TE and TSC induction were performed as previously described15 (link),26 (link). Briefly, for generating TE, human naïve PSCs were dissociated into single cells with a 1:1 mixture of 0.5 mM EDTA and TrypLE Express and plated at a density of 100,000 cell/well of 6-well plate on Geltrex in TE induction medium (1:1 mix of Neurobasal medium (Gibco) and DMEM/F12 (HyClone) supplemented with N2 (Gibco) and B27 (Gibco), penicillin-streptomycin (HyClone), Glutamax (Gibco), β-mercaptoethanol (Sigma), 1 µM PD0325901 (Axon) and 1 µM A83-01 (Selleck)) for 5 days. TELCs at day 5 were used for downstream analysis throughout this study. For generating expandable TSCs, TELCs at day 5 were dissociated into single cells with TrypLE and plated on Geltrex-coated 6-well plates at a 1:4-1:8 split ratio in TSC medium (DMEM/F12 supplemented with N2 and B27, penicillin-streptomycin, Glutamax, β-mercaptoethanol, 1.5 µg/ml L-ascorbic acid, 50 ng/ml EGF (PeproTech), 0.5 µM A83-01 (Selleck), 1 µM SB431542 (Selleck), 2 µM CHIR99021 (Axon), 0.8 mM VPA (Vetec) and 5 µM Y-27632 (Axon) supplemented with 10 µM Y-27632.
A83 01
Manufactured by Selleck Chemicals
Sourced in United States
A83-01 is a selective inhibitor of the Transforming Growth Factor-beta (TGF-β) pathway. It functions by blocking the activation of the ALK5 receptor, which is a key component in the TGF-β signaling cascade. A83-01 is commonly used in cell culture and research applications to study the role of the TGF-β pathway in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by Selleck Chemicals (4 mentions)
Sb431542, by Selleck Chemicals (4 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Pd0325901, by Selleck Chemicals (2 mentions)
Iwr 1 endo, by Selleck Chemicals (2 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (2 mentions)
Activin a, by Sino Biological (2 mentions)
Basic fibroblast growth factor (bfgf), by Selleck Chemicals (2 mentions)
Ly294002, by Selleck Chemicals (2 mentions)
Basic fibroblast growth factor (bfgf), by Sino Biological (2 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Thiazovivin, by Selleck Chemicals (1 mentions)
Vascular endothelial growth factor (vegf), by Sino Biological (1 mentions)
Thrombopoietin, by Sino Biological (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium f12, by Thermo Fisher Scientific (1 mentions)
Accutase, by Merck Group (1 mentions)
8 protocols using a83 01
1
Efficient Stem Cell Differentiation Protocol
2
Transwell Assay for Cell Migration and Invasion
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Cells were harvested 24 hours after transfections and counted, and 80,000 cells were plated on top of a transwell 8 μM insert in low (0.1% FBS) media. Complete media (10% FBS) was placed in the well below the insert. Each condition was performed in triplicate. Cells were allowed to migrate for 20 hours before fixing and staining with 1% Crystal Violet in 20% methanol for 20 min at room temperature. For migration assays related to TGFB signaling, transfected cells were pretreated with A83-01 overnight at indicated concentrations before being plated for migration assays in low FBS media supplemented with the indicated concentrations of A83-01 (Selleckchem). Double-transfected cells were plated for migration in low FBS media with or without TGFB2 (10 ng/ml; Cell Signaling Technologies). The top layer of the transwell membrane was scraped to remove excess stain and washed with phosphate-buffered saline (PBS). Inserts were allowed to dry before imaging on a Nikon epifluorescence microscope using a 10× objective. At least five fields of view were acquired per insert, and the area of migrated cells was calculated using ImageJ and represented as the mean area of migration. For invasion assays, Matrigel (150 μg/cm2) was added to the top of each insert and allowed to solidify before plating cells. Fixation and imaging were performed as described above.
3
Efficient O-IPSC Differentiation Protocol
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5 × 104 O-IPSC cells were seeded to 24 well plate pre-coated with Matrigel in TSCM2 medium with 1.2 μM Thiazovivin for 2 days. TSCM3 medium was renewed every day from day 3 to day 6. TSCM2 medium consists of: 1 μM PD0325901 (Selleck, S1036), 5 μM A83-01 (Selleck, S7692), 250 μM LPA (Tocris, 325465-93-8), 0.8 mM VPA (Selleck, S3944), 20 ng/mL FGF4 (PEPROTECH, 100-31), 70 μg/mL VC (Sigma-Aldrich, V900134), 2% ITS (gibco, 41400045), 100 μg/mL QsrHSA (Oryzogen, HYC002M01), 32.7 μM Ethanolamine (Selleck, S6210), GlutaMAX-I (100× , gibco, 35050-061), Sodium pyruvate (100× , gibco, 11360-070). TSCM3 was made of TSCM2 medium with addition of 5% KSR and without VPA.
4
Directed Hematopoietic Differentiation of hPSCs
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Before haematopoietic differentiation, hPSCs were dissociated by Accutase (Sigma) and plated on growth factor‐reduced Matrigel (Corning)‐coated plates with thiazovivin (0.1 μM, Selleck). Firstly, at day 0, 40 ng/ml of BMP4 (Peprotech), 30 ng/ml of ACTIVINA (Sino Biological Inc.), 20 ng/ml of bFGF (Sino Biological Inc.), 6 μM CHIR99021 (Selleck) and 10 μM LY294002 (Selleck) were added to the basic medium (BM, mimics of the CustommTeSR1) of Dulbecco's‐modified Eagle's medium/F‐12 (GIBCO) supplemented with 1% insulin–transferrin–selenium (GIBCO), 70 μg/ml of vitamin C (Sigma). Second, 30 ng/ml of BMP, 1 μM A8301 (Selleck) and 2 μM IWR‐1‐endo (Selleck) were added to the BM on day 1. Then, on days 2–4 of differentiation, 40 ng/ml of vascular endothelial growth factor (Sino Biological Inc.) and 50 ng/ml of bFGF were added to the BM. Finally, 40 ng/ml of vascular endothelial growth factor, 50 ng/ml of bFGF, 10 μM SB431542 (Selleck), 10 ng/ml of stem cell factor (Peprotech), 50 ng/ml of thrombopoietin (Sino Biological Inc.), 10 ng/ml ofinterleukin 3 (Sino Biological Inc.) and 50 ng/ml of interleukin 6 (Sino Biological Inc.) were added in the BM at days 4–6 of differentiation and further haematopoietic commitment and maturation.
5
Hematopoietic Differentiation of H1 hESCs
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Hematopoietic differentiation was performed as previously described (Zhu et al., 2020 (link)). Briefly, H1 hESCs were plated onto Growth Factor Reduced Matrigel (1:200 dilution; BD Biosciences)-coated plates at a proper initial density. The hematopoietic differentiation medium was changed every day, supplemented with corresponding cytokines or inhibitors, and floated hESCs-HSPCs were collected from D8 for further assays. The cytokines or inhibitors added each day were as follows: D0–D1, 40 ng/mL BMP4 (PeproTech), 30 ng/mL Activin A (Sino Biological), 20 ng/mL bFGF (Sino Biological), 6 μM CHIR99021 (Selleck), and 10 μM LY294002 (Selleck); D1–D2, 30 ng/mL BMP4, 1 μM A8301 (Selleck), and 2 μM IWR-1-endo (Selleck); D2–D4, 40 ng/mL vascular endothelial growth factor (VEGF) (Sino Biological) and 50 ng/mL bFGF; D4 and later, 40 ng/mL VEGF, 50 ng/mL bFGF, 10 μM SB431542 (Selleck), 10 ng/mL SCF (PeproTech), 50 ng/mL TPO (Sino Biological), 10 ng/mL IL3 (Sino Biological), 50 ng/mL IL6 (Sino Biological), and 50 ng/mL Flt3L (PeproTech).
6
Mouse Primary Hepatocyte Isolation
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Mouse HPs were generated as previously described [31 (link)]. Briefly, primary adult mice hepatocytes were isolated from 8 to 12 weeks old female BALB/c mice by two-step perfusion method. After perfusion with Ca+2 free Hank’s/EGTA solution and digestion with collagenase solution (Millipore Sigma), the digested livers were filtered and the suspension was collected via centrifugation at 50 g at 4 °C. Dead cells were removed, the remaining cells were washed twice and cultured with small hepatocytes basal medium (SHM) [DMEM/F12 (high glucose, Hyclone) supplemented with 5 mM HEPES (Sigma), 10 ng epidermal growth factor (PeproTech), 1% ITS (Gibco), 30 mg/L L-proline (Sigma), 0.05% BSA (Gibco), 10–7 M dexamethasone (Dex) (Selleck), 1 mM ascorbic acid, 10 mM nicotinamide (Stem Cell), and 1% penicillin and streptomycin solution (Life Technologies)] supplemented with 10% FBS (Gibco). Purified mouse hepatocytes were then seeded on collagen-coated plates in SHM with or without the combination of the small molecule inhibitors, Y-27632 (Selleck), 0.5 mM A-83–01 (Selleck) and CHIR99021 (Selleck). One day after seeding, the medium was changed and every other day thereafter. We confirmed all procedures for hepatic and biliary functions described for mouse hepatic progenitors as mentioned in [31 (link)].
7
Episomal Reprogramming of Human Fibroblasts
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Yamanaka episomal plasmids used in experiments, including pCXLE‐hOCT3/4‐SHP53 (#27‐007), PCXLE‐hSK (#27078), pCXLE‐hUL (#27080), and pCXLE‐EGFAP (#27082) (encoding OCT4, SOX2, Lin28, L‐MYC, and KLF4). HDFs (5 × 105) were counted and resuspended in the Nucleofector solution supplied in the Amaxa Nucleofector kit (Lonza, Basel, Switzerland). The episomal plasmids were added to the cell suspensions at 10 μg each per reaction and were cotransfected using the program U‐023 on the Amaxa Nucleofector device. The cells were then transferred to a Matrigel‐coated 60‐mm culture dish and cultured in the fibroblast medium for 3 days, and then changed to N2B27 medium supplemented with bFGF (Invitrogen, Carlsbad, CA, USA), Y‐27632 (Sigma, St. Louis, MO, USA), CHIR99021 (Selleck, Houston, TX, USA), PD0325901 (Selleck), hLIF (Invitrogen), and A‐83‐01 (Selleck). On day 12, the medium was changed to E8. By 20‐30 days post‐transfection, clones were picked and cultured in E8 medium.
8
Culturing Primed Human Pluripotent Stem Cells
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HEK293T cells and ICR mouse embryonic fibroblast-derived feeders were maintained in DMEM (Corning) supplemented with 10% fetal bovine serum (NATOCOR). Primed human PSCs, including H9 ESCs and UH10 iPSCs50 (link),51 (link) were routinely cultured in mTeSRTM1 medium (STEMCELL). For passaging, primed PSCs were washed with DPBS (Hyclone) once and treated with 0.5 mM EDTA (Invitrogen, 15575020) for 5 minutes. Then, EDTA was removed and cells were passaged as small clumps using a Pasteur pipette. Human TSCBT were cultured in TSC medium15 (link) (DMEM/F12 supplemented with N2 and B27, penicillin-streptomycin, Glutamax, β-mercaptoethanol, 1.5 µg/ml L-ascorbic acid, 50 ng/ml EGF (PeproTech), 0.5 µM A83-01 (Selleck), 1 µM SB431542 (Selleck), 2 µM CHIR99021 (Axon), 0.8 mM VPA (Vetec) and 5 µM Y-27632 (Axon) supplemented with 10 µM Y-27632. Human H9 ESCs were purchased from WiCell Research Institute, human UH10 iPSCs were provided by Dr. Guangjin Pan (Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, China), and TSCBT were provided by Dr. Hiroaki Okae and Dr. Takahiro Arima (Department of Informative Genetics, Tohoku University Graduate School of Medicine). All cell lines were negative for mycoplasma.
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