hiPSC cells grow in mTeSR were dissociated into single cells using accutase and plated at density of 25–50 k cells/cm2 on matrigel coated cell culture plates and subsequently differentiated into endoderm and mesoderm lineages (Loh et al., 2014 (link); Ang et al., 2018 (link)). For definitive endoderm induction, anterior primitive streak was first specified using 100 ng/ml Activin A (R and D systems, 338-AC-050), 3 μm CHIR (Tocris, 4423) and 20 ng/ml FGF2 (R and D Systems, 233-FB-01M) in CDM2 basal media. After 24 hr, the cells were washed with DMEM/F12 (1x, Thermo Fisher Scientific, 11320033), and definitive endoderm was induced using 100 ng/ml Activin A and 250 nM LDN (Reprocell, Yokohama, Japan; 04–0074) in CDM2 basal media for 24 hr. For lateral mesoderm induction, midprimitive streak was specified using 30 ng/ml Activin, 16 μM CHIR, 20 ng/ml FGF2 and 40 ng/ml BMP (R and D Systems, 314 BP-050) in CDM2 basal media for 24 hr. After 24 hr, cells were washed with DMEM/F12 (1x, Thermo Fisher Scientific, 11320033), and lateral mesoderm was induced using 1 μM A8301 (R and D Systems, 2939) 30 ng/ml BMP and 1 μM C59 (Tocris, Bristol, United Kingdom; 5148) for 24 hr in CDM2 basal media. On the third day, cells were lysed for RNA collection and purification.
A83 01
Manufactured by R&D Systems
Sourced in United States
A83-01 is a small molecule inhibitor that selectively targets the activin/nodal/transforming growth factor-beta (TGF-β) signaling pathway. It functions as a potent and selective inhibitor of the activin-like kinase (ALK) 4, 5, and 7 receptors.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (5 mentions)
Wnt3a, by R&D Systems (5 mentions)
Nicotinamide, by Merck Group (5 mentions)
Sb202190, by Merck Group (4 mentions)
Matrigel, by BD (4 mentions)
Matrigel, by Thermo Fisher Scientific (4 mentions)
Fgf10, by Thermo Fisher Scientific (4 mentions)
N acetylcysteine, by Merck Group (4 mentions)
Matrigel, by Corning (4 mentions)
R spondin 1, by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (4 mentions)
Hepes, by Thermo Fisher Scientific (4 mentions)
Noggin, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fibroblast growth factor (fgf), by Thermo Fisher Scientific (3 mentions)
N acetyl l cysteine, by Merck Group (3 mentions)
Addmem f12, by Thermo Fisher Scientific (3 mentions)
Y 27263, by Merck Group (3 mentions)
Cell recovery solution, by BD (2 mentions)
Prostaglandin e2, by Bio-Techne (2 mentions)
16 protocols using a83 01
1
Directed Differentiation of hiPSCs into Endoderm and Mesoderm
2
Gastric Tumor Organoid Culture Protocol
3
Enteroid Generation from Intestinal Crypts
4
Establishment and Labeling of PDAC Organoids
5
Gastric Tumor Organoid Culture
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Gastric tumours from Tcon mice were harvested and used to generate two cell lines, Tcon3077 and Tcon3944, as previously described.23 (link) For in vitro culture, organoids were mixed with 50 µl of Matrigel (cat. No.354248, BD Bioscience) and plated in 24-well plates. After Matrigel polymerisation, cells were overlaid with DMEM/F12 supplemented with penicillin/streptomycin, 10 mM HEPES, GlutaMAX, 1XB27, 1XN2 (Invitrogen) and 1.25 mM N-acetylcysteine 0.05 µg/mL EGF, 0.1 µg/ml bFGF, 0.01 µM gastrin, 10 mM nicotinamide, 10 µM Y-27632, SB202190 (all from Sigma–Aldrich), 1 µM prostaglandin E2 (Tocris Bioscience), 0.5 µg/mL Recombinant R-spondin1, 0.1 µg/mL mNoggin, 0.1 µg/mL FGF-10 (all from PeproTech) and 0.1 µg/mL Wnt3A, 0.5 µM A83-01 (from R&D Systems). Organoids were passaged weekly at a 1:5–1:8 split ratio by removal from Matrigel with Cell Recovery Solution (BD Biosciences) following the manufacturer’s instructions and transfer to fresh Matrigel.
6
CFPAC-1 Organoid Culture Protocol
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Organoids were established as described before (30 (link)). CFPAC-1 cells were embedded in growth factor-reduced Matrigel (Cat#356231; BD Bioscience, CA, USA), and cultured in human complete medium at 37 C° for 14 days (21 (link), 25 (link)). Human complete medium was AdDMEM/F12(Cat#12634–010; Invitrogen, CA, USA), medium supplemented with 1M HEPES (Invitrogen), GlutaMax (Cat#35050–061; Invitrogen), penicillin/streptomycin (Cat#15140122; Invitrogen), B27 (Cat#17504044; Invitrogen), N-acetyl-l-cysteine (Cat#A9165; Sigma-Aldrich Co.), Wnt-3a (Cat#5036-WN-010; R&D Systems, MN, USA), R-Spondin1 (Cat#120–38; Peprotech, NJ, USA), Noggin (Cat#120-10C; Invitrogen), epidermal growth factor (EGF, Cat#AF-100-15; Peprotech), fibroblast growth factor (FGF, Cat#100–26; Peprotech), nicotinamide (Cat#N0636; Sigma-Aldrich Co.), Y-27263 (Cat# Y0503; Sigma-Aldrich Co.) and A83–01 (Cat#2939/10; R&D Systems).
7
Establishment and Culture of Colorectal Cancer Organoids
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Resection samples from colorectal
cancer patients were provided by the University Cancer Center Frankfurt
(UCT). All materials were collected after prior written informed consent
as part of the interdisciplinary Biobank and Database Frankfurt (iBDF)
and the study was approved by the institutional review board of the
University Hospital Frankfurt (ethics vote: 4/09; project numbers
SGI-12-2018 and SGI-10-2022). Patient-derived organoids were established
and cultured as previously described.40 (link) Briefly, medium containing advanced DMEM/F12 supplemented with 10
mM HEPES, 1× Glutamax, 1× penicillin/streptomycin, 2% B27,
12.5 mM N-acetylcysteine, 500 nM A83-01 (R&D
Systems), 10 μM SB202190 (Sigma-Aldrich), 20% R-spondin 1 conditioned
medium, 10% Noggin conditioned medium, 50 ng/mL human EGF (Peprotech),
and Wnt surrogate (35 ng/mL, # N001-0.5 mg, ImmunoPrecise) was used
for both tumor and normal organoids. Y-27632 (10 μM) was added
to the medium for the first 3 days after passaging.
cancer patients were provided by the University Cancer Center Frankfurt
(UCT). All materials were collected after prior written informed consent
as part of the interdisciplinary Biobank and Database Frankfurt (iBDF)
and the study was approved by the institutional review board of the
University Hospital Frankfurt (ethics vote: 4/09; project numbers
SGI-12-2018 and SGI-10-2022). Patient-derived organoids were established
and cultured as previously described.40 (link) Briefly, medium containing advanced DMEM/F12 supplemented with 10
mM HEPES, 1× Glutamax, 1× penicillin/streptomycin, 2% B27,
12.5 mM N-acetylcysteine, 500 nM A83-01 (R&D
Systems), 10 μM SB202190 (Sigma-Aldrich), 20% R-spondin 1 conditioned
medium, 10% Noggin conditioned medium, 50 ng/mL human EGF (Peprotech),
and Wnt surrogate (35 ng/mL, # N001-0.5 mg, ImmunoPrecise) was used
for both tumor and normal organoids. Y-27632 (10 μM) was added
to the medium for the first 3 days after passaging.
8
Episomal Vector-Based Human iPSC Generation
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For the reprogramming experiments, the hAFCs were electroporated with the episomal expression vectors in the Y4 combination (pCXLE-hOCT3/4-shp53-F, pCXLEhSK, and pCXLE-hUL) using a Nucleofector II device (Lonza AMAXA, Basel, Switzerland) and were cultured in the AFC medium,14 (link) A-83-01 (Tocris/R&D Systems, Minneapolis, MN,USA), 10 μM HA-100 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 10 ng/ml human LIF (Millipore), 100 ng/ml bFGF (R&D, Minneapolis, MN, USA) on the third day to enhance iPSC yield.28 (link) On day 15, the medium was changed to the mTeSR1 (Stem cell Technologies, Vancouver, BC, Canada). Small cell colonies became visible approximately 4–6 weeks after transfection. Single colonies with typical flat human ESC-like morphologies were picked up and replated in a four-well plate for further propagation. All iPSC lines and H9 cell lines were cultured in mTeSR1 medium on Matrigel (BD, San Diego, CA, USA).
9
Intestinal Enteroid Culture Protocol
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Within 24 hr from collection, intestinal crypts were isolated from the samples and enteroid cultures were generated per our previously described protocol used for crypt isolation and enteroid processing.11 (link) Following this, the enteroid cultures were suspended in basement membrane matrix, Matrigel (Corning, #CB-40230C, Corning, NY) domes and grown in 50% L-WRN conditioned media, which is supplemented with 50 ng/mL epidermal growth factor (Millipore Sigma, #GF144, Burlington, MA), 1 mM N-Acetylcysteine (Millipore Sigma, #A9165-5G, Burlington, MA), 500nM A-83-01 (R&D Systems, #2939/10, Minneapolis, MN), 10uM SB202190 (Millipore Sigma, #S7067-5 MG, Burlington, MA), 10 mM Nicotinamide (Millipore Sigma, #N0636-100G, Burlington, MA), and 10nM [leu] 15-gastrin 1 (Millipore Sigma, #G9145-0.1 MG). All enteroids were passaged every 5–7 days and experimented on when at full maturity, between passages 4 and 10 for all experiments.
10
Establishment of Pancreatic Cancer Organoids
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PDAC organoids were established from KPC PCCs, which were established using the outgrowth method as described [21 ]. PCCs were embedded in growth factor-reduced Matrigel (Cat#356231; BD Bioscience, CA, USA), and cultured in human complete medium at 37 C° for 14 days [21 , 25 (link)]. Human complete medium was AdDMEM/F12 (Cat#12634–010; Invitrogen, CA, USA), medium supplemented with 1 M HEPES (Invitrogen), GlutaMax (Cat#35050–061; Invitrogen), penicillin/streptomycin (Cat#15140122; Invitrogen), B27 (Cat#17504044; Invitrogen), N-acetyl-l-cysteine (Cat#A9165; Sigma-Aldrich Co.), Wnt-3a (Cat#5036-WN-010; R&D Systems, MN, USA), R-Spondin 1 (Cat#120–38; Peprotech, NJ, USA), Noggin (Cat#120-10C; Invitrogen), epidermal growth factor (EGF, Cat#AF-100-15; Peprotech), fibroblast growth factor (FGF, Cat#100–26; Peprotech), nicotinamide (Cat#N0636; Sigma-Aldrich Co.), Y-27263 (Cat# Y0503; Sigma-Aldrich Co.) and A83–01 (Cat#2939/10; R&D Systems).
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