hiPSCs (>p20) were split at 1:12 to 1:15 ratios using EDTA as above and grown for 3–4 days at which time they reached ~75% confluence. Media was changed to CDM36 (link), consisting of RPMI 1640 (10–040–CM, Corning), 500 µg ml−1Oryza sativa–derived recombinant human albumin (Oryzogen Sciencell), and 213 µg ml−1L–ascorbic acid 2–phosphate (Sigma–Aldrich). Media was changed every other day (48 h). For days 0–2, media was supplemented with 6 µM CHIR99021 (MedChem Express)19 , 20 . On day 2, media was changed to CDM3 supplemented with 2 µM Wnt–C59 (Biorbyt). Media was changed on day 4 and every other day for CDM3. Contracting cells were noted from day 7. At day 10, media was changed to CDM3L made with using RPMI 1640 no glucose (11879–020, Life Technologies), 500 µg ml−1 recombinant human albumin, and 213 µg ml−1L–ascorbic acid 2–phosphate supplemented with 4 mM L–lactic acid (Sigma–Aldrich). At day 15, media was changed to CDM3M consisting of RPMI 1640 no glucose, 500 µg ml−1 recombinant human albumin, 213 µg ml−1L–ascorbic acid 2–phosphate supplemented with 10 mM D–galactose (Sigma–Aldrich)21 (link), 4 mM L–lactic acid, 1 mM sodium pyruvate (Life Technologies), 20 µg ml−1 insulin (Life technologies), 1 × chemically defined lipid concentrate (Life Technologies), and 200 ng ml−1 tri–iodo–L–thyronine (Sigma–Aldrich)22 (link).
L ascorbic acid 2 phosphate
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Switzerland, Sao Tome and Principe, Canada, Denmark, Israel, Ireland, Japan, Senegal, Italy
L-ascorbic acid 2-phosphate is a stable form of vitamin C. It serves as a precursor for the production of ascorbic acid in cells.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (183 mentions)
β glycerophosphate, by Merck Group (114 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (38 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (33 mentions)
Penicillin, by Thermo Fisher Scientific (27 mentions)
Indomethacin, by Merck Group (26 mentions)
Alizarin red s, by Merck Group (24 mentions)
Insulin, by Merck Group (24 mentions)
Streptomycin, by Thermo Fisher Scientific (22 mentions)
β glycerol phosphate, by Merck Group (20 mentions)
Penicillin streptomycin, by Merck Group (20 mentions)
L proline, by Merck Group (19 mentions)
α mem, by Thermo Fisher Scientific (17 mentions)
Oil red o, by Merck Group (14 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (14 mentions)
Proline, by Merck Group (13 mentions)
Recombinant human albumin, by Merck Group (13 mentions)
Tgf β1, by Thermo Fisher Scientific (12 mentions)
Fetal bovine serum (fbs), by Merck Group (12 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (12 mentions)
383 protocols using l ascorbic acid 2 phosphate
1
Directed Cardiac Differentiation of hiPSCs
2
Cardiomyocyte Differentiation from iPSCs
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The iPSCs (>p20) were passaged using EDTA and grown until the cells reached ∼75% confluency. The medium was changed to CDM3, which consists of RPMI 1640 (Life Technologies), Oryza sativa–derived recombinant human albumin (500 μg ml−1; Sigma-Aldrich), and l -ascorbic acid 2-phosphate (213 μg ml−1; Sigma-Aldrich). Medium was changed every other day (48 hours). At days 0 to 2, medium was supplemented with 6 μM glycogen synthase kinase 3-β inhibitor CHIR99021 (APExBio). On day 2, the medium was changed to CDM3 supplemented with 2 μM Wnt inhibitor Wnt-C59 (Calbiochem Research Biochemicals). The medium was changed on day 4 and every other day for CDM3 cultures. The contracting cells were observed from day 7. At day 10, the medium was changed to CDM3L, which consists of RPMI 1640 no glucose (Life Technologies), recombinant human albumin (500 μg ml−1), and l -ascorbic acid 2-phosphate (213 μg ml−1) supplemented with 4 mM l -lactic acid (Sigma-Aldrich). CM formation was further confirmed by staining for NKX2.5 (Cell Signaling Technology no. 8792S) and cardiac troponin (catalog no. AB8295, Abcam).
3
Hepatocyte-like Cell Differentiation from iPSCs
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Differentiation to hepatocyte-like cells from iPSC-K (HLC-KI): To generate embryoid bodies (EBs), iPSC-K (1 × 104 cells/well) were dissociated into single cells with Accutase (A1110501, Gibco; Thermo Fisher Scientific, MA, USA) after exposure to ROCK inhibitor, Y-27632 (A11105-01, Fujifilm Wako Pure Chemicals, Osaka, Japan), and cultivated in 96-well plates (174925, Thermo Fisher Scientific, MA, USA) in EB medium [75% KO-DMEM, 20% KO-SR, 2 mM GlutaMAX-I, 0.1 mM NEAA, 1% penicillin–streptomycin and 50 µg/mL L-ascorbic acid 2-phosphate (A8960, Sigma-Aldrich, MO, USA)] for 10 days. The EBs were transferred to the 24-well plates coated with collagen type I and cultivated in XF32 medium [85% Knockout DMEM, 15% Knockout Serum Replacement XF CTS (XF-KSR: 12618013, Gibco; Thermo Fisher Scientific, MA, USA), 2 mM GlutaMAX-I, 0.1 mM NEAA, 1% penicillin–streptomycin, 50 µg/mL L-ascorbic acid 2-phosphate, 10 ng/mL heregulin-1β, 200 ng/mL recombinant human IGF-1 (LONG R3-IGF-1: 85580C, Sigma-Aldrich, MO, USA) and 20 ng/mL human bFGF] for 35 days. For iPSC-O differentiation to hepatocyte-like cells (HLC-O): Hepatic differentiation of iPSC-O was performed by Cellartis Hepatocyte Differentiation Kit (Y30050, Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. In this study, cells were used after 30 days of differentiation induction.
4
Multilineage Differentiation of DFAT Cells
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For osteogenic differentiation, DFAT cells were cultured for 3 weeks in DMEM containing 10% FBS, 100 nM dexamethasone, 10 mM β-glycerophosphate (Sigma-Aldrich, MO, USA), and 50 μM L-ascorbic acid-2-phosphate (Sigma-Aldrich). Differentiated cells were stained with Alizarin red S (Sigma-Aldrich) after being fixed in 4% paraformaldehyde. For chondrogenic differentiation, DFAT cells were seeded at a density of 2 × 105 cells per pellet, in 15 cm3 conical tubes. Cells were gently centrifuged at 150 g for 5 min. DFAT cells were cultured for 3 weeks in DMEM containing 1% FBS, 50 μM L-ascorbic acid-2-phosphate, 40 μg/ml proline (Sigma-Aldrich), 100 μg/ml pyruvate (Sigma-Aldrich), 10 ng/ml transforming growth factor (TGF)-β3 (R&D Systems, MN, USA), and 1x insulin-transferrin-selenium-X (ITS) (Invitrogen). The pellet was fixed in 4% paraformaldehyde and then observed under a stereomicroscope. For adipogenic differentiation, DFAT cells were incubated for 3 weeks in 10% FBS DMEM, 1 μM dexamethasone (Sigma-Aldrich), 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), 1x ITS. Differentiated cells were stained with Oil red O (Sigma-Aldrich) after being fixed in 4% paraformaldehyde.
5
Human iPSC Differentiation Protocol
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The differentiation protocol started when human iPS cells reached 80–90% of confluency (day 0). Cells were fed with RPMI-1640-Medium-GlutaMAX Supplement-HEPES (Gibco, 72400-021) supplemented with 0.5 mg ml−1 human recombinant albumin (Sigma-Aldrich, A9731), 0.2 mg ml−1l -ascorbic acid 2-phosphate (Sigma-Aldrich, A8960) and 4 μM CHIR99021 (Sigma-Aldrich, 361559). After 48 h (day 2), medium was replaced by RPMI-1640-Medium-GlutaMAX Supplement-HEPES supplemented with 0.5 mg ml−1 human recombinant albumin (Sigma-Aldrich, A9731), 0.2 mg ml−1l -ascorbic acid 2-phosphate and 5 μM IWP2 (Sigma-Aldrich, 681671). On day 4 and day 6, cells were refreshed with RPMI-1640-Medium-GlutaMAX Supplement-HEPES supplemented with 0.5 mg ml−1 human recombinant albumin and 0.2 mg ml−1l -ascorbic acid 2-phosphate. From day 8 onwards, the medium of the cells was refreshed every 3–4 days with RPMI-1640-Medium-GlutaMAX Supplement-HEPES supplemented with B-27 Supplement (50×)-serum free (Gibco, 17504001).
6
Purification and Characterization of SANLCs
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Purification of SANLCs referenced a metabolic-selection method of cardiomyocytes as previous described [2 (link), 9 (link)]. Briefly, SANLCs induced by three small molecule chemicals were performed in RPMI 1640 medium without d-glucose (11,879,020, Life Technologies) supplemented with 213 µg/ml l-ascorbic acid 2-phosphate (66,170–10-3, Sigma-Aldrich), 500 µg/ml O. sativa-derived recombinant human albumin (Sigma-Aldrich) for 2 days and then in RPMI 1640 medium without d-glucose supplemented with 213 µg/ml l-ascorbic acid 2-phosphate, 500 µg/ml O. sativa-derived recombinant human albumin, and 5 mM sodium dl-lactate (L4263, Sigma-Aldrich) for 3–4 days. Medium was changed to CDM3 medium for maintenance of SANLCs for 2 days. The markers of pacemaker cells were evaluated by analysis of RT-PCR and flow cytometry at day 32. Electrophysiological characteristics were analyzed using action potential (AP) recording at day 32.
7
Differentiation of hiPSC-derived MSCs
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HiPSC-derived MSCs were grown on poly-D-lysine (1 mg/ml)–pretreated culture dishes coated with fibronectin (0.1%; Sigma-Aldrich). For differentiation, IPS cells were plated in mTesR medium at day 1. At day 2, the medium was switched to a mixture composed of TesR and Knock-Out DMEM (KO-DMEM) basal media (1:1). KO-DMEM was supplemented by FBS (15%; Life Technologies), GlutaMAX (1×; Life Technologies), nonessential amino acids (1×; Life Technologies), antibiotics (penicillin, streptomycin), and β-mercaptoethanol (50 μM). Medium was supplemented with thiazovivin (5 μM; StemCell), β-FGF (10 ng/ml; Peprotech), and L-ascorbic acid-2-phosphate (1 mM; Sigma-Aldrich). At day 3, cells were switched to a KO-DMEM medium supplemented with 15% FBS, thiazovivin (5 μM; StemCell), basic FGF (10 ng/ml; Peprotech), and L-ascorbic acid-2-phosphate (1 mM; Sigma-Aldrich). Cells were split at 90% confluency with accutase (StemCell).
8
Lineage-Specific Differentiation of hMSCs
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Expanded hMSCs with shControl and shCTR9#3/#5 were cultured in osteogenic, adipogenic, or chondrogenic medium to induce the lineage-specific differentiation in vitro. For osteogenesis, hMSCs were induced in osteogenic medium composed of 1× MEMα (Corning), 10% FBS, 1% antibiotics, 10 mM β-glycerophosphate, l -ascorbic acid-2 phosphate (50 mg/ml), and 0.1 mM dexamethasone (Sigma-Aldrich) for up to 28 days.
For adipogenesis, hMSCs were induced in adipogenic medium consisting of 1× MEMα, 10% FBS, 1% antibiotics, 1 mM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and insulin (1 mg/ml) (Sigma-Aldrich) for up to 21 days. For chondrogenesis, 1.5 to 3 × 105 cells were centrifuged at 600g for 5 min to form a high cell density pellet before induction with chondrogenic medium containing 1× Dulbecco’s modified Eagle’s medium ([DMEM], high glucose), 1% antibiotics, ITS Premix (Corning), 0.9 mM sodium pyruvate,l -ascorbic acid-2 phosphate (50 mg/ml), l -proline (40 mg/ml), 0.1 mM dexamethasone (Sigma-Aldrich), and TGF-β1 (10 ng/ml) (PeproTech) for 21 days during which the medium was changed every 3 days.
For adipogenesis, hMSCs were induced in adipogenic medium consisting of 1× MEMα, 10% FBS, 1% antibiotics, 1 mM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and insulin (1 mg/ml) (Sigma-Aldrich) for up to 21 days. For chondrogenesis, 1.5 to 3 × 105 cells were centrifuged at 600g for 5 min to form a high cell density pellet before induction with chondrogenic medium containing 1× Dulbecco’s modified Eagle’s medium ([DMEM], high glucose), 1% antibiotics, ITS Premix (Corning), 0.9 mM sodium pyruvate,
9
Vascular and Osteogenic Cell Culture
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Vascular Medium (VM) consisted of endothelial basal medium-2 (Lonza), 6% FBS, 1% penicillin/streptomycin, 10 ng/mL vascular endothelial growth factor-165 (VEGF; PeproTech), 1 ng/ml FGF-2, and 1 µg/mL L-ascorbic acid-2-phosphate (Sigma). For monolayer osteogenic differentiation experiments, Control Medium consisted of low glucose DMEM (GIBCO Invitrogen), 6% FBS, and 1% penicillin/streptomycin; Osteogenic Medium (OM) consisted of Control Medium plus 10 mM β-glycerophosphate (Sigma) and 50 µM L-ascorbic acid-2-phosphate. To support both vascular growth and osteogenic differentiation, OM was further supplemented with 10 /ml VEGF and 1 ng/ml FGF-2 for studies within fibrin gels and PCL scaffolds.
10
Multilineage Differentiation Protocols
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For adipogenesis, 2x10 5 cells/well in a 6-well plate were incubated with DMEM supplemented with 10% FCS, 1% antibiotics, 1µM dexamethason (Sigma, Sigma-Aldrich Chemie B.V, Zwijndrecht, The Netherlands), 0.1mM indomethacin, 0.5 mM 3isobutyl-1methylzanthine (IBMX, Sigma) and 10µM insulin (Sigma). Medium was replaced 2 times per week for 3 weeks, after which the cells were fixed with 10% formaldehyde for 20 minutes at RT and stained with Oil-red-O (Sigma). For chondrogenesis, 4x10 5 cells were pelleted in a 15 ml tube and cultured in DMEM supplemented with 1% antibiotics, ITS-premix (BD Biosciences, Becton Dickinson B.V., Breda, The Netherlands), 0,1 µM dexamethasone, 86.5µM L-ascorbic acid 2phosphate (Sigma) and 10ng/ml TGF-ß1 (Peprotech, London, UK). For microscopy, pellets were sectioned and stained for 10 minutes with 0.2% Toluidine Blue. For osteogenesis, 50x10 3 cells/well were cultured in a 6-well plate with DMEM supplemented with 10% FCS, 1% antibiotics, 0.1µM dexamethasone, 10mM ßglycerophosphate (Sigma) and 173µM L-ascorbic acid 2-phosphate. Cells were fixed for 15 minutes in 10% formaldehyde and subsequently stained with Alizarin-Red-S (20mg/ml, BDH, United Kingdom).
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