Basal medium consisted of Dulbecco’s modified Eagle’s medium-high glucose (Sigma-Aldrich, USA) supplemented with 5 mM sodium bicarbonate (Cinética Química Ltda, Brazil), penicillin (100 units/mL; Sigma-Aldrich), streptomycin (0.1 mg/mL; Sigma-Aldrich), amphotericyn B (0.25 μg/mL; Sigma-Aldrich), gentamicin (60 mg/L; Schering-Plough, USA), and 10% of either aHS or FBS (Cripion Biotecnologia Ltda, Brazil). Adipogenic medium consisted of basal medium with 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), 200 μM indomethacin (Sigma-Aldrich), 1 μM dexamethasone (Aché, Brazil), and 10 μM insulin (Eli Lilly and Company, USA). Osteogenic medium consisted of basal medium with 50 μg/mL ascorbate-2-phosphate (Ecibra, Brazil), 10 mM β-glycerophosphate (Sigma-Aldrich), and 0.1 μM dexamethasone (Aché). Chondrogenic medium consisted of basal medium with 1 mM dexamethasone (Aché), 125 μg/mL bovine serum albumin (PAA, Austria), 1 mM pyruvate (Sigma-Aldrich), 200 U/mL insulin (Eli Lilly and Company), 3.25 μg/mL transferrin (Wako, Brazil), 0.01 μg/mL transforming growth factor-β1 (Sigma-Aldrich), 5 mg/mL ascorbate-2-phosphate (Ecibra) and a reduced concentration of serum supplements - 1% aHS or 1% FBS [5 (link)].
Transforming growth factor β1
Manufactured by Merck Group
Sourced in United States
Transforming growth factor-β1 (TGF-β1) is a cytokine that belongs to the transforming growth factor beta family. It is involved in the regulation of cellular processes, including cell growth, differentiation, and apoptosis. TGF-β1 plays a crucial role in various biological processes and can be used as a laboratory research tool.
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Lab products found in correlation
Pyruvate, by Merck Group (2 mentions)
Isobutylmethylxanthine, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Indomethacin, by Merck Group (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Insulin, by Merck Group (2 mentions)
Transferrin, by Fujifilm (1 mentions)
Dulbecco s modified eagle s medium high glucose, by Merck Group (1 mentions)
Gentamicin, by Merck & Co (1 mentions)
Amphotericyn b, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Insulin, by Eli Lilly (1 mentions)
Horse serum, by Euroclone (1 mentions)
6 well multidish, by Thermo Fisher Scientific (1 mentions)
4 hydroxytamoxifen, by Merck Group (1 mentions)
Matrigel, by BD (1 mentions)
Fibroblast growth factor 2 (fgf2), by Merck Group (1 mentions)
Bmp 2, by Merck Group (1 mentions)
8 protocols using transforming growth factor β1
1
Optimized Stem Cell Culture Media
2
Skeletal Muscle Differentiation Assay
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The ability of C2C12 cells, GRMD MABs and SKM cells to differentiate into skeletal muscle was assessed in vitro by exposing the cells upon reaching 80% of confluence to a specific differentiation medium composed of DMEM supplemented with 2% horse serum (Euroclone), 1% P/S, 1% L-glutamine for 4 to 10 days (53 ). C2C12 cells were induced to differentiate on a 6-well multidish (Nunc), while GRMD MABs and SKM cells were seeded onto matrigel (BD Biosciences) coated dishes. When cells were transduced with the tamoxifen-inducible MyoD-ER lentiviral vector, 1 μM 4-hydroxy-tamoxifen (Sigma-Aldrich) was added in the growth medium that was replaced 24 h later by differentiation medium supplemented with 1 μM 4-hydroxy-tamoxifen. Half of the medium was replaced with fresh differentiation medium every other day. Immunofluorescence (IF) staining for myosin heavy chain (MyHC) was performed to confirm multinucleated myotubes (see specific section for ‘IF staining’). Differentiation of GRMD MABs in smooth muscle-like cells was induced by treating the cells for 10 days with differentiation medium supplemented with 5 ng/ml transforming growth factor β1 (Sigma-Aldrich) (62 (link)). Smooth muscle differentiation was confirmed by IF staining for α-smooth muscle actin (αSMA; see specific section for ‘IF staining’).
3
Odontoblastic Differentiation of hDPSCs
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To induce odontoblast-like differentiation, hDPSCs were treated with odontoblastic differentiation medium; growth medium with 10 nM dexamethasone, 50 ng/mL BMP2, 20 ng/mL transforming growth factor-β1, and 5 ng/mL fibroblast growth factor-2 (all from Sigma-Aldrich, St. Louis, MO, USA), for 3–10 days with or without PEMF [33 (link)]. Control cultures were placed elsewhere to avoid exposure to PEMF and were incubated with the same medium as described for the experimental cultures.
4
TGF-β1 Signaling Pathway Modulation
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Transforming growth factor-β1 (cat #: T1654), UA (cat #: 03240595), and protein A agarose (cat #: P1406) were purchased from Sigma–Aldrich (St. Louis, MO, United States). Matrigel (cat #: 354263) was purchased from Corning Incorporated (New York, NY, United States). Puromycin (cat #: REVG1001) was purchased from GENECHEM (Shanghai, China). The anti-NOX4 (cat #: ab109225), anti-RhoA (cat #: ab187027), anti-ROCK1 (cat #: ab205829), anti-α-SMA (cat #: ab32575), anti-Collagen-I (cat #: ab6308), anti-p67phox (cat #: ab109366), anti-Rac1 (cat #: ab33186), anti-TIMP1 (cat #: ab61224), anti-MMP1 (cat #: ab137332), anti-MMP2 (cat #: ab37150), anti-MMP9 (cat #: 119906), and anti-F-actin (cat #: ab205) antibodies were purchased from Abcam (Cambridge, MA, United States). The anti-GAPDH antibody was purchased from OriGene (cat #: TA802519) (Rockville, MD, United States). The full-length human recombinant NOX4 (cat #: H00050507-G01) and RhoA (cat #: H00000387-P01) proteins were purchased from Abnova (Walnut, CA, United States).
5
Canine Bone Marrow-Derived Mesenchymal Stem Cell Induction
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BM-MSCs were obtained from dogs (2 years old, provided by the Institutional Review Board of the Chinese PLA General Hospital) and isolated as described [10 (link)]. BM-MSCs (passage 3) were cultured and induced with conditioned medium (10 ng/mL fibroblast growth factor, 10 ng/mL transforming growth factor β1, 50 mg/L vitamin C, 10−7 mol/L dexamethasone, 1% insulin-transferrin iron selenium, and 10% fetal bovine serum, all from Sigma). The biphasic scaffolds were placed into 6-well plates and induced for 2 weeks.
6
Chondrogenic Differentiation of Mesenchymal Stem Cells
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Passage two MSCs (5×105 cells/dish) were collected in 5-ml conical polypropylene tubes and then pelleted for 6 minutes at 1,600 rpm. The cell pellet was incubated in chondrogenic culture medium [growth medium containing 50 µg/ml ascorbic phosphate (Sigma-Aldrich), 2 mM pyruvate (Sigma-Aldrich), 10 mg/ml transforming growth factor-β1 (Sigma-Aldrich), and 10 µg/ml insulin (Sigma-Aldrich)]. After 21 days, the pellets were washed with PBS and then fixed with 4% PFA for 4 hours.
7
Differentiation of CFU-Fs Into Osteogenic, Adipogenic, and Chondrogenic Lineages
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The differentiation capacity of CFU-Fs was assessed at P2–P4 as previously described [24 (link)]. Briefly, to induce osteogenic differentiation, adherent cells were incubated in α-minimal essential medium (Euroclone), 10% FBS (Euroclone), 50 µg/mL penicillin, 50 mg/mL streptomycin and 2 mmol/L L-glutamine, supplemented with 107 mol/L dexamethasone, 50 mg/mL-ascorbic acid and 5 mmol/L glycerol phosphate (all from Sigma-Aldrich).
To induce adipogenic differentiation, the medium described above was supplemented with 100 mg/mL insulin, 50 mmol/L isobutyl methylxanthine and 0.5 mmol/L indomethacin (all from Sigma-Aldrich). For chondrogenic differentiation, the cells were cultured in DMEM F12-HAM containing 1% FBS, 100 nM dexamethasone, 0.05 mM ascorbic acid, 10 ng/mL transforming growth factor-β1, 1% insulin-transferrin-sodium selenite (Sigma-Aldrich). Extracellular matrix protein accumulation was stained with 1% Alcian blue 8GX in 3% acetic acid, pH 2.5 (Bio-Optica, Milan, Italy).
To induce adipogenic differentiation, the medium described above was supplemented with 100 mg/mL insulin, 50 mmol/L isobutyl methylxanthine and 0.5 mmol/L indomethacin (all from Sigma-Aldrich). For chondrogenic differentiation, the cells were cultured in DMEM F12-HAM containing 1% FBS, 100 nM dexamethasone, 0.05 mM ascorbic acid, 10 ng/mL transforming growth factor-β1, 1% insulin-transferrin-sodium selenite (Sigma-Aldrich). Extracellular matrix protein accumulation was stained with 1% Alcian blue 8GX in 3% acetic acid, pH 2.5 (Bio-Optica, Milan, Italy).
8
Hanging-Drop Technique for Generating Mesenchymal Tissue Constructs
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Passages 3 to 5 of the hADSCs were employed to generate MTs. As we previously described, the hanging-drop method was utilized to form MTs consisting of hADSCs.[9 (link)] Briefly, 5 × 105 hADSCs were suspended in 1 mL of culture medium or smooth muscle inductive medium (SMIM; culture medium supplemented with 10 ng/mL of transforming growth factor-β1; Sigma-Aldrich, USA) to form MTs based on gravity-forced cell assembly. The MTs (10,000 cells in 20 μL) generated in the culture medium were defined as non-induced MTs (NI-MTs), while the MTs generated in SMIM were defined as ID-MTs. Both NI-MTs and ID-MTs were kept for 3 days in a hanging-drop array at 37°C in a humidified atmosphere of 5% CO2.
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