iPSCs were induced to differentiate into CMs using previously described methods (50 (link)). iPSCs were treated with CHIR 99021 (Selleckchem) in RPMI 1640 (Thermo Fisher Scientific) supplemented with CDM3 for 2 days (days 1 and 2). The medium was changed to RPMI supplemented with WNT-C59 (Selleckchem) for 2 days (days 3 and 4). iPSC-CMs were maintained in RPMI 1640 supplemented with B27-supplement (Thermo Fisher Scientific). iPSC-CMs were purified by metabolic selection in RPMI 1640 without glucose (Thermo Fisher Scientific), supplemented with 5 mM sodium dl -lactate and CDM3 supplement for 6 days (days 10 to 16). After metabolic selection, iPSC-CMs were replated into six-well plates using TrypLE Express (Thermo Fisher Scientific). The iPSC-CMs were >95% pure as determined by cTnT staining (Proteintech, 15513–1-AP, 1:250). CMs were used for experiments on days 35 to 40 after differentiation. Each experiment was performed in replicate using independent differentiated iPSC-CMs. Regarding the ABE- and PE-corrected iPSC-CMs, single clones of iPSCs were isolated and differentiated into iPSC-CMs for assays.
Wnt c59
Manufactured by Selleck Chemicals
Sourced in United States
Wnt-C59 is a small molecule compound that inhibits the Wnt signaling pathway. It functions by blocking the interaction between the Wnt ligand and its receptor, thereby suppressing the downstream signaling cascades.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (11 mentions)
Chir99021, by Selleck Chemicals (10 mentions)
Rpmi 1640, by Thermo Fisher Scientific (9 mentions)
Chir99021, by Bio-Techne (4 mentions)
Tryple express, by Thermo Fisher Scientific (4 mentions)
L ascorbic acid 2 phosphate, by Merck Group (3 mentions)
B27 without insulin, by Thermo Fisher Scientific (3 mentions)
Chir99021, by LC Laboratories (3 mentions)
Chir99021, by Merck Group (2 mentions)
O sativa derived recombinant human albumin, by Merck Group (2 mentions)
Wnt3a, by R&D Systems (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Chir 98014, by Selleck Chemicals (2 mentions)
Recombinant human albumin, by ScienCell (2 mentions)
Rpmi 1640 basal medium, by Thermo Fisher Scientific (2 mentions)
Chir99021, by STEMCELL (2 mentions)
Oryza sativa derived recombinant human albumin, by Merck Group (2 mentions)
Matrigel, by Corning (2 mentions)
Rpmi 1640 without glucose, by Thermo Fisher Scientific (1 mentions)
Gemcitabine, by AbMole (1 mentions)
37 protocols using wnt c59
1
Cardiac Differentiation of Induced Pluripotent Stem Cells
2
Cardiac Organoid Generation from hiPSCs
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hiPSC-based cardiac organoids were generated by modifying the protocol previously published (Lewis-Israeli et al., 2021 (link)). Briefly, hiPSC aggregates were formed by seeding hiPSCs at 10,000 cells/well on ultra-low attachment V-shaped 96-well plate (Sbio, MS-9096-VZ), previously rinsed with anti-adherence rinse solution (Stem Cell Technology, 07010). The plate was then centrifuged at 100 g for 3 min and placed in the CO2 incubator (day −2). After 24 h, 80 µL of media was changed to fresh Essential 8 medium. On day 0, media was changed to RPMI 1640 (Welgene, LM 011-01) based differentiation media supplemented with B27 minus insulin (Gibco, A1517001) containing 5 µM CHIR99021 (TOCRIS, 4,423) and incubated for 48 h. After 48 h (day 2), media was changed to RPMI-1640 + B27 minus insulin differentiation media containing 2 µM Wnt-C59 (Selleckchem, S7037) and incubated for 48 h. On day 4, the media was changed to fresh RPMI-1640 + B27 minus insulin differentiation media. From day 6 to day 30, the media was replenished with fresh RPMI-1640 + B27 with insulin (Gibco, 17504-44) differentiation media every other day. Spontaneously beating organoids start arising from day 9. Contractility measurement was performed at day 30. The organoids were generated from the C2N3 iPSC line (Figures 2 , 3 ) and the SCVI-111 (Figure 5 ).
3
Exploring TNBC Chemoresistance Mechanisms
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TNBC cell lines (TNBCC), MDA-MB-231 and MDA-MB-468 were purchased (ATCC, CA, U.S.A.). The Gemcitabine-resistance cell lines (GRC), MDA-MB-231-R and MDA-MB-468-R were produced by treating TNBCC with increasing Gemcitabine concentrations (0.1–15 nm) for 12 months (GI50 < 7.0) [18 (link)]. Gemcitabine was bought from AbMole (Beijing, China). Cells were cultured in DMEM (PM150210) containing 4500 mg/l glucose, 10% fine FBS (164220-100) and 100 U/ml penicillin–streptomycin (PB180122) in 5% CO2, 95% O2 condition at 37°C (Procell, Wuhan, China).
Each cell line was also incubated with NLRP3 agonist Nigericin sodium salt (NSS) and antagonist CY-09 separately (Selleck, Shanghai, China) to differentiate NLRP3 expression in both TNBCC and GRC. Wnt inhibitor Wnt-C59 was added into subgroups of GRC so as to inactivate signaling pathway (Selleck, Shanghai, China).
All the cell lines were exposed to the 0, 1, 3, 5 nM Gemcitabine, respectively, for 72 h for the following assays.
Each cell line was also incubated with NLRP3 agonist Nigericin sodium salt (NSS) and antagonist CY-09 separately (Selleck, Shanghai, China) to differentiate NLRP3 expression in both TNBCC and GRC. Wnt inhibitor Wnt-C59 was added into subgroups of GRC so as to inactivate signaling pathway (Selleck, Shanghai, China).
All the cell lines were exposed to the 0, 1, 3, 5 nM Gemcitabine, respectively, for 72 h for the following assays.
4
Cardiomyocyte Differentiation from hiPSCs
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hiPSCs were seeded at 65k/cm2 in mTeSR+ with 10 μM ROCK inhibitor on plates coated with 80 μg/mL Matrigel (Day −2). After 48 h (Day 0), media was replaced with RBA media [RPMI with L-glutamine (Invitrogen, 11875-119), 500 μg/mL bovine serum albumin (BSA; Sigma, A9418-50G), 213 μg/mL ascorbic acid (Sigma, A8960-5G)] supplemented with 5 μM Chiron 99021 (Cayman Chemical, 13122). After 48 h (Day 2), media was replaced with RBA media supplemented with 2 μM Wnt C59 (SelleckChem, S7037). After 48 h (Day 4), media was replaced with unsupplemented RBA media. After 48 h (Day 6), media was replaced with RPMI-based cardiomyocyte media [RPMI with L-glutamine, B27 supplement with insulin (Invitrogen, 175044, Lot 2181371), 50 U/mL penicillin/streptomycin] and replaced every other day until Day 20. hiPSC-CMs were dissociated with 0.5% trypsin (Invitrogen, 15090046) in 500 μM EDTA with 25 μU DNAse I (Sigma, 260913-25MU) and replated at 65k/cm2 in RPMI-based cardiomyocyte media with 5% FBS on 20 μg/mL Matrigel-coated plates. hiPSC-CMs were metabolically enriched for 5 d with daily feeding with DMEM without glucose or L-glutamine (Invitrogen, F530S) supplemented with 4 mM Sodium L-lactate (Sigma, 71718-10G). hiPSC-CMs were frozen at Day 25 in Cryostor at −80C or immediately used for EHTs.
5
Culturing and Treating GC Cell Lines
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GC cell lines were purchased from the American Type Culture Collection (ATCC) and Korean Cell Line Bank (KCLB). Mycoplasma test was done using MycoAlert™ Mycoplasma Detection Kit (Lonza; LT07-118). Cells were grown in Dulbecco’s modified essential medium or RPMI1640 supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator with 5% CO2. Reagents were sourced commercially as follows: DY131 (#2266; TOCRIS, Bristol, UK), GSK5182 (#AOB1629; Aobious, Gloucester. MA), ICG-001 (#S2662), XAV-939 (#S1180), and Wnt-C59 (#S7037; Selleckchem, Houston, TX), and CHX (#01810; Sigma-Aldrich, St Louis, MO).
6
Efficient Cardiomyocyte Differentiation and Purification
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ESCs were split and cultured as described above. When cells reached ~ 90% confluence, cardiomyocyte differentiation was initiated by changing the culture medium to differentiation medium CDM3 [20 (link)]. During cardiomyocyte differentiation, cells were treated with 5 μM of the glycogen synthase kinase 3-β inhibitor CHIR99021 (Sigma, USA) on days 0–2 and 2 μM of the Wnt pathway inhibitor Wnt-C59 (Selleck Chemicals, USA) on days 4–6. The medium was changed daily, and spontaneous beating was noted from day 9. For cardiomyocyte purification, the cells were replated and cultured in CDM3L medium, which consisted of glucose-free RPMI 1640 (Thermo Fisher, USA) and 5 mM sodium dl -lactate (Sigma, USA). Cardiomyocytes were then split at 1:4 with 0.25% trypsin (Sigma, USA) containing 0.1 mM EDTA and seeded on 0.1% gelatin-coated dishes in CDM3.
7
Directed Cardiomyocyte Differentiation from hiPSCs
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Cardiomyocyte differentiation was performed in CardioEasy medium (Cellapy China) by temporal modulation of the canonical WNT signaling pathway with GSK3 inhibitor and WNT inhibitor, as described in previous protocol [2 (link)]. CardioEasy medium, consisting of RPMI 1640 medium, 500 µg/ml O. sativa-derived recombinant human albumin (Sigma-Aldrich), and 213 µg/ml l-ascorbic acid 2-phosphate (Sigma-Aldrich), is abbreviated as CDM3 medium. Briefly, at day 0, 80–90% confluent hiPSCs were cultured in CDM3 medium with 6 µM CHIR99021 (SML1046, Sigma, USA) for 48 h. At day 2, medium was changed to CDM3 medium supplemented with 2 µM WNT-C59 (S7037, Selleck Chemicals) and continued to incubate for 48 h. Medium was refreshed on day 4 and every other day for CDM3 medium. The contracting cardiomyocytes can be noted from day 7.
8
Cardiomyocyte Differentiation from Human iPSCs
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Human iPSCs at 60 to 80% confluency were differentiated into cardiomyocytes as previously described (61 (link)). Briefly, cells were cultured in CDM3 media supplemented with 4 to 6 μM CHIR99021 (Selleckchem) for 48 hours (days 1 to 2) and then CDM3 media supplemented with 2 μM WNT-C59 (Selleckchem) for 48 hours (days 3 to 4). Starting on day 5, cells were cultured in basal media [RPMI 1640 (Gibco) supplemented with B-27 supplement (Thermo Fisher Scientific)] for 6 days (days 5 to 10). On day 10, cells were cultured in selective media [RPMI 1640, without glucose (Gibco), supplemented with B-27 supplement] for 10 days (days 11 to 20) and then basal media thereafter. Cardiomyocytes were used for experiments on day 30 and harvested using TrypLE Express (Thermo Fisher Scientific).
9
Directed Differentiation of hiPSC Cardiomyocytes
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Previously established healthy control (25 (link), 26 (link)), LQTS (25 (link)), and SQTS (30 (link)) hiPSC lines were used. Undifferentiated hiPSCs colonies were propagated using mTeSR-1 medium. For CM differentiation, a modification of a monolayer-directed differentiation system was used (27 (link), 28 (link)). In brief, the differentiation RPMI/B27 medium containing RPMI-1640, 2%-B27 supplement minus insulin (Invitrogen), 1% penicillin/streptomycin, and 6 mM CHIR99021 (Stemgent) was used for 2 days. Medium was changed to RPMI/B27 (without CHIR) supplemented with 2 μM Wnt-C59 (Selleckchem) on days 3–4. Beating monolayers (20–60 days) were enzymatically dissociated into small clusters or single CMs using TrypLE (Thermo Fisher Scientific) and plated on matrigel-coated MatTek plates for the different studies.
10
Spinal Cord Injury and Wnt Signaling
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Experiments were performed on late larval sea lampreys (Petromyzon marinus, 10–13 cm; ~5–7 years old), in accordance with institutional IACUC regulations and included in experimental protocols approved by The Marine Biological Laboratory and The Feinstein Institute for Medical Research. Spinal cord transections, behavioral recovery scoring, and tissue histochemistry were performed as described previously18 (link),27 (link). To block Wnt signaling, Wnt-C59, (Selleckchem; Houston, TX) was added (10μM in 0.1% DMSO/PBS) at the time and site of spinal injury via a small piece of Gelfoam (Pfizer, NY, NY). Animals undergoing spinal cord injury and treated with vehicle (0.1% DMSO/PBS) were used as controls. These manipulations slowed the trajectory of functional recovery (Fig. 7b -vehicle), compared to recovery without any manipulations (Fig. 1c ), which is due to insertion of the Gelfoam.
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