The neuroectoderm differentiation was based on Douvaras and Fossati (2015 (link)). In brief, 90% confluent hESCs were subjected to neural induction for 8 days using 100 nM RA (Sigma-Aldrich), 10 μM SB431542 (Tocris), and 250 nM LDN193189 (STEMCELL Technologies). The induction of cardiac progenitor differentiation was based on Lin and Zou (2020 ). hESCs at 80%–90% confluence were treated with 5 mM CHIR99021 (Tocris) for 24 h, after which the cells were cultured in medium with 0.6 U/mL heparin (Sigma-Aldrich) for 24 h. Subsequently, the medium was supplemented with 0.6 U/mL heparin and 3 mM IWP2 (Tocris) for another 3 days. The 8-day depatoblast differentiation was based on Boon et al. (2020 (link)). Differentiation was initiated at 40%–50% confluency, and the hESCs were treated with with 50 ng/mL activin A (STEMCELL Technologies), 50 ng/mL WNT3A (PeproTech), and 6 μL/mL DMSO (Sigma-Aldrich) for 48 h. The cells were incubated for an additional 48 h in the same medium without WNT3A. Then, the medium was changed to contain 50 ng/mL BMP4 (STEMCELL Technologies) and 6 μL/mL DMSO for the following 4 days. More details on the differentiation and the media composition can be found in the supplemental information .
Heparin
Manufactured by Bio-Techne
Sourced in United States
Heparin is a naturally occurring anticoagulant used as a laboratory reagent. It is a glycosaminoglycan that inhibits several steps in the blood coagulation cascade.
Automatically generated - may contain errors
Lab products found in correlation
Hydrocortisone, by Bio-Techne (5 mentions)
Stemxvivo serum free tumorsphere media, by R&D Systems (2 mentions)
Stemxvivo serum free media, by R&D Systems (2 mentions)
Heparin, by Merck Group (1 mentions)
Chir99021, by Bio-Techne (1 mentions)
Sb431542, by Bio-Techne (1 mentions)
Ldn193189, by STEMCELL (1 mentions)
Activin a, by STEMCELL (1 mentions)
Wnt3a, by Thermo Fisher Scientific (1 mentions)
Wnt3a, by Merck Group (1 mentions)
Bone morphogenetic protein 4 (bmp4), by STEMCELL (1 mentions)
Dimethyl sulfoxide (dmso), by STEMCELL (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
T75 nunclon sphera easyflasks, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
12 protocols using heparin
1
Directed Differentiation of hESCs
2
Tumorsphere Formation Assay for Adherent Cell Lines
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Four adherent LA-derived primary cell lines were seeded for tumorsphere formation. StemXVivo serum-free tumorsphere media (cat# CCM012, R&D Systems, Minneapolis, MN, USA) was supplemented with 2U/mL Heparin (cat# 2812, Tocris, Auckland, New Zealand) and 0.5 µg/mL Hydrocortisone (cat# 4093, Tocris Bioscience, Bristol, UK). T75 Nunclon™ Sphera™ EasYFlasks (cat# 174952, ThermoFisher Scientific, Waltham, MA, USA) were seeded with 24 mL of media containing 1 × 104 live cells per mL as determined by cell counting using the Trypan Blue Exclusion method. They were incubated at 37 °C with 5% CO2 for 10 days or until initial signs of dark necrotic centers were observed. Feeding occurred every three to four days by the addition of 12 mL of tumorsphere media. Daily measuring was performed from day three by taking three representative images for each cell line. Five typical sphere-like structures from each image were measured in the longest and shortest dimension, and measurements used to establish a nominal average size for each sphere.
A cell line was considered positive for initial tumorsphere formation if the average sphere size was greater than 50 µm, and if at least 50% of measured spheres were greater than 50 µm, according to previously established criteria [44 (link)].
A cell line was considered positive for initial tumorsphere formation if the average sphere size was greater than 50 µm, and if at least 50% of measured spheres were greater than 50 µm, according to previously established criteria [44 (link)].
3
Isolation and Culture of Mouse NSCs
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NSCs were isolated from mouse embryonic cortex (E13.5) and cultured using the method of Reynolds and Weiss [32 (link), 33 (link)], with slight modifications. Briefly, forebrain cortices were cut into small pieces, incubated with accutase (Invitrogen, Carlsbad, CA, USA) and triturated through a 26-gauge needle. The pellets were suspended in growth medium [Dulbecco’s modified Eagle’s medium (DMEM):F-12 medium (DMEM/F12) (Invitrogen) supplemented with epidermal growth factor (EGF, 20 ng/ml), basic fibroblast growth factor (bFGF, 20 ng/ml), leukaemia inhibitory factor (LIF, 10 ng/ml; both from PeproTech, Rocky Hill, NJ, USA), heparin (2.5 μg/ml, Tocris Bioscience, Minneapolis, MN, USA), 2% B27 supplement, l -glutamine (1 mM), penicillin and streptomycin (both 100 U/ml) (all from Invitrogen)]. The cells were incubated in a humidified incubator at 37 °C with 5% CO2, and half the medium was exchanged every 2–3 days. After 5–7 days in vitro, the cells proliferated as free-floating neurospheres. Floating neurospheres were collected and passaged after enzymatic dissociation with accutase and mechanically triturated with fire-polished glass pipettes.
4
Tumorsphere Formation Assay
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Single cell was plated in ultralow attachment 6-well plates (Corning) at 25,000 cells per well for primary tumorsphere formation. After incubation for 7 days, mammospheres were collected and dissociated by trypsin. As the secondary tumorsphere formation, 200 cells per well were plated in ultralow attachment 96-well plates. Cells were grown in StemXVivo Serum-Free Media (R&D) containing 2 U/ml Heparin (Tocris) and 0.8 μg/ml Hydrocortisone (Tocris). Mammospheres were harvested 7 days later, and tumorsphere was calculated under inverted phase-contrast light microscope (Olympus). The experiment was repeated three times. Statistical difference on secondary tumorsphere formation was assessed using student T-test with p ≤ 0.05 as the significance criterion.
When the effect of IL6 on tumorsphere formation was examined, recombinant human IL6 (Peprotech) was supplemented at concentration of 0, 100, 200 ng/μl in the beginning of the primary and secondary tumorsphere formation stages.
When the effect of IL6 on tumorsphere formation was examined, recombinant human IL6 (Peprotech) was supplemented at concentration of 0, 100, 200 ng/μl in the beginning of the primary and secondary tumorsphere formation stages.
5
Neuroscience Reagent Acquisition Protocol
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The following reagents were purchased from the indicated sources: 1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), D-2-amino-5-phospho-valeric acid (D-APV) and 3-Aminopropyl(diethoxymethyl)phosphonic acid (CGP55845) from the Molecular, Cellular, and Genomic Neuroscience Research Branch (MCGNRB) of the National Institute of Mental Health (NIMH, Bethesda, MD, USA). STO609, U0126, U73122 and wortmannin from Labnet (Madrid, Spain). Leptin, heparin, isoguvacine, nifedipin, QX314, ruthenium red and SKF96365 from Tocris Cookson (Bristol, UK). Tetrodotoxin (TTX) from Abcam (Bristol, UK). 1.2- bis(2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and thapsigargin from Sigma (St Louis, MN, USA).
6
Tumorsphere Formation Assay
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The cells were suspended in StemXVivo Serum-Free Tumorsphere Media (R&D, USA), supplemented with 2 U/mL heparin (Tocris, USA) and hydrocortisone (Tocris, USA), then seeded at 3×104/well in 6-well ultralow adhesion culture plates (Costar, USA) and cultured at 37 °C with 5% CO2 for 7 days to form tumorspheres.
7
Colony Forming Assay for Cell Stemness
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Cells were plated in 6-well plates at a density of 4×105 cells/well in 2 mL of complete culture medium, and incubated with PAM for 24 h, harvested and dosed. 200 cells were plated in 200 μL StemXVivo Serum-Free Media (R&D) containing 2 U/mL heparin (Tocris), 0.8 μg/mL hydrocortisone (Tocris), 100 U/mL penicillin (Solarbio, Catalog# P1400), and 100 μg/mL streptomycin (Solarbio, Catalog# P1400) in ultra-low adherent 96-well plates (Corning). Colonies were quantified after 7 days.
8
Isolation and culture of endothelial progenitor cells
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For the isolation of mononuclear cells (MNCs), ~100 ml of circulating blood was obtained from each patient with DVT or healthy controls using BD Vacutainer EDTA tube (BD Biosciences) and immediately stored in the dark. The samples were processed following collection as follows: MNCs were extracted by density gradient centrifugation using Biocoll (Biochrom, Ltd.) at 5,003 x g at 4˚C for 20 min and washed three times using PBS. Isolated cells were plated on culture dishes that were pre-coated with human fibronectin (Sigma-Aldrich; Merck KGaA) and cultured in endothelial cell growth medium (GE Healthcare Life Sciences) supplemented with bovine brain extract (12 mg/ml), human epidermal growth factor (10 ng/ml), human insulin-like growth factor-1 (50 ng/ml), hydrocortisone (1 mg/ml) and streptomycin (100 µg/ml) and penicillin (100 U/ml; GE Healthcare Life Sciences) at 37˚C. Heparin (10 U/ml; Tocris Bioscience) was used to avoid platelet coagulation and cells were maintained in a humidified 5% CO2 atmosphere at 37˚C. After 3 days, floating cells were aspirated and the culture medium was replaced. EPC colonies formed after 1-2 weeks of culture. Medium was replenished every day for the first 7 days and every other day for the following 7 days. The medium was then replenished every 2-3 days. A total of two batches of cells were used in the subsequent experiments.
9
Prostate Cancer Tumorsphere Formation
10
Characterization of Platelet Activation
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ADP (Adenosine 5′-diphosphate sodium), prostacyclin PGI 2 , apyrase, PAF, ACD (acidcitrate-dextrose) anticoagulant and p-chlorophenylalanine (PCPA) were obtained from Sigma Aldrich, TNP (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) -BSA from Santa Cruz, thrombin from Roche, anti-GPIbα (polyclonal rat IgG) and isotype control from Emfret Analytics and heparin from Tocris. Human albumin and fibrinogen were provided by LFB Biomédicaments (2014-01029). Anti-FcγRIIA (mouse IgG2b,
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