A83 01

Manufactured by Bio-Techne

Sourced in United Kingdom, United States, Germany

A83-01 is a small molecule inhibitor of the ALK5 (TGF-beta type I receptor) kinase. It functions by selectively binding to and inhibiting the activity of the ALK5 receptor, which plays a role in the TGF-beta signaling pathway.

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TGF-β/Alk inhibitor A83-01 TGFβ inhibitor A83-01 ALK4/5/7 inhibitor A83-01 TGF-β type I receptor inhibitor A83-01

236 protocols using a83 01

Directed Differentiation of hPSCs to AME-E and AME-L

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For differentiation to AME-E-like cells, partially primed hPSC were dissociated to single cells using TrypLE Express and counted. The cells were plated to Geltrex-coated tissue culture plates at a seeding density of 1x10⁵/cm² in differentiation medium with 10μM ROCK inhibitor, and further cultured for 5 days. Differentiation medium was prepared as following: N2B27 basal medium, 1μM PD0325901 and 1μM A8301 (Cat. 2939, Tocris Bio-Techne). 100nM LDN193189 (alternative name DM3189, Cat. 1509, Axon Medchem) or 20ng/ml BMP4 (Miltenyi Biotec) were optionally added to the medium. The medium was changed daily. When the spheres appeared, the medium was refreshed by careful exchange of half volume of the medium, and the volume of the medium per well was increased.
Differentiation to TE-like cells was performed according to Guo et al. (2021) (link) and Io et al. (2021) (link). For differentiation to AME-L-like cells, primed hPSC were dissociated to single cells using TrypLE Express and counted. The cells were plated to Geltrex-coated tissue culture plates at a seeding density of 1x10⁵/cm² in differentiation medium with 10μM ROCK inhibitor, and further cultured for 5 days. Differentiation medium was prepared as following: N2B27 basal medium, 1μM PD0325901, 1μM A8301 (Cat. 2939, Tocris Bio-Techne) and 20ng/ml BMP4 (Miltenyi Biotec). The medium was changed daily.

Rostovskaya M., Andrews S., Reik W, & Rugg-Gunn P.J. (2022). Amniogenesis occurs in two independent waves in primates. Cell Stem Cell, 29(5), 744-759.e6.

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Detailed Enteroid Culture Protocol

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All enteroid media were prepared as reported previously (32 (link)). Advanced Dulbecco’s modified Eagle’s medium (DMEM)–F-12 medium supplemented with 1× GlutaMAX (Gibco), 10 mM HEPES (Quality Biologicals), and 100 Units/ml penicillin-streptomycin (Gibco) was used as the basal medium.
Complete medium with growth factor (CMGF⁺) is comprised of 22.3% (vol/vol) basal medium, 50% (vol/vol) Wnt3a-conditioned medium, 15% (vol/vol) R-spondin-1-conditioned medium, 10% (vol/vol) noggin-conditioned medium, 1× B27 supplement (Gibco), 1 mM N-acetylcysteine (Sigma), 1× Primocin (InvivoGen), 50 ng/ml human epidermal growth factor (R&D Systems), 10 nM [Leu-15]-gastrin (AnaSpec), 500 nM A83-01 (Tocris), and 10 μM SB202190 (Tocris).
Differentiation medium is comprised of 86.2% (vol/vol) basal medium, 10% (vol/vol) noggin-conditioned medium, 1 mM N-acetylcysteine (Sigma), 50 ng/ml human epidermal growth factor (R&D Systems), 10 nM [Leu-15]-gastrin (AnaSpec), and 500 nM A83-01 (Tocris).

Ranganathan S., Doucet M., Grassel C.L., Delaine-Elias B., Zachos N.C, & Barry E.M. (2019). Evaluating Shigella flexneri Pathogenesis in the Human Enteroid Model. Infection and Immunity, 87(4), e00740-18.

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Cell Culture Conditions for Transitional Carcinoma Lines

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The transitional carcinoma cell lines NBT-II, T24, J82, UMUC3 and human embryonic kidney cells HEK293T were obtained from the American Type Culture Collection (ATCC; catalogue no. CRL-1655 and CRL-1749). Cells were routinely cultured in Dulbecco’s modified eagle medium (DMEM, Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS, HyClone Thermo Scientific), and 100 units/ml penicillin–streptomycin (1 × pen–strep, Invitrogen). Cultured cells were regularly tested for mycoplasma contamination. Cells were grown in a 5% CO₂ atmosphere incubator at 37 °C. When appropriate HGF (5 ng/ml, Calbiochem), TGF-β1 (2.5 ng/ml, R&D), SB431542 (5 μM, Tocris), A83-01 (8 μM, Tocris), A83-01 is a small molecule inhibitor specifically targeting TβRI (ALK5), Activin receptor type-1B/ALK4 and Activin receptor type-A/ALK-7, the three of which contain highly structurally related kinase domains, PD0325901 (1 μM, Selleck Chemicals), LY2157299 (1 μM, Selleck Chemicals), AZD0530 (2 µM, Selleck Chemicals), JNJ38877605 (4 µM, Selleck Chemicals) or MG132 (2 μM; Sigma) was added. All cell lines were independently genotyped and validated (please refer to source data).

Sim W.J., Iyengar P.V., Lama D., Lui S.K., Ng H.C., Haviv-Shapira L., Domany E., Kappei D., Tan T.Z., Saei A., Jaynes P.W., Verma C.S., Kumar A.P., Rouanne M., Ha H.K., Radulescu C., ten Dijke P., Eichhorn P.J, & Thiery J.P. (2019). c-Met activation leads to the establishment of a TGFβ-receptor regulatory network in bladder cancer progression. Nature Communications, 10, 4349.

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Airway Epithelial Cell Culture Protocol

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Filter-grown airway epithelial cells were measured under open or short circuit conditions as detailed in previous reports [4 (link), 29 (link)]. In brief, PBE cells were grown on Millipore filters in an air-liquid interface (ALI) in Advanced DMEM/F12 media (Thermo Fisher Scientific, USA) supplemented with 0.5 μg/mL hydrocortisone, 100 nM triiodothyronine, and 0.5 μg/mL epinephrine (all from Sigma-Aldrich, Missouri, USA); 0.25 μg/mL human epidermal growth factor (PeproTech, UK); 100 nM TTNPB (Cayman, USA); and 50 nM A83-01 (Tocris Bioscience, Bristol, UK) for 14–21 days. For the first week of ALI, 500 nM A83-01 was supplemented, and for the second week, 10 μM of DAPT (Tocris Bioscience, Bristol, UK) was added.

Benedetto R., Centeio R., Ousingsawat J., Schreiber R., Janda M, & Kunzelmann K. (2020). Transport properties in CFTR−/− knockout piglets suggest normal airway surface liquid pH and enhanced amiloride-sensitive Na+ absorption. Pflugers Archiv, 472(10), 1507-1519.

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Optimized Cell Culture Conditions

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All media were prepared as reported previously^{57 (link)}. Advanced Dulbecco’s modified Eagle’s medium (DMEM)–F-12 medium supplemented with 1× GlutaMAX (Gibco), 10 mM HEPES (Sigma-Aldrich), and 100 Units/ml penicillin-streptomycin (Sigma-Aldrich) was used as the basal medium. Expansion medium (EM) is basal medium supplemented with 50% (vol/vol) Wnt3A-conditioned medium, 20% (vol/vol) R-spondin-1-conditioned medium, 10% (vol/vol) Noggin-conditioned medium, 1× B27 supplement (Gibco), 1 mM N-acetylcysteine (Sigma), 1× Primocin (InvivoGen), 50 ng/ml human epidermal growth factor (R&D Systems), 10 nM [Leu-15]-gastrin (AnaSpec), 500 nM A83-01 (Tocris), and 10 μM SB202190 (Tocris). Differentiation medium is comprised of basal medium (no penicillin-streptomycin added), 10% (vol/vol) Noggin-conditioned medium, 1 mM N-acetylcysteine (Sigma), 50 ng/ml human epidermal growth factor (R&D Systems), 10 nM [Leu-15]-gastrin (AnaSpec), 500 nM A83-01 (Tocris), and 10% fetal bovine serum (Sigma Aldrich). Wnt3A (American Type Culture Collection, Manassas, VA), R-spondin1 (kindly provided by Dr. Calvin Kuo, Stanford University, Stanford, CA), and Noggin^{58 (link)} (kindly provided by Dr. Marcel Bijvelds, Erasmus University, Rotterdam, the Netherlands) cell lines were maintained to produce conditioned media.

Liu L., Saitz-Rojas W., Smith R., Gonyar L., In J.G., Kovbasnjuk O., Zachos N.C., Donowitz M., Nataro J.P, & Ruiz-Perez F. (2020). Mucus layer modeling of human colonoids during infection with enteroaggragative E. coli. Scientific Reports, 10, 10533.

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Differentiation of Pluripotent Cells into NSCs

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Pluripotent cells were differentiated in neural stem cells (NSCs), using a modified dual SMAD inhibition protocol [20 (link)]. In short, 18,000 cells/cm² were plated on Matrigel-coated cell culture dishes in mTeSR-1 medium in the presence of 10 µM Y27632. When cells reached 90% confluency, the medium was switched to differentiation medium (KnockOut DMEM (Gibco), 15% KnockOut serum replacement (Gibco), 2 mM l-glutamine (Gibco), MEM non-essential amino acids (Sigma), 0.1 mM β-mercaptoethanol, 100U/ml penicillin and 100 µg/ml streptomycin) supplemented with 2 µM A 83-01 (Tocris) and 2 µM Dorsomorphin (Sigma-Aldrich). At day 6, medium was changed to an equal ratio of differentiation medium and NSC medium (KnockOut DMEM-F12 (Gibco), 2 mM l-glutamine (Gibco), 20 ng/ml bFGF (Peprotech), 20 ng/ml EGF (Peprotech), 2% StemPro Neural supplement (Gibco), 100U/ml penicillin and 100 µg/ml streptomycin) supplemented with 2 µM A 83-01 (Tocris) and 2 µM Dorsomorphin (Sigma-Aldrich). At day 10, cells were passaged (NSC p = 0) using Accutase (Sigma) and maintained in NSC medium. We used commercially available H9-derived NSCs (Gibco) as a control (a kind gift from Raymond Poot, Rotterdam).

Perenthaler E., Nikoncuk A., Yousefi S., Berdowski W.M., Alsagob M., Capo I., van der Linde H.C., van den Berg P., Jacobs E.H., Putar D., Ghazvini M., Aronica E., van IJcken W.F., de Valk W.G., Medici-van den Herik E., van Slegtenhorst M., Brick L., Kozenko M., Kohler J.N., Bernstein J.A., Monaghan K.G., Begtrup A., Torene R., Al Futaisi A., Al Murshedi F., Mani R., Al Azri F., Kamsteeg E.J., Mojarrad M., Eslahi A., Khazaei Z., Darmiyan F.M., Doosti M., Karimiani E.G., Vandrovcova J., Zafar F., Rana N., Kandaswamy K.K., Hertecant J., Bauer P., AlMuhaizea M.A., Salih M.A., Aldosary M., Almass R., Al-Quait L., Qubbaj W., Coskun S., Alahmadi K.O., Hamad M.H., Alwadaee S., Awartani K., Dababo A.M., Almohanna F., Colak D., Dehghani M., Mehrjardi M.Y., Gunel M., Ercan-Sencicek A.G., Passi G.R., Cheema H.A., Efthymiou S., Houlden H., Bertoli-Avella A.M., Brooks A.S., Retterer K., Maroofian R., Kaya N., van Ham T.J, & Barakat T.S. (2019). Loss of UGP2 in brain leads to a severe epileptic encephalopathy, emphasizing that bi-allelic isoform-specific start-loss mutations of essential genes can cause genetic diseases. Acta Neuropathologica, 139(3), 415-442.

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Directed Differentiation of Human PSCs

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Except where specified, reset cells were ʻre-primed' before initiating differentiation. Cells were plated on Geltrex in t2iLGö and after 48 h the medium was changed to E8. Cultures were maintained in E8, passaging at confluence. Lineage-specific differentiation was initiated between 25 and 44 days.
Definitive endoderm was induced according to Loh et al. (2014) (link). Cells were cultured in CDM2 medium (in-house according to Loh et al., 2014) supplemented with 100 ng/ml activin A (produced in-house), 100 nM PI-103 (Bio-Techne, 2930), 3 µM CHIR99021, 10 ng/ml FGF2, 3 ng/ml BMP4 (Peprotech) for 1 day. For the next 2 days the following supplements were applied: 100 ng/ml activin A, 100 nM PI-103, 20 ng/ml FGF2, 250 nM LDN193189.
For lateral mesoderm induction (Loh et al., 2016 (link)), cells were treated with CDM2 supplemented with 30 ng/ml activin A, 40 ng/ml BMP4 (Miltenyi Biotech, 130-098-788), 6 µM CHIR99021, 20 ng/ml FGF2, 100 nM PI-103 for 1 day, then with 1 µM A8301, 30 ng/ml BMP4 and 10 µM XAV939 (Sigma-Aldrich).
For neural differentiation via dual SMAD inhibition (Chambers et al., 2009 (link)), cells were treated with N2B27 medium supplemented with 500 nM LDN193189 (Axon, 1509) and 1 μM A 83-01 (Bio-Techne, 2939) for 10 days, then passaged to plates coated with poly-L-ornithine and laminin and further cultured in N2B27 without supplements.

Guo G., von Meyenn F., Rostovskaya M., Clarke J., Dietmann S., Baker D., Sahakyan A., Myers S., Bertone P., Reik W., Plath K, & Smith A. (2017). Epigenetic resetting of human pluripotency. Development (Cambridge, England), 144(15), 2748-2763.

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Establishment and Differentiation of Human Colonoids

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Establishment of human colonoids was performed as previously described by Fujii et al.(Fujii et al., 2015 (link)). The crypts were cultured in growth medium containing advanced Dulbecco’s modified Eagle’s medium/F12, penicillin/streptomycin (100 units/mL), 10 mM HEPES, 2 mM Glutamax, supplements N2 (1x) and B27 (1x), 50 ng/mL mouse epidermal growth factor (all from Life Gibco), 1 mM N-acetylcysteine (Sigma-Aldrich), 50% v/v Wnt3a conditioned medium, 10% v/v R-spondin-1 conditioned medium, 100 ng/mL murine recombinant noggin protein (Peprotech),10 nM gastrin (Sigma-Aldrich), 500 nM A83-01 (Bio-techne), 10 μM SB202190 (Sigma-Aldrich) and 10 mM Nicotinamide (Sigma-Aldrich). 10 μM Y-27632 (Sigma-Aldrich) was added to the culture medium for the initial 3 days. Medium was changed every 2 days. Differentiation towards a mature epithelium in human colonoids was achieved with reduction of Wnt3a to 15% v/v and withdraw of SB202190 and Nicotinamide for 5–7 days. During the last 24 h in differentiation medium human colonoids were treated with human recombinant IL17A (50 ng/mL), TNFα (10 ng/mL), IFNγ (20 ng/mL), IL13 (10 ng/mL) and IL9 (10 ng/mL). Cytokine concentrations were determined based on previous in house experience and published literature.

Pavlidis P., Tsakmaki A., Treveil A., Li K., Cozzetto D., Yang F., Niazi U., Hayee B.H., Saqi M., Friedman J., Korcsmaros T., Bewick G, & Powell N. (2022). Cytokine responsive networks in human colonic epithelial organoids unveil a molecular classification of inflammatory bowel disease. Cell Reports, 40(13), 111439.

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Cell culture maintenance and differentiation

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HEK293T and BEAS-2B cells were maintained in DMEM (Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Pittsburgh, PA, USA). THP1, a human monocyte cell line, was cultured in RPMI 1640 medium with 10% fetal bovine serum (Thermo Fisher Scientific) and differentiated by preincubation in culture medium with 100 ng·mL⁻¹ phorbol 12-myristate 13-acetate overnight. nHBEs were cultured in Small Airway Epithelial Cell Medium (Promocell, Heidelberg, Germany) with 1.0 μM A8301 (Tocris Bioscience, Bristol, UK), 0.5 μM CHIR99021 (Sigma-Aldrich, St Louis, MO, USA) and 5 μM Y27632 (Sigma-Aldrich) on plates precoated with laminin-enriched 804G-conditioned medium [16 (link), 17 (link)].

Liu T., Liu S., Rui X., Cao Y., Hecker J., Guo F., Zhang Y., Gong L., Zhou Y., Yu Y., Krishnamoorthyni N., Bates S., Chun S., Boyer N., Xu S., Park J.A., Perrella M.A., Levy B.D., Weiss S.T., Mou H., Raby B.A, & Zhou X. (2024). Gasdermin B, an asthma-susceptibility gene, promotes MAVS-TBK1 signalling and airway inflammation. The European Respiratory Journal, 63(5), 2301232.

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Expansion of Human Liver Organoids

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After 1 week of seeding the organoids, isolation media were changed to human liver expansion media (EM; Ad+++ supplemented with 1X B27 supplement without retinoic acid (Gibco), 1X N2 supplement (Gibco), 1.25 mM N-acetyl-L-cysteine (Sigma), 20% (vol/vol) Rspo-1 conditioned medium, 1.25% (vol/vol) Wnt3a conditioned medium [Barker et al., 2010 (link)], 10 mM nicotinamide (Sigma), 10 nM recombinant human (Leu15)-gastrin I (Sigma), 50 ng/ml recombinant human EGF (Peprotech), 100 ng/ml recombinant human FGF10 (Peprotech), 25 ng/ml recombinant human HGF (Peprotech), 10 μM forskolin (Sigma), and 5 μM A8301 (Tocris)) (Huch et al., 2015 (link)).
EM was changed twice a week, and cultures were split every 7–10 days according to organoid density. For passaging (1:4-1:8, depending on growth rate of the culture), organoids were resuspended in 10 ml Ad+++, incubated in ice for 10 min, and collected by centrifugation (5 min at 200 xg). Subsequently, organoids were incubated for 1–2 min in TrypLE Express at RT and mechanically disrupted by pipetting. After a further wash in Ad+++, cells were resuspended in BME solution and seeded in 24- or 48-well suspension plates. After BME solution had solidified, wells were filled with 500 µl (24 wells) or 250 µl (48 wells) of human liver organoid expansion medium.

De Crignis E., Hossain T., Romal S., Carofiglio F., Moulos P., Khalid M.M., Rao S., Bazrafshan A., Verstegen M.M., Pourfarzad F., Koutsothanassis C., Gehart H., Kan T.W., Palstra R.J., Boucher C., IJzermans J.N., Huch M., Boj S.F., Vries R., Clevers H., van der Laan L.J., Hatzis P, & Mahmoudi T. (2021). Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma. eLife, 10, e60747.

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