hMSCs were seeded into four-well plates and exposed to adipogenic induction media composed of DMEM, 10% fetal bovine serum (FBS), 10% horse serum (Gibco, U.S.A.), 100 µM dexamethasone (Sigma, U.K.), 1 µM Rosiglitazone (BRL) (Novo Nordisk Bagsvaerd, Denmark), 3 µg/ml insulin (Sigma, U.K.), 450 µM isobutylmethylxanthine (IBMX) (Sigma, U.K.), and 1% penicillin-streptomycin (Sigma, U.K.) supplemented with FAK inhibitors (PF-573228 and PF-562271) or IGF-1R/InsR inhibitors (NVP-AEW541 and GSK1904529A), which were purchased from Selleckchem Inc. (Selleckchem Inc., Houston, TX, U.S.A.). Inhibitors were used at 5 µM throughout all experiments. Adipocyte induction medium (AIM) was changed every 2 days and for 7 days. Previous published work from our group indicated day 7 as a time point on which adipogenic markers were up-regulated significantly in an enriched population of 70–80% of adipogenic populations [8 (link)].
Rosiglitazone
Manufactured by Novo Nordisk
Sourced in Denmark, United Kingdom, United States
Rosiglitazone is a pharmaceutical compound used in the development and testing of laboratory equipment. It is a member of the thiazolidinedione class of drugs. Rosiglitazone functions as a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, which plays a role in the regulation of glucose and lipid metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Insulin, by Merck Group (8 mentions)
Dexamethasone (dex), by Merck Group (7 mentions)
Horse serum, by Merck Group (5 mentions)
Isobutylmethylxanthine, by Merck Group (4 mentions)
Pen strep, by Merck Group (3 mentions)
Calcitriol, by Merck Group (3 mentions)
L ascorbic acid, by Fujifilm (2 mentions)
β glycerophosphate, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Nvp aew541, by Selleck Chemicals (1 mentions)
Gsk1904529a, by Selleck Chemicals (1 mentions)
Pf 562271, by Selleck Chemicals (1 mentions)
Isobutylmethylxanthine ibmx, by Merck Group (1 mentions)
Pf 573228, by Selleck Chemicals (1 mentions)
Dexamethasone (dex), by Fujifilm (1 mentions)
6 well plate, by Corning (1 mentions)
Alpha modification of eagle s medium, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
9 protocols using rosiglitazone
1
Adipogenic Differentiation of hMSCs
2
Adipogenic Differentiation of Cells
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Cells were grown in standard DMEM growth medium in six-well plates at 0.3 × 106 cells/ml. At 90–100% confluence, cells were cultured in DMEM supplemented with adipogenic induction mixture containing 10% FBS, 10% horse serum (Sigma), 1% Pen–strep, 100 nm dexamethasone, 0.45 mm isobutyl methyl xanthine57 (link) (Sigma), 3 μg/ml insulin (Sigma), and 1 μM Rosiglitazone58 (Novo Nordisk, Bagsvaerd, Denmark). The media were replaced three times per week.
3
Dermal Spheroid Differentiation Protocols
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Individual dermal spheroids were collected and cultured in medium containing adipogenic- or osteogenic- or neurogenic-differentiation factors and compared with spheroids cultured in plain culture medium without differentiation factors [25 (link),27 (link)]. Adipogenic differentiation medium comprised DMEM, 10% FBS, 10% horse serum, 1% penicillin-streptomycin, 100 nM dexamethasone, 0.45 mM isobutyl methyl xanthine, 3 mg/mL insulin, and 1 mM rosiglitazone (Novo Nordisk, Bagsvaerd, Denmark). Osteogenic differentiation medium comprised DMEM, 10% FBS, 1% penicillin-streptomycin, 50 mg/mL L-ascorbic acid (Wako Chemicals, Neuss, Germany), 10mM b-glycerophosphate, 10 nM calcitriol (1a, 25-dihydroxyvitamin D3), and 10 nM dexamethasone. While, neurogenic differentiation medium comprised serum-free DMEM-F12 and 10 mM all-trans retinoic acid (R2625, Sigma-Aldrich, St. Louis, MI, USA(.
4
Adipocytic Differentiation Protocol
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To induce adipocytic differentiation, cells were initially grown in standard DMEM growth medium in 6-well plates at a density of 0.3 × 106 cells/ml. Once 90–100% confluence was reached, the medium was replaced with DMEM supplemented with adipogenic induction mixture, containing 10% FBS, 10% horse serum (Sigma), 1% pen/strep, 100 nM dexamethasone, 0.45 mM isobutyl methylxanthine [41 (link)] (Sigma), 3 μg/ml insulin (Sigma), and 1 μM rosiglitazone [42 (link)] (Novo Nordisk, Bagsvaerd, Denmark). The medium was replaced 3 times per week, and cells cultured in parallel in standard culture medium were used as controls.
5
Dental Pulp Stem Cell Differentiation
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DPSCs from human dental pulp tissue was purchased from Lonza (cat. no. PT-5025. Basel, Switzerland)) and identified by DPSC’s biomarkers. Cryopreserved human DPSCs were recovered from liquid nitrogen using an AccuVital cell recovery kit (AccuRef Scientific, Goshen, NY, USA) and cultured as previously reported. Single-cell suspensions (0.01 to 1 × 105/well) of dental pulp were seeded into 6-well plates (Costar, USA) with alpha modification of Eagle’s medium (Gibco, BRL, USA) supplemented with 20% FBS, 1% Pen-Strept, 10 nM dexamethasone, 50 μg/ml L-ascorbic acid and 10 mM β-glycerophosphate,10 nM calcitriol (Sigma, USA) for 14 d to induce osteogenic differentiation and then incubated at 37°C in 5% CO2. For the induction of adipogenesis, alpha-minimum essential medium (MEM) supplemented with 10% FBS, 10% horse serum, 1% Pen-Strept, 100 nM dexamethasone, 0.45 mM isobutyl methyl xanthine, 3 μg/ml insulin (all purchased from Sigma, USA), and 1 μM rosiglitazone (BRL49653; Novo Nordisk, Denmark) was added to culture DPSCs for 14 d. For hypoxia treatment, DPSCs were cultured under 5% O2 in a hypoxia chamber (STEMCELL Technologies, Vancouver, USA) for 24 h at 37°C. For TNF-α treatment, cells were treated with 20 ng/ml TNF-α for 24 h before collection. The addition of Taxifolin is associated with hypoxia and TNF-α treatment.
6
Osteogenic and Adipogenic Differentiation of Stem Cells
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Cells at the fourth passage were cultured on 6-well plates. At 60–70 % confluency, osteogenic differentiation was induced using osteoinduction medium prepared according to the protocol of Vishnubalaji et al. [60 (link)], and composed of DMEM supplemented with 10 % FBS, 1 % Pen-Strept, 50 μg/ml L-ascorbic acid (Wako Chemicals GmbH, Germany), 10 mM glycerol phosphate disodium salt (β-glycerophosphate), 10 nM dexamethasone, and 10 nM calcitriol (1α,25-dihydroxyvitamin D3) (Sigma, UK). Cells maintained in the regular culture medium served as controls. The resultant osteogenesis was evaluated after 14 days through cytochemical staining for ALP.
Adipogenic differentiation was also induced using standard adipogenic medium [60 (link)], composed of DMEM supplemented with 10 % FBS, 10 % horse serum, 1 % Pen-Strept, 100 nM dexamethasone, 0.45 mM isobutyl methyl xanthine, 3 μg/ml insulin (all purchased from Sigma, UK), and 1 μM rosiglitazone (BRL49653; Novo Nordisk, Denmark). The resultant differentiation was assessed at 14 days through the use of oil red O staining.
Adipogenic differentiation was also induced using standard adipogenic medium [60 (link)], composed of DMEM supplemented with 10 % FBS, 10 % horse serum, 1 % Pen-Strept, 100 nM dexamethasone, 0.45 mM isobutyl methyl xanthine, 3 μg/ml insulin (all purchased from Sigma, UK), and 1 μM rosiglitazone (BRL49653; Novo Nordisk, Denmark). The resultant differentiation was assessed at 14 days through the use of oil red O staining.
7
Adipocytic and Osteoblastic Differentiation Protocol
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Adipocytic and Osteoblastic differentiation were performed as previously described43 (link),48 (link). In brief, cells were cultured in basal medium till 70–80% confluence. Osteogenic induction medium consisting of DMEM containing 10% FBS, 1% P/S, 50 μg/mL L-ascorbic acid (Wako Chemicals GmbH, Neuss, Germany), 10 mM β-glycerophosphate (Sigma), and 10 nM calcitriol[(1α,25-dihydroxy vitamin D3) (sigma)], 10 nM dexamethasone (Sigma) was added. Adipogenic-induction mixture containing 10% FBS, 10% Horse Serum (Sigma), 1% P/S, 100 nM dexamethasone, 0.45 mM isobutyl methyl xanthine [Sigma], 3 μg/mL insulin (Sigma), and 1 μM Rosiglitazone [(BRL49653) (Novo Nordisk, Bagsvaerd, Denmark)] were added to adipogenic cultures. Both induction media were replaced every 3 days.
8
Adipogenic Differentiation of Cells
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Cells were grown in standard DMEM growth medium in 6-well plates at 0.3 × 106 cells/ml. At 90–100% confluence, cells were switched to DMEM supplemented with adipogenic induction mixture containing 10% FBS, 10% horse serum (Sigma), 1% Pen-strep, 100 nM dexamethasone, 0.45 mM isobutyl methyl xanthine36 (link) (Sigma)], 3 μg/ml insulin (Sigma), and 1 μM rosiglitazone37 (link) (Novo Nordisk, Bagsvaerd, Denmark). The media were replaced 3 times per week. Cells cultured in standard culture medium were considered as the negative control. For dose response experiments, cells were seeded and treated at D -2 with 10 ng/ml TGF-β1 for 2 days commitment stage, then differentiation stage was initiated by adding adipocytic induction medium at D0, while TGF-β1 treatment was continued till day 7 in TGF-β -continuous treated cells.
9
Adipogenic Differentiation Protocol
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Cells were grown in standard DMEM growth medium in 6-well plates at 0.3 × 106 cells/ml. When a 90–100% cell confluence was reached, the cells were cultured in DMEM supplemented with adipogenic induction mixture containing 10% FBS, 10% Horse Serum (Sigma-Aldrich, St. Louis, MO), 1% Pen-strep, 100 nM dexamethasone, 0.45 mM isobutyl methyl xanthine18 (link) (Sigma, US), 3 μg/mL insulin (Sigma, US), and 1 μM Rosiglitazone19 (link) (Novo Nordisk, Bagsvaerd, Denmark). The media used was replaced 3 times per week.
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