hPDCs at passage 3-5 were evaluated for their ability to differentiate into adipogenic, chondrogenic, or osteogenic lineages in vitro using previously described techniques 12 (link),16 (link). Briefly, cells were cultured in lineage‐specific media for 21 days, with the media changed every 3 days. Adipogenic medium contained 1 μM dexamethasone, 10 μM insulin, 100 μM indomethacin, and 500 μM isobutylmethylxanthine. For the detection of lipid droplets, differentiated cells were stained by oil red O solution for 30 min. Chondrogenic medium consisted of 90% StemPro Osteocyte/Chondrocyte Differentiation Basal Medium (Invitrogen, CA, USA) and 10% StemPro Chondrogenesis Supplement (Invitrogen). Chondrogenesis was evaluated by Alcian blue staining. Osteogenic induction medium was composed of DMEM, supplemented with 10% FBS, 50 μg/mL L-ascorbic acid 2-phosphate, 10 nM dexamethasone, and 10 mM β-glycerophosphate. Osteogenesis was confirmed by alizarin red S and von Kossa staining. During osteoblastic differentiation, hPDCs were sampled and the culture media was collected 48 h after 0, 10, and 21 days of culture.
Stempro chondrogenesis supplement
Manufactured by Thermo Fisher Scientific
Sourced in United States
StemPro® Chondrogenesis Supplement is a cell culture medium supplement designed to support chondrogenesis, the differentiation of stem cells or progenitor cells into chondrocytes, the cells that make up cartilage tissue.
Automatically generated - may contain errors
Lab products found in correlation
Stempro osteocyte chondrocyte differentiation basal medium, by Thermo Fisher Scientific (3 mentions)
Stempro chondrocyte differentiation basal medium, by Thermo Fisher Scientific (2 mentions)
Osteogenic stimulatory supplement, by STEMCELL (1 mentions)
Adipogenic stimulatory supplement, by STEMCELL (1 mentions)
Dexamethasone (dex), by STEMCELL (1 mentions)
β glycerophosphate, by STEMCELL (1 mentions)
Mesencult msc basal medium, by STEMCELL (1 mentions)
Ascorbic acid, by STEMCELL (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Stempro osteocyte chondrocyte basal medium, by Thermo Fisher Scientific (1 mentions)
Bright ot5000 cryostat, by Avantor (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Ti e microscope, by Nikon (1 mentions)
Alcian blue 8gx dye, by Merck Group (1 mentions)
9 protocols using stempro chondrogenesis supplement
1
Multilineage Differentiation of hPDCs
2
Differentiation of Dental-Derived MSCs
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Dental-derived MSCs were differentiated at passage four into adipocytes, and chondrocytes using suitable culture conditions as previously described [18 (link), 19 (link)]. For adipocyte differentiation, cells were cultured in DMEM containing 10% FBS, 100 µM indomethacin, 10 µM insulin, and 1 µM dexamethasone for 21 days. The accumulation of lipid droplets was evaluated by Oil Red O staining. For chondrogenesis, cells were cultured in StemPro chondrocyte differentiation basal medium and 10% StemPro chondrogenesis supplement (Invitrogen, Carlsbad, CA, USA) for 21 days. The deposition of proteoglycans was confirmed by Alcian blue staining.
3
Differentiation of human BM-derived MSCs
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To qualify isolated cells from human BM as hMSCs, cells were induced to differentiation into adipogenic, osteogenic and chondrogenic lineages. Briefly, for adipogenic differentiation, 5x103 hMSCs/cm2 were exposed to Complete MesenCult Adipogenic Medium containing MesenCult MSC Basal Medium (Stemcell) and 10% Adipogenic Stimulatory Supplement (Stemcell) for 3 weeks. For osteogenic differentiation, 2x105 cells/cm2 incubated with Complete MesenCult Osteogenic Medium including MesenCult MSC Basal Medium, Osteogenic Stimulatory Supplement, β-Glycerophosphate, Dexamethasone, Ascorbic acid (all from Stemcell) for 5 weeks. For chondrogenic differentiation, 7.5x106 cells/cm2 were cultured for 3 weeks with Stempro Chondrocyte Differentiation Basal Medium (Gibco) containing 10% Stempro Chondrogenesis Supplement (Gibco). Standard histochemical staining methods were applied. Osteogenic, adipogenic and chondrogenic differentiation were confirmed by Toluidine Blue, Oil Red O, Alcian Blue staining, respectively.
4
Multilineage Differentiation of DPSCs
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The in vitro differentiation potential of DPSCs (into adipocytes, osteocytes, and chondrocytes) was evaluated using a previously published protocol with minor modifications (Jang et al. 2018 (link)). Briefly, adipogenesis was carried out in the adipogenic medium with 10 µM insulin, 1 µM dexamethasone, 100 µM indomethacin, and 500 µM isobutylmethylxanthine (IBMX). Adipogenesis was confirmed by the accumulation of oil droplets upon staining of the differentiated cells with Oil red O solution. Osteogenesis was performed in the osteogenic medium consisting of 50 µM ascorbate-2-phosphate, 0.1 µM dexamethasone, and 10 mM glycerol-2-phosphate. Osteogenesis was then confirmed by the accumulation of calcium nodules upon staining of the differentiated cells with alizarin red and von Kossa. Chondrogenesis was carried out using the commercial chondrogenic medium (StemPro® Osteocyte/Chondrocyte Differentiation Basal Medium; StemPro® Chondrogenesis supplement, Gibco, Life technologies). Chondrogenesis was confirmed by staining the differentiated cells with Safranin O and Alcian blue.
5
Multilineage Differentiation of Human Dental Pulp Stem Cells
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The in vitro differentiation ability into adipocyte, osteocyte, and chondrocyte lineages was assessed as previously described 10 (link), 12 . Briefly, hDPSCs-fresh and hDPSCs-cryo were cultured in ADMEM containing lineage-specific constituents for 21 days with a change to fresh medium every 3 days. The adipogenic medium was composed of 1 µM dexamethasone, 10 µM insulin, 100 µM indomethacin, and 500 µM isobutylmethylxanthine (IBMX). Adipogenesis was confirmed based on the accumulation of lipid droplets by staining with Oil red O solution. The osteogenic medium was composed of 0.1 µM dexamethasone, 50 µM ascorbate-2-phosphate, and 10 mM glycerol-2-phosphate. Osteogenesis was confirmed via Alizarin Red and Von Kossa staining. The commercial chondrogenic medium (StemPro® Osteocyte/Chondrocyte Differentiation Basal Medium; StemPro® Chondrogenesis supplement, Gibco Life Technologies, Gaithersburg, MD, USA) and differentiation was confirmed via Alcian blue staining (Fig. 3 A). Cytochemical staining with Oil red O, Alizarin Red, Von Kossa, and Alcian Blue were conducted as detailed by the manufacturer as previously reported 10 (link),12 ,15 . The in vitro differentiated cells were analyzed for lineage-specific marker expressions by using real-time quantitative PCR (RT-qPCR) (Figs. 3 B & C).
6
Chondrogenic Differentiation of hMSCs
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For chondrogenic differentiation, the hMSC line was harvested at p3 and seeded into 24-well plates at a density of 7.5 × 105 cells/cm2. To stimulate chondrogenic differentiation, the culture medium was replaced with Stempro chondrocyte differentiation basal medium (Gibco) containing 10% Stempro chondrogenesis supplement (Gibco). After 21 days of cultivation, the chondrogenic pellet was stained with Alcian Blue.
7
In vitro Differentiation of hDPSCs
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In vitro differentiation into mesenchymal lineages (adipocytes, osteocytes, and chondrocytes) was performed using previously described protocols [21 (link),24 (link)]. Briefly, third-passage hDPSCs-cryo at 70% confluence were induced to lineage specific growth conditions for 21 days. Adipogenic medium consisted of 1 µM dexamethasone, 10 µM insulin, 100 µM indomethacin, and 500 µM isobutyl methyl xanthine (IBMX). For the confirmation of adipogenesis, differentiated cells were analyzed for the accumulation of lipid droplets by staining with Oil red O solution for 30 min. Osteogenic medium consisted of 50 µM ascorbate-2-phosphate, 10 mM glycerol-2-phosphate, and 0.1 µM dexamethasone. Osteogenesis was confirmed by staining differentiated cells with Alizarin red and von Kossa for the detection of mineralization and calcium deposition, respectively. Chondrogenesis was induced by using commercial chondrogenic medium (StemPro® Osteocyte/Chondrocyte Differentiation Basal Medium; StemPro® Chondrogenesis supplement, Gibco® by life technologies, Grand Island, NY, USA) and differentiation was analyzed by Alcian blue and Safranin O staining. RT-qPCR was used to evaluate the expression level of lineage-specific marker genes.
8
Chondrogenic Differentiation of Mesenchymal Cells
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1 × 106 cells were cultured as pellet culture in StemPro Osteocyte/Chondrocyte basal medium (Life Technologies) supplemented with StemPro Chondrogenesis supplement (Life Technologies) in a humidified incubator at 37°C and 5% CO2. Fresh medium was provided every 2–3 days. After 21 days, medium was aspirated and cell pellets were washed with PBS before fixation in 4% PFA for 60 min. After 3 washings steps with ddH2O, Alcian Blue staining solution was added [0.1 mg/ml Alcian Blue dissolved in 6 parts EtOH and 4 parts of acetic acid (Sigma-Aldrich)] and incubated over night at room temperature under exclusion of light. Cell pellets were embedded in O.C.T compound (TAAB Laboratory Equipment Ltd., Reading, UK) and frozen in super-cooled ethanol at −80°C. Cryostat sections (10 μm) were prepared (Bright OT5000 cryostat), mounted on poly-lysine-coated glass slides (VWR) and visualized using a Nikon TiE microscope (Nikon UK Ltd., Kingston upon Thames, UK).
9
Murine MSC Chondrogenic Differentiation
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Murine MSCs were stimulated for 3 weeks in StemPro Osteocyte/Chondrocyte differentiation basal medium supplemented with StemPro Chondrogenesis Supplement (Life Technologies). After fixation of the cells with 4% paraformaldehyde, proteoglycan aggrecan in the cartilage extracellular matrix was stained by an Alcian Blue 8GX dye (Sigma Aldrich) according to PromoCell's application note for “chondrogenic differentiation and analysis of MSC” (PromoCell).
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