Monolayer-directed differentiation of hiPSC into CMs was performed as described previously with slight modifications [20 (link)]. Four days before initiation of cardiac differentiation, hiPSCs were split in a 1:8 ratio. On day 0 of cardiac differentiation, hiPSCs with 90% confluence were incubated with RPMI-B27 without insulin (Life Technologies, CA, USA) supplemented with 6 μM CHIR99021 (LC Laboratories, MA, USA) for 48 h. After withdrawn of CHIR99021, cells were kept in RPMI-B27 without insulin for another 48 h, and followed by IWR1 (LC Laboratories) treatment in the same basal medium for 48 h. Media were then switched to RPMI-B27 supplemented with insulin for further culturing. Beating hiPSC-CMs were expected to be observed from day 9 post initiation of cardiac differentiation. On day 11, derived hiPSC-CMs were metabolically purified by 4-day culture in RPMI-B27 without d -glucose, and then maintained in RPMI-B27 supplemented with insulin.
Chir99021
Manufactured by LC Laboratories
Sourced in United States
CHIR99021 is a small molecule that functions as a highly potent and selective inhibitor of glycogen synthase kinase 3 (GSK3).
Automatically generated - may contain errors
Lab products found in correlation
Pd0325901, by LC Laboratories (11 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (4 mentions)
Activin a, by Thermo Fisher Scientific (3 mentions)
Xav939, by Merck Group (3 mentions)
Wnt c59, by Selleck Chemicals (3 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Activin a, by R&D Systems (2 mentions)
Horse serum, by Thermo Fisher Scientific (2 mentions)
Recombinant bmp4, by Thermo Fisher Scientific (2 mentions)
Hek293t cell, by American Type Culture Collection (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
α tubulin sc 23948, by Santa Cruz Biotechnology (2 mentions)
Oil red o, by Merck Group (2 mentions)
Acc antibody 3662, by Cell Signaling Technology (2 mentions)
Polyclonal sin1 antibody a300 910a, by Fortis Life Sciences (2 mentions)
Mk 2206, by Active Motif (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
29 protocols using chir99021
1
Monolayer-directed Cardiac Differentiation of hiPSCs
2
Directed Differentiation of iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
iPSCs were dissociated using 0.5 mM EDTA and transferred to ultra-low attachment culture dishes in E8 medium (without TGF-β1 and FGF2) for embryoid body (EB) formation. After 2 days, EBs were transferred to Matrigel-coated dishes. The medium was changed to a differentiation medium: DMEM/F12 containing 20% FBS, L-glutamine, and non-essential amino acids for mesoderm and endoderm induction; or DMEM/F12 containing 50% neurobasal medium, B27, N2, and L-glutamine for ectoderm induction. After 14 days, the cells were fixed and analyzed by immunofluorescence staining with antibodies against TUJ1, SMA, and AFP. For the directed differentiation of endoderm, cells were treated with RPMI supplemented with 2% FBS, 100 ng/mL activin A (R&D systems, Minneapolis, MN, USA) and 5 µM CHIR 99021 (LC laboratories, Woburn, MA, USA) for the first day, followed by the same media without CHIR99021 for 2 days. For mesoderm, cells were treated with advanced RPMI (Thermo Scientific) supplemented with Glutamax (Thermo Scientific) and 5 µM CHIR 99021 for 2 days. For ectoderm, cells were treated with DMEM (Thermo Scientific) supplemented with 1 µM dorsomorphin (Merck), 10 µM SB431542 (Merck), and 1 µM PD0325901 (Tocris, Bristol, UK) for 8 days. Germ layer-specific primers listed in Supplementary Table S1 were used to determine the gene expressions by ΔΔCt method.
3
Expansion of Feeder-free Mouse ESCs
4
Culture of Mouse Embryonic Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mouse ESC line AB2.2 was cultured on mouse embryonic fibroblast (MEF) feeder cells with ES cell medium supplemented with 15% fetal bovine serum (Hyclone, Logan, UT, USA, cat. no. SH30396.03), 1000 U/mL leukemia inhibitory factor (LIF, Millipore, Burlington, MA, USA, cat. no. ESG1107), 3 μM CHIR99021 (LC Laboratories, Woburn, MA, USA, cat. no. C-6556), and 1 μM PD0325901 (LC Laboratories, cat. no. P-9688). Fifty millimolar lactate (Sangon Biotech, Shanghai, China, cat. no. A604046) was added and maintained for 24 h before experimental procedures. Cells were cultured in a humidified chamber at 37 °C with 5% CO2. For culture of mouse ESC lines, the medium was refreshed daily, and cells were routinely passaged every 2 days.
5
Skeletal Muscle Differentiation of ESCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ESCs were cultured as previously described (Shakya et al., 2015a) with 2i conditions: ERK inhibitor PD0325901 (1 μM, LC Laboratories) and GSK3 inhibitor CHIR99021 (3 μM, LC Laboratories). Cultures were maintained on irradiated feeders (ThermoFisher). Prior to all experiments ESCs were plated on gelatin to deplete the feeders. For MD differentiation, ESCs were plated on gelatin and cultured as previously described (Chal et al., 2015) . Briefly, parental and cKO cells were cultured in N2B27 medium supplemented with recombinant Bmp4 (Peprotech) for 2 d. After 48 hr, media was changed to RDL (Rspo3, DMSO, LDN) medium. Cells were harvested 24 hr (day 3) or 96 hr (day 6) later. For muscle differentiation, cells were switched to HIFL (Hgf, Igf, Fgf, Ldn) medium and cultured for 48 hr (day 8) after which medium was switched to 2% horse serum (ThermoFisher). Cells were harvested on day 11 (overexpression experiments) or 19 (RT-qPCR).
6
Feeder-Free Expansion of Embryonic Stem Cells
7
Maintenance of hEPSCs in N2B27-LCDM Medium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hEPSCs were maintained in an N2B27‐LCDM medium under 20% O2 and 5% CO2 at 37 °C. A total of 500 mL of N2B27‐LCDM medium was prepared, containing 241 mL of DMEM/F12 (Thermo Fisher Scientific, 11330‐032), 241 mL of Neurobasal medium (Thermo Fisher Scientific, 21103‐049), 2.5 mL of N2 supplement (Thermo Fisher Scientific, 17502‐048), 5 mL of B27 supplement (Thermo Fisher Scientific, 12587‐010), 1% nonessential amino acids (Thermo Fisher Scientific, 11140‐050), 0.1 × 10−3m β‐mercaptoethanol (Sigma, M3148), 1% GlutaMAX (Thermo Fisher Scientific, 35050–061), and 5% KnockOut serum replacement (optional) (Thermo Fisher Scientific, A3181502). We added recombinant human LIF (10 ng mL−1; Peprotech, 300–05), CHIR 99021 (1 × 10−6m ; LC Laboratories, C‐6556), IWR‐1‐endo (1 × 10−6m ; Abmole, M2782), DiM (2 × 10−6m ; Tocris, 1425) and MiH (2 × 10−6m ; Tocris, 3268), Y‐27632 (2 × 10−6m ; LC Laboratories, Y‐5301) to the N2B27 medium. hEPSCs were cultured on MEF feeder cells and passaged with 0.05% trypsin for 3 min at 37 °C.
8
Expansion of Feeder-free Mouse ESCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ES cells were cultured in ESC media (15% FBS, 25 mM HEPES, 1× GlutaMAX, 1× MEM Non-essential Amino Acids Solution, 1× Penicillin/Streptomycin, and 0.055 mM β-Mercaptoethanol in DMEM High Glucose (4.5 g/L)) containing 2i (1 μM PD0325901, LC Laboratories; and 3μM CHIR99021, LC Laboratories) and LIF (1300 U/mL, in-house) on cell culture plates coated with 0.2% gelatin under feeder-free conditions. The expanded ES colonies were dissociated using 0.25% trypsin-EDTA solution for passaging.
9
Doxycycline-inducible Dux ESC Line
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Doxycycline (Dox)-inducible Dux ESC line was generated using the PiggyBac Transposon System in our laboratory (hereafter referred to as ESCDUX), in which Dux expression was induced in the presence of Dox21 (link). The ESCDUX was cultured in ESC medium (10% FBS, 1× sodium pyruvate, 1× GlutaMAX, 1× MEM non-essential amino acids solution, 1× penicillin/streptomycin and 0.055 mM β-mercaptoethanol in DMEM high glucose (4.5 g liter−1)) containing 2i (1 µM PD0325901, LC Laboratories; and 3 µM CHIR99021, LC Laboratories) and LIF (1,000 U ml−1, in-house) on cell-culture plates coated with 0.1% gelatin under feeder-free conditions. The expanded colonies were dissociated using 0.05% trypsin-EDTA solution for passaging.
10
Cardiomyocyte Differentiation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We began the cardiomyocyte differentiation protocol when cells were 80% confluent using the protocol developed by Feaster et al [28 (link)]. Briefly, differentiation was initiated (day 0–2) by replacing TeSR E8 medium with RPMI 1640 medium (Lonza, cat 12-702F) supplemented with B27 (minus insulin, Gibco, cat A1895601) and CHIR99021 (6 μM, LC Laboratories, cat C-6556), a GSK3 inhibitor. On days 3–4, CHIR99021 was removed and replaced with RPMI 1640 medium supplemented with B27 (minus insulin) and IWR-1 (500 μM, Sigma, cat I0761), a Wnt signaling inhibitor. On days 5–9, cells were maintained in RPMI 1640 medium supplemented only with B27 (minus insulin). From days 10–15, a metabolic selection protocol was employed using RPMI 1640 without glucose (Life Technologies, cat 11879) plus B27 without insulin. Following metabolic selection, cells were maintained in RPMI 1640 supplemented with B27 (Gibco, cat 17504-044) and 1% pen strep (Gibco, cat 10378-016). Beating cardiomyocytes were fed daily until day 20 when functional assays were carried out as described below.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!