HiPSCs were split at 1:8 to 1:12 ratios using EDTA, and grown for 3‐4 days until they reached ~85% confluence. The media were changed to CDM3,19 which consisted of RPMI 1640 (Corning), 500 µg/mL Oryza sativa‐derived recombinant human albumin (Oryzogen Sciencell), and 213 µg/mL L‐ascorbic acid 2‐phosphate (Sigma‐Aldrich). The media were changed every 48 hours. On day 0, the media were changed to CDM3 supplemented with 6 µmol/L CHIR99021 (MedChem Express).20 , 21 On day 2, the media were changed to CDM3 supplemented with 2 µmol/L Wnt–C59 (Biorbyt). The media were changed on day 4 and every other day for CDM3. Contracting cells were observed from day 8. On day 10, the media were changed to a purification medium made using RPMI 1640 (no glucose) (Corning), 500 µg/mL recombinant human albumin, and 213 µg/mL L‐ascorbic acid 2‐phosphate. The medium was replaced with RPMI 1640 (Corning) supplemented with 500 µg/mL recombinant human albumin 48 hours before the experiment in order to avoid antioxidant effects.
Chir99021
Manufactured by MedChemExpress
Sourced in United States, China
CHIR99021 is a selective and potent inhibitor of glycogen synthase kinase-3 (GSK-3).
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Y 27632, by MedChemExpress (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
A83 01, by MedChemExpress (3 mentions)
Xav 939, by MedChemExpress (3 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (3 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (3 mentions)
Wnt c59, by Biorbyt (2 mentions)
L ascorbic acid 2 phosphate, by Merck Group (2 mentions)
Rpmi 1640, by Corning (2 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (2 mentions)
Repsox, by MedChemExpress (2 mentions)
Activin a, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Tgf β1, by Thermo Fisher Scientific (2 mentions)
Forskolin, by MedChemExpress (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Pd0325901, by MedChemExpress (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
31 protocols using chir99021
1
Cardiac Differentiation of hiPSCs
2
Directed Differentiation of hESCs into Germ Cells
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hESC were plated on collagen I (Corning)–coated 6-well plates (Corning), 24-well plates (Corning), or glass coverslips (15 mm). Cells were cultured in EpiLife medium until 70 to 80% confluence. The culture medium was replaced by the chemical induction medium consisting of DMEM/F12 (Invitrogen, #10565018), 0.5% N2 (Gibco, #17502048), 0.5% B27 (Gibco, #17504044), and 1% P/S, supplemented with 5 μM Repsox (MedChem Express, #HY-13012), 3 μM CHIR99021 (MedChem Express, #HY-10182), bFGF (10 ng/ml; PeproTech, #100-18B-50), and BMP4 (10 ng/ml; PeproTech, #120-05ET-10). The control group was treated with the basal medium containing 1% dimethyl sulfoxide in the absence of small molecules and growth factors. The chemical induction medium was refreshed every 2 days. After 6 to 8 days of induction, hESC were induced into GCs, and the induced GCs were maintained and passaged in the chemical induction medium for the following use. Other small molecules used in the study included 3 μM Kenpaullone (MedChem Express, #HY-12302), 5 μM A83-01 (MedChem Express, #HY-10432), and 5 μM SB431542 (Selleck, #S1067), and 5 μM DAPT (MedChem Express, HY-13027 ).
3
Generation of RPE cells from iPSCs
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RPE cells were generated from iPSCs following the previously reported method [26 (link)]. Briefly, iPSCs were grown to 80% confluence in mTeSRTMPlus medium and switched to RPE differentiation medium containing Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA), 20% knockout serum replacement (KSR; Thermo Fisher Scientific, Waltham, MA, USA), 1× non-essential amino acids (NEAA; Sigma, San Luis, MO, USA), 50 μM β-mercaptoethanol (Gibco), 1% penicillin–streptomycin (Gibco) and 10 mM nicotinamide (MedChemExpress, Princeton, NJ, USA) for 0–7 days. Then, nicotinamide was replaced with 100 ng/mL Activin A (Peprotech, Rocky Hill, NJ, USA) for another week. On day 14 of differentiation, Activin A was removed from the medium, and 3 μM CHIR99021 (MedChemExpress) was added. On day 42, RPE cells were collected by incubating in TrypLE Express (Gibco) for 30 min at 37 °C and then seeded on a Matrigel-coated 6-well plate with a density of approximately 1 × 106 cells/well. Meanwhile, RPE differentiation medium was replaced with RPE maintenance medium consisting of DMEM, 4% KSR, 1× NEAA, 1% penicillin–streptomycin and 50 μM β-mercaptoethanol without molecule compound. At this point, the cells were defined as passage 1 (P1). Only RPE cells at P2 and P3 were used in this study.
4
Reactivating Wnt/β-catenin Signaling
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CHIR-99021 was purchased from MedChemExpress (United States). The transfected MHCC97H and HepG2 cells were treated with 2 μmol/ml CHIR99021 to reactivate Wnt/β-catenin signal as per the supplier’s specification.
5
Mouse Embryonic Stem Cell Differentiation
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Mouse ESCs (V6.5; Novus Biologicals) were maintained on a 0.1% gelatin (MilliporeSigma)-coated dish, under 2i + LIF conditions: N2B27 medium supplemented with 1 × GlutaMAX (Gibco), 1000 U/ml mouse LIF (MilliporeSigma), 3 μM CHIR99021 (MedChem Express) and 1 μM PD0325901 (Selleckchem). Cells were passaged every 3 days.
Mouse ESCs were induced to differentiate into EpiLCs and PGCLCs, as previously described (7 (link)). Briefly, three hundred thousand cells were plated on six-well culture plates coated with 16.7 mg/ml fibronectin (MilliporeSigma) and grown in N2B27 medium supplemented with 1% knockout serum replacement (KSR; Gibco), bFGF (12 ng/ml; Life Technologies), and activin A (20 ng/ml; R&D Systems) for 2 days. The medium was changed every day. The PGCLCs were induced under a floating condition by plating two thousand day 2 EpiLCs in a single well from a 96-well Lipidure-Coat Plate (Amsbio) and grown in serum-free GK15 medium (GMEM [Gibco], KSR [15%; Gibco], nonessential amino acids [Gibco], sodium pyruvate [1 mM; Gibco], 2-mercaptoethanol [0.1 mM; Gibco], L-glutamine [2 mM; Gibco], BMP4 [500 ng/ml; Miltenyi Biotec], SCF [100 ng/ml; Miltenyi Biotec], BMP8b [500 ng/ml; R&D Systems], EGF [50 ng/ml; Pepprotech]) and LIF (MilliporeSigma) for 4 days.
Mouse ESCs were induced to differentiate into EpiLCs and PGCLCs, as previously described (7 (link)). Briefly, three hundred thousand cells were plated on six-well culture plates coated with 16.7 mg/ml fibronectin (MilliporeSigma) and grown in N2B27 medium supplemented with 1% knockout serum replacement (KSR; Gibco), bFGF (12 ng/ml; Life Technologies), and activin A (20 ng/ml; R&D Systems) for 2 days. The medium was changed every day. The PGCLCs were induced under a floating condition by plating two thousand day 2 EpiLCs in a single well from a 96-well Lipidure-Coat Plate (Amsbio) and grown in serum-free GK15 medium (GMEM [Gibco], KSR [15%; Gibco], nonessential amino acids [Gibco], sodium pyruvate [1 mM; Gibco], 2-mercaptoethanol [0.1 mM; Gibco], L-glutamine [2 mM; Gibco], BMP4 [500 ng/ml; Miltenyi Biotec], SCF [100 ng/ml; Miltenyi Biotec], BMP8b [500 ng/ml; R&D Systems], EGF [50 ng/ml; Pepprotech]) and LIF (MilliporeSigma) for 4 days.
6
Modulation of NDRG1 and GSK3B Signaling
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NDRG1 or GSK3B genes were inhibited by transient transfection with siRNA (siNDRG1 and siGSK3β) from Santa Cruz (50 ng/ml) and lipofectamine RNAiMAX (Invitrogen) following the manufacturer's instructions for 48h after stimulation with TGFβ1 (10 ng/ml) (PeproTech) for 8h or 14 days. TGFβ1 was replenished every 72h. Scrambled siRNA (SCR) was used as negative control 20 (link). Galunisertib (LY2157299, TGFβR1 inhibitor) (5 μM), CHIR99021 (GSK3α/β inhibitor) (10 μM), LY294002 (PI3K inhibitor) (10 μM), MK2206 (Akt inhibitor) (1 μM), GSK650394 (SGK1/2 inhibitor) (10 μM), and Rapamycin (mTOR inhibitor) (10 μM) were from MedChemExpress. Cells were pre-stimulated with TGFβ1 for 8h and treated with each inhibitor for 24, 48, and 72h.
7
Screening Stem Cell Signaling Modulators
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A screening of small molecules from the MedChemExpress stem cell signaling library (catalogue No. HY-LO17) was performed using a basic cocktail containing forskolin (5 μM), (MedChemExpress, Monmouth Junction, NJ, USA), CHIR99021 (2 μM), (MedChemExpress, Monmouth Junction, NJ, USA), RepSox (2 μM) (MedChemExpress Monmouth Junction, NJ, USA), and LDN193189 (0.5 μM), (MedChemExpress, Monmouth Junction, NJ, USA) in Dulbecco’s modified Eagle medium: F12 (DMEM: F12) (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing N2 (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) as the only supplement. Each of the small molecules from the library was added to the basic cocktail at a final concentration of 5 μM and investigated for its ability to generate a neuronal-like morphology and Tuj1+ cells.
8
Directed Cardiac Differentiation of hiPSCs
9
CHIR-99021 Yolk Sac Injection Protocol
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CHIR-99021 (CHIR-99021 monohydrochloride, MedChemExpress, Shanghai, China) was dissolved in 0.9% NS. Beforehand, we injected ink to train the accuracy of the yolk sac injection and to determine a needle insertion distance. In the CHIR-99021 injected experiment, first, the center of large end of each egg was located under the egg candler. An alcohol swab was used to disinfect the target injection site, then a small hole was drilled at the injection site (Figure S1). The volume of 100 μL of the CHIR-99021 solution was rapidly injected into the egg yolk sac using a 0.8 mm × 38 mm needle. After paraffin sealing, the eggs were returned to the incubator. The injection operation process of each egg was controlled in about 15 min outside the incubator environment for standardization.
10
Astrocyte Reprogramming via Small Molecules
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Astrocytes were grown to confluence before reprogramming. Cells were cultured on poly-L-lysine (PLO)/laminin substrates and media was changed to high glucose Dulbecco's Modified Eagle Medium (DMEM) with 2 mM Glutamax (Gibco), 1% N2/B27 (ThermoFisher Scientific), 1% Human Serum Replacement 3 (Sigma Aldrich), and 1% penicillin/streptomycin (ThermoFisher Scientific) for cellular reprogramming experiments. 15 μg/mL ASCL-IPTD (iProgen Biotech) was supplemented for all 12 days, with 50% media changes every 2 days. For days 1-2, 5μM SB431542 (Stemcell Technologies) and 0.25 μM LDN193189 (Stemcell Technologies) were added to cultures. For days 3–12 the following small molecules were added: forskolin (F, 10 μM, Medchem Express), CHIR99021 (C, 1.5 μM, Medchem Express), ISX9 (I, 20 μM, Medchem Express), DAPT (D, 5 μM, Medchem Express). The combinations tested were C, F, D, I, CF, CI, CD, FI, FD, ID, FCI, FCD, CID, FID, CFID. For days 3–6, 0.5 μM valproic acid (Stemcell Technologies) was also added to improve chromatin accessibility.
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