hiPSCs were maintained and differentiated to cardiomyocytes as previously described [35 (link), 36 (link)]. Briefly, hiPSCs (P25-P50) derived from normal human epidermal keratinocytes at Duke University were cultured in mTESR1 plus media (Stemcell Technologies) and passaged every 4–5 days with 0.5 mM EDTA in PBS. To initiate cardiomyocyte differentiation, hiPSCs were detached with accutase (Stemcell Technologies) treatment and seeded in 10-cm dishes at a density of 6.4×105/cm2. Cells in monolayer were allowed to grow for 3 days and media switched to RPMI-1640 media supplemented with B27 minus insulin (RB−, Stemcell Technologies). Cells were treated with CHIR 99021 (12 μM, Tocris Bioscience), recombinant activin A (60 ng/ml, R&D Systems) and ascorbic acid (50 μg/ml, Sigma-Aldrich) for 24 hours, followed by endo-IWR 1 (5 μM, Tocris Bioscience) for 5 days, of which ascorbic acid (50 μg/ml) was included during the first 3 days. Thereafter, cells were incubated in RPMI-1640 and insulin-supplemented B27 (RB+, Stemcell Technologies). Spontaneous contractions of hiPSC-CMs usually appeared on the 5th day after initiation of differentiation. hiPSC-CM purity determined by flow cytometry for cardiac troponin T (cTnT) ranged between 75% and 89%. No metabolic selection [38 (link)] was applied.
Endo iwr 1
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
Endo-IWR-1 is a small molecule that inhibits Wnt/β-catenin signaling pathway. It functions by preventing the phosphorylation and subsequent degradation of β-catenin.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by Bio-Techne (2 mentions)
Mtesr1 plus media, by STEMCELL (1 mentions)
Recombinant activin a, by R&D Systems (1 mentions)
Accutase, by STEMCELL (1 mentions)
Ascorbic acid, by Bio-Techne (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
Forskolin, by Merck Group (1 mentions)
Dmem f12, by Welgene (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
B27 without vitamin a, by Thermo Fisher Scientific (1 mentions)
Sb431542, by Bio-Techne (1 mentions)
Ldn193189, by Bio-Techne (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Sb431542, by Merck Group (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Mb100b, by R&D Systems (1 mentions)
S31 201, by Merck Group (1 mentions)
Acetic acid, by Corning (1 mentions)
Db100b, by R&D Systems (1 mentions)
10 protocols using endo iwr 1
1
Cardiomyocyte Differentiation from hiPSCs
2
Cyst Formation in Mouse IMCD Cells
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Mouse inner medullary collecting duct (IMCD) cells were cultured in Dulbecco’s modified Eagle’s medium F/12 (DMEM F/12; Welgene) supplemented with 10% fetal bovine serum (FBS). For in vitro analysis of cysts, 2.0 to 3.0 × 105 cells/mL of DMEM F/12 were plated on Matrigel (Corning, no. 354230) at a 1:1 ratio and cultured by addition of DMEM F/12 without FBS for 5 to 6 d. The drugs, including forskolin (Sigma-Aldrich, no. F3917), TAZ modulator (Merck, no. 530959), Exo IWR1 (Tocris, no. 3947), and Endo IWR1 (Tocris, no. 3532), were used at optimal concentrations. Exo- and Endo-IWR1 were provided by E.J. For 3D culture, drugs were added to the medium at least 2 d after seeding, and the treatment was continued until the in vitro cysts had grown sufficiently.
3
Differentiation of iPSCs into NPCs
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Differentiation of iPSCs into NPCs was initiated, as previously described (Maguire et al, 2016 (link)) with indicated modification. Briefly, cultures were treated with daily media changes containing SB431542 (10 μM; Tocris), LDN193189 (1 μM, Tocris), and endo‐IWR1 (1.5 μM; Tocris) and supplemented with B27 without vitamin A (Invitrogen) and passaged at days 4 and 8 of differentiation. From days 8 to 14 of differentiation, NPCs were expanded in Invitrogen neural expansion media, containing Neural Induction Supplement in Advanced DMEM/F12 and Neurobasal Medium (Invitrogen, per manufacturer instructions). On Day 14, NPCs were cryopreserved after confirmation of NPC identity with > 90% expression of Forse‐1.
4
Laminin-521 Coated Cell Culture for Stemcell Differentiation
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Optical-quality plastic tissue culture dishes (ibidi, Martinsried, Germany) were coated with 10 µg/mL Laminin-521 in PBS +/+ for 2 hr at 37°C or overnight at 4°C. Single cells were collected as described for micropatterned cell culture and dishes were seeded with 50,000 single cells resuspended in 2 mL E7 supplemented with 10 µM Rock-inhibitor. The samples were incubated overnight. On the following day, the medium was changed to E7 supplemented with 10 µM Rock-inhibitor and ligands or small molecules. For the 2 day protocol in which cells were switched from E7 ±100 ng/mL WNT3A to E7 ±10 ng/mL ACTIVIN A, the samples were washed with PBS +/+ before adding fresh medium. Live imaging was carried out in E7 imaging medium. Small molecules were used at the following concentrations and were replaced every 24 hr: 10 µM SB431542 (Stemgent), 1 µM IWP-2 (Stemgent), and 1 µM endo-IWR-1 (Tocris).
5
Trophoblast Stem Cell Culture and Manipulation
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Mouse TS cells (blastocyst-derived TS EGFP line, a kind gift of Dr Janet Rossant, Toronto, Canada), proven to exhibit full developmental competence as they colonize all trophoblast layers in chimeras, were cultured as described previously44 . Briefly, TS cells were grown in a standard TS medium (RPMI 1640 supplemented with 20% fetal calf serum, 2 mM L-Glutamine, 2 mM sodium pyruvate and 100 mM 2-mercaptoethanol) containing 70% mouse embryonic fibroblast -conditioned medium and 25 ng ml−1 Fgf2 and 1 μg ml−1 heparin. Cells were split every third day using trypsin. Transfections were performed for 6 h in OptiMEM media supplemented with Fgf2 and heparin using 1% Lipofectamine 2000 (Life Technologies) on nonadherent dishes. After 24 h, cells were selected with 300 μg ml−1 G418. Inhibitors used were as follows: 1 μM LDN 193189 trihydrochloride (Axon, 1509); 2 μM endo-IWR-1 (Tocris, 3532); 2 μM PD0325901; 3 μM CHIR99021; 50 nM Gsk-Lsd1 (N-[(1R,2S)-2-phenylcyclopropyl]-4-piperidinamine, dihydrochloride), kindly provided by the Structural Genomics Consortium (http://www.thesgc.org ); 10 μM SB431542; and 15 μM DES (Sigma).
6
NTAPP Exposure Alters Wnt/β-catenin Signaling
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Human dermal papilla cells (hDP cells) were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and antibiotic/antimycotic solution (Gibco BRL, Gaithersburg, MD, USA) containing penicillin and streptomycin. Cells were incubated at 37 °C in a 5% CO2 incubator. All cultures used for experiments were in the third passage. In order to expose NTAPP on cells, 6.5 × 104 cells were seeded in a 35-mm culture dish and incubated for 24 h. The cells were exposed to 3.6 kV, 4.2 kV, and 5.0 kV of NTAPP for 1 min, the NTAPP-exposed cells were further incubated for 24 h before harvesting. The distance between the device and cells was fixed to 1 cm, and 1. 5 ml of medium was used.
To understand which step of Wnt/β-catenin signaling is affected by NTAPP, experiments using two inhibitors for Wnt/β-catenin signaling pathway were performed. The hDP cells were pre-treated with or without 10 μM endo-IWR1 or 10 μM IWP2 (both from Tocris Bioscience, Bristol, UK) for 24 h before other experiments.
To understand which step of Wnt/β-catenin signaling is affected by NTAPP, experiments using two inhibitors for Wnt/β-catenin signaling pathway were performed. The hDP cells were pre-treated with or without 10 μM endo-IWR1 or 10 μM IWP2 (both from Tocris Bioscience, Bristol, UK) for 24 h before other experiments.
7
Modulating Notch and Wnt Pathways
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Vessel fragments were cut and placed in the bottom of 24 well plates. 1 ml of filtered seawater containing the Notch-signaling inhibitor DAPT (Tocris, 0.2–2 μM), the Wnt-signaling inhibitor endo-IWR1 (Tocris, 0.1–1 μM) or control drugs Exo-IWR (1uM) and the Notch-sparing gamma-secretase modulator E2012 (1 μM) were added to filtered seawater and replaced every other day. The number of colonies that reached stage 4 was counted at 14 days post injury (n = 16 for each condition). Vehicle-treated controls were subclones from the same colonies as the experimental samples.
8
Inhibition of ERK and WNT pathways
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U0126 (Erk inhibitor, EMD4Biosciences) and IWR-1-endo (WNT1 inhibitor, Tocris), were dissolved in DMSO and used at the concentration of 10 and 0.2 µM, respectively from day 2 after differentiation until use.
9
Quantifying MCP-1 and TGF-β in Kidney Cell Secretome
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MCP-1 and TGF-β in 24-hour–conditioned medium from RPTECs or human kidney cells (HK2, ATCC, CRL-2190) were quantified using an MCP-1 ELISA kit (Abcam, ab100721) or a TGF-β1 ELISA kit (R&D Systems, DB100B or MB100B) following the manufacturer’s instructions. Briefly, cells were plated in 6-well plates (3 × 105 to 4 × 105 cells/well) in complete medium. Twenty-four hours later, cells were incubated with serum-free medium containing 20 mM acetic acid or collagen I (50 μg/mL in 20 mM acetic acid, Corning). In some experiments, cells were incubated with the DDR1 inhibitor Cmp-1 (3 μM) synthesized as previously described (16 (link)), or the WNT/β-catenin inhibitor IWR1-endo (30 μM, Tocris), or the STAT3 inhibitor S31-201 (10 μM, MilliporeSigma) for 30 minutes prior to 24 hours of treatment with acetic acid or collagen I.
10
Directed Differentiation of hiPSCs into Cardiac Myocytes
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HiPSCs (19-9-7T and 6-9-9, WiCell, Madison, WI, USA) were plated in feeder-free conditions using matrigel-coated culture dishes and chemically defined medium, mTeSR™ 1 (Stemcell Technologies, Inc., Cambridge, MA, USA). Cardiac myocytes were generated using a directed differentiation protocol.12 (link) Briefly, differentiation of confluent (80%–90%) cells was initiated by adding RPMI/B27 medium (Thermo Fisher Scientific, Waltham, MA, USA) lacking insulin and containing the CHIR99021 (Tocris, Minneapolis, MN, USA) for 24 hours, followed by RPMI/B27 media with an inhibitor of Wnt signaling, IWR-1-endo (Tocris). Differentiated hiPSCs were replated on a coverslip prior to transfection and action potential (AP) recordings.
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