroblast growth factor (4 ng/mL, bFGF, R&D Systems), and 1 μM retinoic acid (RA, Sigma). After 5 days of culture, the differentiated cells grew into approximately 80% confluence and the medium was replaced with corneal endothelial differentiation medium (CDM) consisting of DMEM/F12 (Gibco), 0.2% BSA, bFGF (8 ng/mL), PDGF-BB (10 μg/mL, R&D Systems), DKK-2 (10 μg/ml, R&D Systems), insulin-transferring-selenium (Gibco), 2 mM L-GlutaMAX (Gibco), NEAA (0.1 mM), ascorbic acid (50 μg/mL, Sigma), Heregulin β-1 (10 ng/mL, Peprotech), IGF-1 (200 ng/mL, Peprotech), 50 × B27 (Gibco), β-mercaptoethanol (0.01 mM, Sigma), Y27632 (10 μM, Sigma), and SB431542 (1 μM, Millipore). The NCCs were induced into CEPs in the following 3 days, and toward CECs within 14 days. All cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ and the medium was changed every day.

Li Z., Duan H., Jia Y., Zhao C., Li W., Wang X., Gong Y., Dong C., Ma B., Dou S., Zhang B., Li D., Cao Y., Xie L., Zhou Q, & Shi W. (2022). Long-term corneal recovery by simultaneous delivery of hPSC-derived corneal endothelial precursors and nicotinamide. The Journal of Clinical Investigation, 132(1), e146658.

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Muscle Stem/Progenitor Cell Culture Protocols

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Primary human muscle stem/progenitor cells were cultured with F10 basal medium (Gibco, cat#11550043) containing 20% FBS (Gibco, cat#10-013-CV), 2.5 ng/mL bFGF (R&D, cat#233-FB-025). Mouse muscle stem cells were cultured with F10 basal medium (Gibco, cat#11550043) containing 20% FBS (Gibco, cat#10-013-CV), 2.5 ng/mL bFGF (R&D, cat#233-FB-025), 5 ng/mL IL-1α (Peprotech, cat#211-11 A), 5 ng/mL IL-13 (Peprotech, cat#210-13), 5 ng/mL IFN-γ (Peprotech, cat#315-05), 5 ng/mL TNF-α (Peprotech, cat#315-01 A), and 1% penicillin-streptomycin (Gibco, cat#15140-122) in collagen-coated dishes at 37 °C in 5% CO₂ as described previously^{31 (link)}. The differentiation medium was DMEM (Gibco, cat#11965118) containing 2% horse serum (HyClone, cat#HYCLSH30074.03HI), and 1% penicillin-streptomycin (Gibco, cat#15140-122).

Shao X., Fu X., Yang J., Sui W., Li S., Yang W., Lin X., Zhang Y., Jia M., Liu H., Liu W., Han L., Yu Y., Deng Y., Zhang T., Yang J, & Hu P. (2023). The asymmetrical ESR1 signaling in muscle progenitor cells determines the progression of adolescent idiopathic scoliosis. Cell Discovery, 9, 44.

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Cardiac Differentiation of Human Pluripotent Stem Cells

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KIND1 is an in-house derived hES cell line derived at our laboratory in Mumbai [40 (link)] and the HES3 cell line (WiCell Research Institute Inc.) was available from Dr Prabha Sampath’s laboratory for the present study.
Undifferentiated feeder-free KIND1 hES cells were cultured in Stempro hES SFM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 8 ng of bFGF (Peprotech, NJ, USA) as described earlier [7 (link)], while the HES3 cell line was grown in mTeSR™1 medium (STEMCELL Technologies Inc., Canada) at 37 °C and 5% CO₂. For subjecting the confluent pluripotent KIND1 and HES3 hES cells to cardiac differentiation, they were transitioned from growth medium into RPMI 1640 containing 5% B-27 and 1% glutamax (basal medium), and the differentiation protocol was followed as reported by our group earlier [41 (link)]. In brief, cells were first exposed to basal medium supplemented with 100 ng/ml Activin A (Peprotech) and 5 ng/ml of bFGF (R&D Systems, MN, USA) for 24 h. This was followed by 15 ng/ml BMP4 (R&D Systems) and 5 ng/ml bFGF (R&D Systems) in basal medium for another 4 days. Finally, the cells were treated with WNT pathway blocker DKK1 (Peprotech) at 150 ng/ml concentration for the next 4 days. From day 9 onward, the cells were maintained in basal medium until day 20 wherein the media were changed on every alternate day.

Pursani V., Bhartiya D., Tanavde V., Bashir M, & Sampath P. (2018). Transcriptional activator DOT1L putatively regulates human embryonic stem cell differentiation into the cardiac lineage. Stem Cell Research & Therapy, 9, 97.

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