Rw4 murine (male,129X1/SvJ) embryonic stem cells (mESCs) were cultured feeder-free in 0.1% gelatin-coated dishes. Serum condition: Knockout DMEM (Life Technologies), 2 mM Glutamax (GIBCO), 0.1 mM non-essential amino acids (GIBCO), 15% ESC-grade fetal bovine serum (FBS) (GIBCO), 0.1 mM β-mercaptoethanol, and leukemia inhibitory factor (LIF) (Millipore), 2i/Serum condition: above medium supplemented with 2i; 1 μM MEK inhibitor PD0325901 (Sigma) and 3 μM GSK3 inhibitor CHIR99021 (Sigma). 2i condition: serum free ESGRO Complete Basal medium (Millipore) with 0.1mM LIF and 2i as described above. For drug treatment, mESCs were grown in respective medium supplemented with 10 μM GSK-J4 (Sigma) for 96 h, 50 μM Zebularin for 96 h or 10 μM EPZ-6438 (BioVision) for 7 days. For all conditions cells were passaged in 48 h intervals, using accutase (Sigma) for detachment. Cell line was tested for mycoplasma contamination.
Gsk3 inhibitor chir99021
Manufactured by Merck Group
Sourced in United Kingdom, United States, China
CHIR99021 is a selective glycogen synthase kinase 3 (GSK3) inhibitor. It inhibits both isoforms of GSK3, GSK3α and GSK3β, with high potency. CHIR99021 is commonly used in research applications as a tool compound to study the effects of GSK3 inhibition.
Automatically generated - may contain errors
Lab products found in correlation
Mek inhibitor pd0325901, by Merck Group (2 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Leukemia inhibitory factor, by Merck Group (1 mentions)
Gsk j4, by Merck Group (1 mentions)
Esgro complete basal medium, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Knockout dmem, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Accutase, by Merck Group (1 mentions)
Sodium lactate, by Merck Group (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (1 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (1 mentions)
Cal 101, by Selleck Chemicals (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
Rapamycin, by Cell Signaling Technology (1 mentions)
Platelet derived growth factor, by R&D Systems (1 mentions)
Maraviroc, by Apexbio (1 mentions)
Fms related tyrosine kinase 3 ligand, by R&D Systems (1 mentions)
Azd8055, by Selleck Chemicals (1 mentions)
5 protocols using gsk3 inhibitor chir99021
1
Epigenetic Regulation in Mouse Embryonic Stem Cells
2
Chemically-Defined Cardiomyocyte Differentiation
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Blood cord-derived human induced pluripotent stem cells (hiPSCs) were obtained from Life Technologies (Episomal human iPSC line, A18944, Warrington, UK). hiPSC colonies were expanded on Geltrex (Gibco, Warrington, UK) coated plates in Essential 8 cell culture medium (Thermo Scientific, Warrington, UK) until at least passage 20 before differentiation.
Chemically-defined differentiation to cardiomyocytes was achieved following a previously described protocol [24 (link)]. Briefly, when ~90% confluent hiPSCs were stimulated with differentiation medium containing RPMI 1640 + HEPES + GlutaMax + B27 supplement—insulin (Invitrogen, Warrington, UK), along with 6 µM GSK3 inhibitor Chir99021 (Sigma, Gillingham, UK), for 48 h. The media were then replaced with a differentiation medium containing 2 µM Wnt inhibitor C59 (Selleckchem, Houston, TX, USA) for a further 48 h. Cells were maintained in differentiation medium from days 4 to 8, before changing to a maintenance medium (RPMI/HEPES/GlutaMax/B27 + insulin supplement) once spontaneously-beating colonies were established for prolonged culture. Cardiomyocytes were selected through the addition of 5 mM sodium lactate (Sigma, Gillingham, UK) to the media from day 15, before plating for individual assays.
Chemically-defined differentiation to cardiomyocytes was achieved following a previously described protocol [24 (link)]. Briefly, when ~90% confluent hiPSCs were stimulated with differentiation medium containing RPMI 1640 + HEPES + GlutaMax + B27 supplement—insulin (Invitrogen, Warrington, UK), along with 6 µM GSK3 inhibitor Chir99021 (Sigma, Gillingham, UK), for 48 h. The media were then replaced with a differentiation medium containing 2 µM Wnt inhibitor C59 (Selleckchem, Houston, TX, USA) for a further 48 h. Cells were maintained in differentiation medium from days 4 to 8, before changing to a maintenance medium (RPMI/HEPES/GlutaMax/B27 + insulin supplement) once spontaneously-beating colonies were established for prolonged culture. Cardiomyocytes were selected through the addition of 5 mM sodium lactate (Sigma, Gillingham, UK) to the media from day 15, before plating for individual assays.
3
Cytokine-Driven Cellular Signaling Assay
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Recombinant human proteins were obtained from the following the manufacturers: basic fibroblast growth factor, platelet-derived growth factor, Fms-related tyrosine kinase 3 ligand and vascular endothelial growth factor (all R&D Systems), Epidermal growth factor (Sigma), stem cell factor (Peprotech, Rocky Hill, NJ, USA), CCL3 (R&D Systems), CCL5 (R&D Systems) and CCL23 (BioLegend, San Diego, CA, USA).
The following inhibitors were used: mTOR inhibitors AZD8055 (Selleckchem, Houston, TX, USA) and rapamycin (Cell Signaling, Boston, MA, USA), PI3K inhibitor CAL101 (Selleckchem), caspase inhibitor Q-VD-OPh (Apexbio, Houston, TX, USA), GSK3 inhibitor CHIR99021 (Sigma), CCR1 inhibitor BX471 (Sigma), CCR2 inhibitor INCB3284 (Tocris Bioscience, Bristol, UK) and CCR5 inhibitor Maraviroc (Apexbio).
The following inhibitors were used: mTOR inhibitors AZD8055 (Selleckchem, Houston, TX, USA) and rapamycin (Cell Signaling, Boston, MA, USA), PI3K inhibitor CAL101 (Selleckchem), caspase inhibitor Q-VD-OPh (Apexbio, Houston, TX, USA), GSK3 inhibitor CHIR99021 (Sigma), CCR1 inhibitor BX471 (Sigma), CCR2 inhibitor INCB3284 (Tocris Bioscience, Bristol, UK) and CCR5 inhibitor Maraviroc (Apexbio).
4
Retinoic Acid-Induced Differentiation Assay
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Retinoic acid (RA) differentiation experiments were performed as previously described in Semrau et al., 2017. Explicitly, prior to differentiation cells were grown for at least 2 passages in 2i medium plus LIF (2i/L): DMEM/F12 (Gibco) supplemented with 0.5x N2 supplement, 0.5x B27 supplement, 0.5 mM L-glutamine, 20 µg/ml human insulin (Sigma-Aldrich), 1 × 100 U/ml penicillin/streptomycin, 0.5x MEM Non- Essential Amino Acids, 0.1 mM 2-Mercaptoethanol, 1 µM MEK inhibitor (PD0325901, Sigma-Aldrich), 3 µM GSK3 inhibitor (CHIR99021, Sigma-Aldrich), 1000 U/ml mouse LIF (Peprotech). Cells were seeded at a density of 2.5 × 105 per 10 cm dish and grown over night (12 h). The next day cells were washed twice with PBS and medium was replaced with basal N2B27 medium (2i/L medium without inhibitors, LIF and the additional insulin) supplemented with 0.25 μM all-trans retinoic acid (Sigma-Aldrich). Medium was being refreshed every 48H.
5
Directed Differentiation of hiPSCs
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The information of puchased reagents and cell line used in the study: LHpb-YaabC3 hiPSC (HNF-P30-P11, OSINGLAY BIO, China), BJ human foreskin broblast cell line (CRL2522, ATCC, USA), HN4 human embryonic stem cell (hESC) line (HES-P20-P9, OSINGLAY BIO, China), Cell culture media RPMI/1640 (11875093, Thermo Fisher Scienti c, USA), DMEM/F12 media (11320082, Thermo Fisher Scienti c, USA) , medium for hiPSC BioCISO (BC-PM0001, OSINGLAY BIO, China), GSK3 inhibitor CHIR99021 (S1263, Sigma, USA), FGF inhibitor PD (3044, Tocris Bioscience, UK), Wnt inhibitor IWP2 (3533, Tocris Bioscience, UK), ROCK inhibitor Y27632 (1254, Tocris Bioscience, UK) , BMP activator BMP4 (120-05ET, PEPROTECH, USA), RA inhibitor BMS(SML1149, Sigma, USA), B-27 supplement with (17504-044, Thermo Fisher Scienti c, USA) or without insulin, Matrigel (354277, Corning, UK), TRIzol Reagent (15596026, Thermo Fisher Scienti c, USA), Real-time PCR reagents (208056, Qiagen, Germany). All primers/oligos were synthesized by Shenggong Biotech, China and listed in supplemental Table 1. All other reagents, unless speci ed otherwise, were products of Sigma.
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