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Ldn193189

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LDN193189 is a small molecule inhibitor that targets the transforming growth factor-beta (TGF-β) signaling pathway. It functions by selectively inhibiting the TGF-β type I receptor ALK5 and related activin-like kinase (ALK) receptors, thereby modulating TGF-β signaling.

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Lab products found in correlation

Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (6 mentions) Igf 1, by Thermo Fisher Scientific (4 mentions) Knockout serum replacement (ksr), by Thermo Fisher Scientific (4 mentions) Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (2 mentions) Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (2 mentions) Sb431542, by Bio-Techne (2 mentions) Ascorbic acid, by Merck Group (2 mentions) N2 supplement, by Thermo Fisher Scientific (2 mentions) Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Streptomycin, by Merck Group (2 mentions) Penicillin, by Merck Group (2 mentions) Igf 1, by Merck Group (2 mentions) Fibroblast growth factor 2 (fgf2), by Miltenyi Biotec (2 mentions) Ldn193189, by Bio-Techne (2 mentions) Ldn 193189, by Miltenyi Biotec (2 mentions) Chir99021, by Miltenyi Biotec (2 mentions) Dmem f12, by Thermo Fisher Scientific (2 mentions) Knockout dmem, by Thermo Fisher Scientific (2 mentions) Mem non essential amino acid, by Thermo Fisher Scientific (2 mentions)

11 protocols using ldn193189

1

Mouse ESC Differentiation into Myogenic Lineage

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Mouse ESCs were cultured as previously described (46 (link)) 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 Oct1 deletion, cells were treated with 4-hydroxytamoxifen and a completely yellow sub-colony was picked and expanded as published (15 (link)). For MD differentiation, ESCs were plated on gelatin and cultured as previously described (20 (link)). Briefly, parental and cKO cells were cultured in N2B27 medium supplemented with recombinant Bmp4 (Peprotech) for 2 d. After 48 hr, media supplementation was changed to RDL (Rspo3, R&D Biosystems; DMSO, MilliporeSigma; LDN193189, Peprotech). Cells were harvested 24 hr (day 3) or 96 hr (day 6) later. For muscle differentiation, media supplementation was switched to HIFL (HGF, Peprotech; IGF-1, Peprotech; FGF-2, Peprotech; LDN193189) and cells cultured for 48 hr (day 8), after which medium was switched to 2% horse serum (ThermoFisher). Cells were harvested on day 11 (for imaging and Oct1 complementation) or day 19 (for RT-qPCR).
Perovanovic J., Wu Y., Abewe H., Shen Z., Hughes E.P., Gertz J., Chandrasekharan M.B, & Tantin D. (2023). Oct1 cooperates with the Smad family of transcription factors to promote mesodermal lineage specification. Science signaling, 16(781), eadd5750.
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2

Differentiation of hiPSCs into Dopaminergic and Motor Neurons

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For neural induction, a modified method previously described was used (Chambers et al., 2009 (link)). hiPSCs were cultured in neural induction medium combining dual ALK inhibition (SB + LDN:10 μm SB431542 (Tocris Bioscience, Bristol, United Kingdom) and 0.2 μm LDN193189 (Stemgent, United Kingdom) in KSR media. For differentiating neuronal cells into neurons, increasing amounts of N2 (Gibco, Waltham, MA, United States) (25, 50, and 75%) was added to the KSR media maintaining LDN193189. On day 5–9 sonic hedgehog (SHH; Curis, Lexington, MA United States) was added in N2 media followed by the addition on day 9 of BDNF (PeproTech, London, United Kingdom), ascorbic acid (Sigma-Aldrich, St. Louis, MO, United States) and FGF8 (PeproTech, London, United Kingdom) and maturated on days 12–30 with BDNF, ascorbic acid, GDNF (PeproTech, London, United Kingdom), TGFβ3 (Sigma-Aldrich, St. Louis, MO, United States), cAMP (Sigma-Aldrich, St. Louis, MO, United States) for doparminergic subtype and retinoic acid (Sigma-Aldrich, St. Louis, MO, United States) for motor neuron cells.
Spitalieri P., Talarico R.V., Murdocca M., Fontana L., Marcaurelio M., Campione E., Massa R., Meola G., Serafino A., Novelli G., Sangiuolo F, & Botta A. (2018). Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2. Frontiers in Physiology, 9, 967.
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3

Directed Differentiation of iPSCs to DA Neurons

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Human iPSCs were cultured using standard protocol on inactivated mouse embryonic fibroblast. DA neuron differentiation was done as previously described (Kriks et al., 2011 (link)). Briefly, iPSCs were cultured on Matrigel (Corning)-coated plate at a density of 4×104 cells/cm2 in SRM media containing growth factors and small molecules including FGF8a (100ng/mL, R&D Systems), SHH C25II (100ng/mL, R&D Systems), LDN193189 (100nM, Stemgent), SB431542 (10μM, R&D Systems), CHIR99021 (3μM, Stemgent), and purmorphamine (2μM, Sigma) for the first five days. For the next six days, cells were maintained in Neurobasal medium (Gibco) containing B-27 supplement minus vitamin A (Gibco), N-2 supplement (Gibco) along with LDN193189 and CHIR99021. In the final stage, colonies were made into single cell suspension and seeded at density of 4×106 cells/cm2 on poly-ornithine and laminin coated plate in neurobasal media containing B27 minus vitamin A, BDNF (20ng/mL, Peprotech), GDNF (20ng/mL, Peprotech), TGFβ (1ng/mL, R&D Systems), ascorbic acid (0.2mM, Sigma), dibutyryl-cAMP (0.5mM, Sigma) and DAPT (10μM, Stemgent) until maturation. DA neurons were cultured for >60 differentiation days before measurements.
Kim J.W., Yin X., Jhaldiyal A., Khan M.R., Martin I., Xie Z., Perez-Rosello T., Kumar M., Abalde-Atristain L., Xu J., Chen L., Eacker S.M., Surmeier D.J., Ingolia N.T., Dawson T.M, & Dawson V.L. (2020). Defects in mRNA translation in LRRK2-mutant hiPSC-derived dopaminergic neurons leads to dysregulated calcium homeostasis. Cell stem cell, 27(4), 633-645.e7.
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4

Paraxial Mesoderm Induction from Human iPSCs

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Paraxial mesoderm was induced from human iPS cells as described18 (link),19 (link). Human iPS cells were dissociated with TrypLE™ Select (Gibco) and plated as single cells per well on iMatrix-coated 6-well plates in mTeSR1 supplemented with 10 μM ROCK inhibitor (Y-27632, Wako) for 1 d. The medium was then changed to DMEM/F12 supplemented with 1% v/v Insulin-Transferrin-Selenium (ITS, Gibco), 3 μM CHIR-99021 (Tocris), and 0.5 μM LDN-193189 (stemgent) (CL medium). At day 3, 20 ng/ml FGF-2 (Pepro Tech) was added (CLF medium) for an additional 3 d. After 6 d of differentiation, the medium was changed to DMEM/F12 supplemented with 10 ng/mL HGF (Pepro Tech), 2 ng/mL IGF-1 (Pepro Tech), 20 ng/mL FGF-2 (Pepro Tech), and 0.5 μM LDN-193189 (HIFL medium) for 2 d. After 8 d differentiation, cells were cultured in DMEM/F12, 15% KSR (gibco), supplemented with 2 ng/mL IGF-1 for 4 d.
Sakai-Takemura F., Narita A., Masuda S., Wakamatsu T., Watanabe N., Nishiyama T., Nogami K., Blanc M., Takeda S, & Miyagoe-Suzuki Y. (2018). Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors. Scientific Reports, 8, 6555.
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5

Differentiation of hiPS Cells into Progenitors

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hiPS cells were seeded onto flasks coated with Matrigel at a density of 0.5–1 × 104 cells per cm2 in primed hiPS cell medium (KSR/FGF2). After 24 h, medium was changed to DMEM/F12-based medium supplemented with ITS (insulin + transferrin + selenium; Sigma-Aldrich) with 1% penicillin and streptomycin (Gibco, ThermoFisher Scientific), 3 µM CHIR99021 (Miltenyi Biotec), 0.5 µM LDN193189 (Tocris Bioscience, Bio-Techne) for 3 days. On days 4–6, the medium was changed to DMEM/F12-based medium supplemented with ITS and 3 µM CHIR99021, 20 ng ml−1 FGF2 (Miltenyi Biotec), 0.5 µM LDN193189. On days 7–8, the medium was changed to DMEM/F12-based medium supplemented with 20 ng ml−1 FGF2, 0.5 µM LDN193189, 2 ng ml−1 IGF1 (Peprotech). On days 9–30, the medium was changed to DMEM/F12-based medium supplemented with 15% knockout serum replacement (Gibco, ThermoFisher Scientific), 1% penicillin and streptomycin, 0.05 mg ml−1 BSA (Sigma-Aldrich), 2 ng ml−1 IGF1.
Buckberry S., Liu X., Poppe D., Tan J.P., Sun G., Chen J., Nguyen T.V., de Mendoza A., Pflueger J., Frazer T., Vargas-Landín D.B., Paynter J.M., Smits N., Liu N., Ouyang J.F., Rossello F.J., Chy H.S., Rackham O.J., Laslett A.L., Breen J., Faulkner G.J., Nefzger C.M., Polo J.M, & Lister R. (2023). Transient naive reprogramming corrects hiPS cells functionally and epigenetically. Nature, 620(7975), 863-872.
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6

Stepwise Hepatic Differentiation of hADSCs

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hADSCs between passage 3 and 7 were planted at a density of 2–3 × 104 cells/cm2 on collagen I-coated dishes (Invitrogen) and cultured in expansion medium at 37 °C with 5 % CO2. Once the cells reached 100% confluence, they were incubated with 10 % FBS RPMI-1640 (Gibco) medium containing 1 μM ATRA for 24 h. The cells were then incubated with serum-free RPMI-1640 medium containing 100 nM IDE1, 3 μM CHIR99021, and 10 μM LY294002 (Selleckchem) for 24 h. Next, the cells were then incubated with serum-free RPMI-1640 medium containing 100 nM IDE1, 10 μM LY294002, 250 nM LDN-193189, and 20 ng/mL FGF4 (PeproTech) for 2 days and then changed to serum-free RPMI-1640 medium containing 100 nM IDE1, 10 μM LY294002, and 20 ng/mL FGF4 for 24 h. The medium was changed to Williams’ E (Gibco) supplemented with 150 ng/mL hepatocyte growth factor (HGF; Sino Biological), 20 ng/mL FGF4, 30 ng/mL oncostatin M (OsM; PeproTech), 2 × 10−5 mol/L dexamethasone (Dex, Sigma-Aldrich), and 1% ITS (Sigma-Aldrich). The differentiation medium was changed every 2 days.
Fu Y., Deng J., Jiang Q., Wang Y., Zhang Y., Yao Y., Cheng F., Chen X., Xu F., Huang M., Yang Y., Zhang S., Yu D., Zhao R.C., Wei Y, & Deng H. (2016). Rapid generation of functional hepatocyte-like cells from human adipose-derived stem cells. Stem Cell Research & Therapy, 7, 105.
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7

Differentiation of hiPS Cells into Progenitors

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hiPS cells were seeded onto flasks coated with Matrigel at a density of 0.5–1 × 104 cells per cm2 in primed hiPS cell medium (KSR/FGF2). After 24 h, medium was changed to DMEM/F12-based medium supplemented with ITS (insulin + transferrin + selenium; Sigma-Aldrich) with 1% penicillin and streptomycin (Gibco, ThermoFisher Scientific), 3 µM CHIR99021 (Miltenyi Biotec), 0.5 µM LDN193189 (Tocris Bioscience, Bio-Techne) for 3 days. On days 4–6, the medium was changed to DMEM/F12-based medium supplemented with ITS and 3 µM CHIR99021, 20 ng ml−1 FGF2 (Miltenyi Biotec), 0.5 µM LDN193189. On days 7–8, the medium was changed to DMEM/F12-based medium supplemented with 20 ng ml−1 FGF2, 0.5 µM LDN193189, 2 ng ml−1 IGF1 (Peprotech). On days 9–30, the medium was changed to DMEM/F12-based medium supplemented with 15% knockout serum replacement (Gibco, ThermoFisher Scientific), 1% penicillin and streptomycin, 0.05 mg ml−1 BSA (Sigma-Aldrich), 2 ng ml−1 IGF1.
Buckberry S., Liu X., Poppe D., Tan J.P., Sun G., Chen J., Nguyen T.V., de Mendoza A., Pflueger J., Frazer T., Vargas-Landín D.B., Paynter J.M., Smits N., Liu N., Ouyang J.F., Rossello F.J., Chy H.S., Rackham O.J., Laslett A.L., Breen J., Faulkner G.J., Nefzger C.M., Polo J.M, & Lister R. (2023). Transient naive reprogramming corrects hiPS cells functionally and epigenetically. Nature, 620(7975), 863-872.
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8

Neural Differentiation of Embryoid Bodies

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Neural differentiation of embryoid bodies (EBs) was performed as previously described [62 (link)] with modifications. Briefly, EB formation was performed by the forced aggregation method. To this goal, PSC lines were cultured in feeder-free conditions as monolayers with E8 medium and passaged every 3 days with TrypLE. For the production of uniform-size EBs, iPSCs grown for 3–10 passages were counted and seeded at 5000 cells per well in 96-well, V-bottom uncoated plates (249,952; NUNC, Rochester, NY). For induction of neural differentiation, EBs were grown in suspension for 7–8 days followed by adherence to Matrigel-coated plates in the Neural Induction Medium (NIM) consisting of DMEM/F12 (GIBCO, 11,320,033), 2 mM l-glutamine, 0.1% bovine serum albumin (Fraction V; Sigma-Aldrich), 1% NEAA, 2% B27 without retinoic acid (GIBCO), 1% N2 supplement (GIBCO), LDN193189 (PeproTech) throughout culture, and 10 μM SB431542 (Tocris Bioscience, Bristol, UK). Numerous rosette structures were formed 2–3 days after the adherent culture of EBs.
Das D., Sonthalia S., Stein-O.’Brien G., Wahbeh M., Feuer K., Goff L., Colantuoni C., Mahairaki V, & Avramopoulos D. (2024). Insights for disease modeling from single-cell transcriptomics of iPSC-derived Ngn2-induced neurons and astrocytes across differentiation time and co-culture. BMC Biology, 22, 75.
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9

Efficient Generation of Neural Stem Cells

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For the generation of NPCs, we followed a previously published protocol19 (link). Briefly, hiPSC colonies were gently disaggregated from the culture plate and plated for 6 hours in non-adherent conditions in DMEM/F12 (Gibco/Invitrogen), 2% B27, 1% N2, 10 μM Y-27632 (Miltenyl-Biotech), 100 nM LDN-193189 (120-10 C; PeproTech), 10 μM SB431542 (S4317-5MG; Sigma-Aldrich) and 2 ng/ml bFGF. Cells were then plated for 10 days on Matrigel-coated dishes in this medium before being detached with accutase and re-plated on Matrigel-coated dishes and cultured in neural induction medium: 1:1 DMEM/F12:neurobasal medium supplemented with 2% B27, 1% N2, 1% ultraglutamine, 10 ng/ml Epidermal Growth Factor (EGF; AF-100-15 Peprotech) and 10 ng/ml bFGF. Culturing cells in this neural induction medium generates homogenous cultures of NSCs (>95% of the cells).
Calatayud C., Carola G., Fernández-Carasa I., Valtorta M., Jiménez-Delgado S., Díaz M., Soriano-Fradera J., Cappelletti G., García-Sancho J., Raya Á, & Consiglio A. (2019). CRISPR/Cas9-mediated generation of a tyrosine hydroxylase reporter iPSC line for live imaging and isolation of dopaminergic neurons. Scientific Reports, 9, 6811.
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10

Neural Induction and Differentiation

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Neural induction was performed as previously reported33 . Briefly, cells were dissociated to single cells using Accutase and plated on gelatin for 10 minutes to remove MEFs. Non-adherent cells were collected and plated on Geltrex-treated dishes at a density of 150-200k cells per well of a 24-well plate in the presence of MEF-conditioned hESC media containing 10 ng/ml of FGF-2 (Life Tech) and 10 uM of Y-27632 (Tocris). Neural differentiation was initiated when cells were confluent using KSR media containing 820 ml of Knockout DMEM (Life Tech), 150 ml Knockout Serum Replacement (Life Tech), 1 mM L-glutamine (Life Tech), 100 uM MEM non-essential amino acids (Life Tech), and 0.1 mM beta- mercaptoethanol (Life Tech) to inhibit SMAD signaling, 100 nM of LDN-193189 (Cat. no. ab142186, Abcam) and 5 uM of SB431542 (Cat. No. 13031, Cayman Chemical) were added on Days 0 through 9. Cells were fed daily, and N2 media (Life Tech) was added in increasing 25% increments every other day starting on Day 4 (100% N2 on Day 10).
Choi J., Lee S., Clement K., Mallard W., Tagliazucchi G.M., Lim H., Choi I.Y., Ferrari F., Tsankov A., Pop R., Lee G., Rinn J., Meissner A., Park P.J, & Hochedlinger K. (2015). A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs. Nature biotechnology, 33(11), 1173-1181.
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