Tgf β3

Manufactured by Lonza

Sourced in United States, United Kingdom

TGF-β3 is a recombinant transforming growth factor-beta 3 protein. It is a member of the transforming growth factor beta superfamily of cytokines, which play a role in the regulation of cell growth, differentiation, and other cellular functions.

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Lab products found in correlation

Its premix, by BD (6 mentions) β glycerol phosphate, by Merck Group (3 mentions) Hmsc chondrogenic differentiation bulletkit, by Lonza (2 mentions) Sodium pyruvate, by Thermo Fisher Scientific (2 mentions) L proline, by Lonza (2 mentions) Hmsc adipogenic bulletkit, by Lonza (2 mentions) α mem, by Lonza (2 mentions) Alizarin red staining, by Merck Group (2 mentions) 4000b dmi, by Leica (1 mentions) Alcian blue, by Merck Group (1 mentions) Pt 3003, by Lonza (1 mentions) Pt 4124, by Lonza (1 mentions) Adipogenic bulletkit, by Lonza (1 mentions) Hmsc chondrogenic singlequots, by Lonza (1 mentions) Ascorbic acid, by Merck Group (1 mentions) Foetal bovine serum (fbs), by Lonza (1 mentions) Hbmsc, by Lonza (1 mentions) Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Lonza (1 mentions)

22 protocols using tgf β3

Multilineage Differentiation of MSCs

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Cells were grown from cryopreserved stocks to confluence in twelve-well-plates and then subjected to specific differentiating conditions using the osteogenic differentiation medium PT-4120, and chondrogenic medium PT-3003 supplemented with 10 ng/mL TGFβ3, PT-4124 (all from Lonza) under conditions described by the manufacturer.
Multilineage differentiation potential of MSCs was assessed by histochemical staining and phase contrast microscopy (Leica 4000b DMI, Leica Microsystems GmbH. Germany).
The degree of mineralization in osteogenic cultures was assessed staining with 2% Alizarin Red S (Sigma-Aldrich-Aldrich). Briefly, cells were washed three times with phosphate buffered saline (PBS) pH 4.2 and fixed 20 minutes with 4% paraformaldehyde. Fixed cells were washed and stained and washed again to remove excess of stain. Similarly, chondrogenic potential was evaluated measuring production ECM proteoglycan produced by chondrocytes after staining for 20 minutes with 1% Alcian blue (Sigma-Aldrich).

Tornero-Esteban P., Peralta-Sastre A., Herranz E., Rodríguez-Rodríguez L., Mucientes A., Abásolo L., Marco F., Fernández-Gutiérrez B, & Lamas J.R. (2015). Altered Expression of Wnt Signaling Pathway Components in Osteogenesis of Mesenchymal Stem Cells in Osteoarthritis Patients. PLoS ONE, 10(9), e0137170.

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Multilineage Differentiation of hMSCs

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For adipogenic differentiation the hMSC Adipogenic BulletKit (PT-3004, Lonza) was used, and for osteogenic differentiation 10–8 M dexamethasone, 0.2 mM ascorbic acid and 10 mM β-glycerolphosphate (Sigma) was added to the media. For chondrogenic differentiation the hMSC Chondrogenic Differentiation BulletKit (PT-3003, Lonza) with TGF-β 3 (PT-4124, Lonza) was applied. Differentiation was assessed with specific stainings (Oil Red O, adipogenic; Alizarin Red, osteogenic; Safranin O, chondrogenic).

Khong S.M., Lee M., Kosaric N., Khong D.M., Dong Y., Hopfner U., Aitzetmüller M.M., Duscher D., Schäfer R, & Gurtner G. (2018). Single-Cell Transcriptomics of Human Mesenchymal Stem Cells Reveal Age-Related Cellular Subpopulation Depletion and Impaired Regenerative Function. Stem cells (Dayton, Ohio), 37(2), 240-246.

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hADSC Chondrogenic Differentiation Protocol

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hADSC chondrogenic differentiation was achieved as previously described (Calabrese et al., 2015 (link)). Briefly, 2.5 × 10⁵ cells were centrifuged to form a three-dimensional aggregate and resuspended in complete chondrogenic medium containing differentiation basal medium (Chondrogenic Basal Medium, Lonza) supplemented with chondrogenic differentiation inducing factors (hMSC Chondrogenic SingleQuots containing Dexamethasone, Ascorbate, ITS+supplement, GA-1000 Sodium Pyruvate, Proline and L-Glutamine, Lonza) and TGF-β3 (Lonza). Pellets were incubated at 37°C in a humidified atmosphere of 5% CO₂. The growth medium was replaced every 2–3 days. The chondrogenic differentiation was completed on day 28 after induction. Pellets were fixed in formalin at three different time points along differentiation (1, 2, and 4 weeks). Subsequently, pellets were paraffin embedded and cut into 3 μm-thick sections for immunohistological processing.

Calabrese G., Forte S., Gulino R., Cefalì F., Figallo E., Salvatorelli L., Maniscalchi E.T., Angelico G., Parenti R., Gulisano M., Memeo L, & Giuffrida R. (2017). Combination of Collagen-Based Scaffold and Bioactive Factors Induces Adipose-Derived Mesenchymal Stem Cells Chondrogenic Differentiation In vitro. Frontiers in Physiology, 8, 50.

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Chondrogenic Differentiation of hMSCs

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To form a cell pellet, 2.5×10⁵ hMSCs in 0.5 ml medium were centrifuged down in a 15 ml conical tube at 150×g for 5 minutes at room temperature [41] (link). Chondrogenic differentiation was induced by chondrogenic medium composed of DMEM-high glucose, supplemented with 1% ITS+Premix (BD Bioscience), 1% penicillin-streptomycin, 100 μg/ml sodium pyruvate (Invitrogen), 50 μg/ml ascorbic acid-2-phosphate, 40 μg/ml L-proline, 0.1 μM dexamethasone, and 10 ng/ml recombinant human transforming growth factor-beta3 (TGF-β3) (Lonza, Walkersville, MD). Pellets were cultured at 37°C, 5% CO₂ with medium changes every 2–3 days for 24 days.

Chen J., Li C, & Wang S. (2014). Periodic Heat Shock Accelerated the Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Pellet Culture. PLoS ONE, 9(3), e91561.

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Characterization of MSC Differentiation

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MSC can be functionally characterised by their in vitro differentiation capacity, and we evaluated the differentiation potential into the adipogenic, osteogenic and chondrogenic lineage as described previously [27] (link). Briefly, for adipogenic and osteogenic differentiation MSC were seeded at a density of 1000 cells per cm² and kept under standard culture conditions until reaching subconfluency. Subsequently, either adipogenic differentiation was induced using the hMSC Adipogenic BulletKit (Lonza), or osteogenic differentiation was induced using “osteogenic medium” composed of α-MEM (Lonza), 1% PS (Lonza), 10% FCS, 10⁻⁸ M dexamethasone, 0.2 mM ascorbic acid and 10 mM β-glycerolphosphate (Sigma-Aldrich). After three weeks under differentiation conditions, lipid vacuoles in adipogenic cultures were stained with Oil Red O and calcium deposits of osteogenic cultures with Alizarin Red S, respectively. For chondrogenic differentiation, 2.5 × 10⁵ MSC were kept in micromass pellet cultures. Differentiation was induced using the hMSC Chondrogenic Differentiation BulletKit (Lonza), supplemented with TGF-β 3 (Lonza) as a growth factor. After four weeks of differentiation, frozen sections of fixed pellets were performed and stained with Alcian blue.

Schäfer R., Schwab M., Siegel G., von Ameln-Mayerhofer A., Buadze M., Lourhmati A., Wendel H.P., Kluba T., Krueger M.A., Calaminus C., Scheer E., Dominici M., Grisendi G., Doeppner T.R., Schlechter J., Finzel A.K., Gross D., Klaffschenkel R., Gehring F.K., Spohn G., Kretschmer A., Bieback K., Krämer-Albers E.M., Barth K., Eckert A., Elser S., Schmehl J., Claussen C.D., Seifried E., Hermann D.M., Northoff H, & Danielyan L. (2020). Modulating endothelial adhesion and migration impacts stem cell therapies efficacy. EBioMedicine, 60, 102987.

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Chondrogenic Differentiation of hBMSCs

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Chemicals were purchased from Sigma–Aldrich (Castle Hill, NSW, Australia) unless stated otherwise. Human bone marrow-derived mesenchymal stromal cells (hBMSCs) from three donors (Donor 1: 34Y,F; Donor 2: 23Y,F; Donor 3: 26Y,F) were purchased from Lonza (Mt Waverley, VIC, Australia). Low glucose cell culture medium was supplied by Life Technologies, USA (Dulbecco's Modified Eagle's Medium (DMEM), containing d-glucose and sodium pyruvate). Life Technologies, UK; Scientifix Life, USA; and Lonza, AU supplied, respectively, penicillin-streptomycin 100 U ml⁻¹ (pen/strep), foetal bovine serum (FBS), and Transforming Growth Factor-β3 (TGF-β3). Dulbecco's Phosphate-buffered saline (DPBS) was purchased from Thermo Fisher (Scoresby, VIC, Australia). All experiments were performed with biological and technical triplicates.

Donderwinkel I., Tuan R.S., Cameron N.R, & Frith J.E. (2023). A systematic investigation of the effects of TGF-β3 and mechanical stimulation on tenogenic differentiation of mesenchymal stromal cells in a poly(ethylene glycol)/gelatin-based hydrogel. Journal of Orthopaedic Translation, 43, 1-13.

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Multilineage Differentiation of Murine MSCs

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We assessed the differentiation potential of murine oMSC and sMSC into the adipogenic, osteogenic and chondrogenic lineage as described previously [22] . Briefly, for adipogenic and osteogenic differentiation, MSC were seeded at a density of 1,000 cells per cm² and kept under standard culture conditions until reaching subconfluency. Subsequently, either adipogenic differentiation was induced using the MSC Adipogenic BulletKit (Lonza), or osteogenic differentiation was induced using “osteogenic medium” composed of α-MEM (Lonza), 1% PS (Lonza), 10% FCS, 10⁻⁸ M dexamethasone, 0.2 mM ascorbic acid and 10 mM β-glycerolphosphate (Sigma-Aldrich). After three weeks under differentiation conditions, lipid vacuoles in adipogenic cultures were stained with Oil Red O and calcium deposits of osteogenic cultures with Alizarin Red S, respectively. For chondrogenic differentiation, 2.5 × 10⁵ MSC were kept in micromass pellet cultures. Differentiation was induced using the MSC Chondrogenic Differentiation BulletKit (Lonza), supplemented with TGF-β 3 (Lonza) as a growth factor. After four weeks of differentiation, frozen sections of fixed pellets were performed and stained with Alcian blue.

Danielyan L., Schwab M., Siegel G., Brawek B., Garaschuk O., Asavapanumas N., Buadze M., Lourhmati A., Wendel H.P., Avci-Adali M., Krueger M.A., Calaminus C., Naumann U., Winter S., Schaeffeler E., Spogis A., Beer-Hammer S., Neher J.J., Spohn G., Kretschmer A., Krämer-Albers E.M., Barth K., Lee H.J., Kim S.U., Frey WH I.I., Claussen C.D., Hermann D.M., Doeppner T.R., Seifried E., Gleiter C.H., Northoff H, & Schäfer R. (2020). Cell motility and migration as determinants of stem cell efficacy. EBioMedicine, 60, 102989.

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Evaluating PURA's Effect on Chondrogenic Differentiation

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To evaluate the effects of PURA on the chondrogenic potential of hASCs, the cells were plated in 24-well plates (1.5 × 10⁴ cells/well) and maintained in culture for 3 days before the differentiation protocol. To assess whether cell confluence could influence chondrogenic differentiation on PURA, another treatment group (PURA2) was added where 7 × 10⁴ cells/well were plated. The hASCs were induced for chondrogenic differentiation for a period of 21 days using Human Mesenchymal Stem Cell (hMSC) Chondrogenic Differentiation Medium BulletKit 10TM (Lonza^®, Walkersville, MD, USA; catalog number PT-3003). The differentiation medium was supplemented with 10 ng/mL of TGF-β3 (Lonza^®, Walkersville, MD, USA; catalog number PT-4124) in each of the medium changes, which was performed every 3–4 days. After 21 days of culturing, the efficiency of chondrogenic differentiation was assessed using Safranin O staining, glycosaminoglycan (GAG) quantification, and expression of chondrogenic genes using real-time quantitative polymerase chain reaction (RT-qPCR).

Nogoceke R., Josino R., Robert A.W, & Stimamiglio M.A. (2023). Evaluation of a Peptide Hydrogel as a Chondro-Instructive Three-Dimensional Microenvironment. Polymers, 15(24), 4630.

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Chondrocyte Differentiation in Pellet Culture

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Cells were differentiated as pellet cultures in 15 ml polypropylene conical tubes with 2.5 × 10⁵ per pellet. The pellets were formed by centrifugation and CSE was added at the time of induction with chondrocyte induction medium supplemented with TGF-β3 (Lonza). After 24 h the medium was changed to fresh TGF-β3 supplemented chondrocyte induction medium followed by new medium every 3 d. After 21 d, the pellets were fixed in formalin, and either paraffin embedded and sectioned (5 μm) or left intact and stained with Alcian blue to visualize glycosaminoglycans followed by a Nuclear Fast Red (Sigma-Aldrich) counterstain.

Wahl E.A., Schenck T.L., Machens H.G, & Egaña J.T. (2016). Acute stimulation of mesenchymal stem cells with cigarette smoke extract affects their migration, differentiation, and paracrine potential. Scientific Reports, 6, 22957.

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Multilineage Differentiation of Mesenchymal Stem Cells

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Twenty‐four hours after NP loading, hAMSC and hCV‐MSC were counted and differentiated toward chondrogenic, adipogenic and osteogenic lineages. For chondrogenic differentiation, 2.5 × 10⁵ cells were grown for 2–3 weeks in tubes with hMSC Chondrogenic bulletkit (Lonza) supplemented with 10 ng/ml TGFβ3 (Lonza). The differentiation was detected with Alcian Blue staining (Sigma‐Aldrich, Italy). For adipogenic differentiation, cells were plated at 5 × 10³/cm² and when 80% confluency was reached, differentiation was induced using hMSC Adipogenic bulletkit (Lonza). Three weeks after the start of differentiation, the cytoplasmatic lipid vacuoles were stained with Oil red solution (Sigma‐Aldrich, Italy). For osteogenic differentiation, cells were plated at 5 × 10³/cm² and when 80% confluency was reached, differentiation was induced using hMSC Osteogenic bulletkit (Lonza). Calcium deposition was detected 3 weeks after the start of differentiation by Alizarin red staining (Sigma‐Aldrich, Italy). CHANG medium C by itself and cells without NPs were used as controls.

Bigini P., Zanier E.R., Saragozza S., Maciotta S., Romele P., Bonassi Signoroni P., Silini A., Pischiutta F., Sammali E., Balducci C., Violatto M.B., Talamini L., Garry D., Moscatelli D., Ferrari R., Salmona M., De Simoni M.G., Maggi F., Simoni G., Grati F.R, & Parolini O. (2016). Internalization of nanopolymeric tracers does not alter characteristics of placental cells. Journal of Cellular and Molecular Medicine, 20(6), 1036-1048.

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