Human pdgf bb

Human pulmonary arteriole samples were obtained based on the protocol approved by the Medical Research and Clinical Technology Application Branch of the Ethics Committee of the First Affiliated Hospital of Fujian Medical University [Approval No. MRCTA, FMU (2001) 483 ECFAH]. These samples were collected from 10 male patients, aged 60 ± 11 years, who had part of their lung lobes removed due to emphysema or lung abscess in the Department of Thoracic Surgery of the First Affiliated Hospital of Fujian Medical University. All participants have understood and signed the written informed consent. Meanwhile, we determined where to take the pulmonary arteries based on the lung lobe to be resected during surgery. Sterile ophthalmic scissors were used to cut the pulmonary arteries of patients into small pieces. Then, small pieces of pulmonary arteries were cultured in DMEM/F12 containing 20% FBS in a humidified environment of 5% CO₂ at 37°C (Wu et al., 2022 (link)). The medium was replaced by DMEM with 0.02% FBS and starved for 24 h when the cells were 70%–80% confluent. Treated with human PDGF-BB (Pepro Tech) (20 ng/mL) for 48 h, HPAMSCs were collected for the following testing.

Glioma-associated stem cells (GASC) were isolated from the three regions, displaying a progressive intensity of fluorescence for 5-aminolevulinc acid and maintained, in vitro, applying, with minor modifications, a protocol optimized for culturing multipotent adult stem cells from normal and neoplastic human tissues. Briefly, GBM fragments were first mechanically disaggregated with scalpels and then enzymatically dissociated, in a 0.025% Collagenase type II solution (Worthington) in Joklik modified Eagle’s Medium (Sigma-Aldrich, St.Louis, MO, USA) for 5 min at 37 °C. Collagenase activity was stopped by adding 10% Fetal Bovine Serum in Joklik modified Eagle’s Medium (Sigma-Aldrich). Cell suspension was centrifuged at 500× g for 10 min after being filtered through a sieve (BD Falcon, Franklin Lakes, NJ, USA) in order to select a population less than 40 μm in diameter. 2.0 × 10⁶ freshly isolated human cells were plated onto 100 mm diameter, human fibronectin (Sigma-Aldrich)-coated dishes (BD Falcon), in an expansion medium composed as follows: 60% low glucose DMEM (Invitrogen), 40% MCDB-201, 1 mg/mL linoleic acid-BSA, 10⁻⁹ M dexamethasone, 10⁻⁴ M ascorbic acid-2 phosphate, 1× insulin-transferrin-sodium selenite (all from Sigma-Aldrich), 2% foetal bovine serum (StemCell Technologies, Cambridge, UK), 10 ng/mL human PDGF-BB, 10 ng/mL human EGF (both from Peprotech EC, London, UK).

The HSJD-DIPG-007 cell line was established from DIPG tumor tissue issued at the Sant Joan de Déu Hospital (Barcelona, Spain). These cells express archetypal histone mutations H3F31 (H3.3), K27M-mutated protein (H3K27M), and the activin A receptor type 1 (ACVR1) R206H mutation frequently observed in patients with DIPG [14 (link),15 (link)]. Cells were cultured in serum-free DMEM-F12/Neurobasal-A (1:1, Gibco, Courtaboeuf, France), supplemented with HEPES (10 mM, Gibco), sodium pyruvate (1 mM, Gibco), non-essential amino acids (Gibco), glutaMAX-I supplement (Gibco), antibiotic-antimycotic (100 U mL⁻¹ penicillin, 100 µg mL^–1 streptomycin, 0.25 µg mL⁻¹ amphotericin B, Life Technologies, Courtaboeuf, France), B-27 supplement (vitamin A), human EGF (20 ng mL⁻¹, Peprotech, Neuilly-sur-Seine, France), human bFGF (20 ng mL⁻¹, Peprotech), human PDGF-AA (10 ng mL⁻¹, Peprotech), human PDGF-BB (10 ng mL⁻¹, Peprotech), and heparin (2 µg mL⁻¹, Sigma, St Quentin, France).

ARQ 092 was synthesized at ArQule, Inc. (Burlington, MA). ARQ 751 was synthesized at Daiichi Sankyo RD Novare Co. Ltd (Daiichi Sankyo Ltd, Japan). MK-2206 was synthesized by SAI Life Sciences (Hyderabad, India) or at Daiichi Sankyo RD Novare Co. Ltd. GDC-0068 was purchased from Selleckchem (Houston, TX). Paclitaxel was purchased from Bristol-Myers Squibb (New York, NY) and trastuzumab was purchased from Chugai Pharmaceutical Co. (Tokyo, Japan). Trametinib was purchased from LC Laboratories (Woburn, MA). AN3CA, BT-474, ZR-75-1, MDA-MB-453, NCI-H1650, NIH 3T3 and 293T cells were purchased from American Tissue and Culture Collection (Manassas, VA). KU-19-19 cells were purchased from Creative Bioarray (Shirley, NY). KPL-4 cells were kindly provided by Dr. J. Kurebayashi (Kawasaki Medical School, Okayama, Japan) [37 (link)]. Human PDGF-BB was purchased from PeproTech (Rocky Hill, NJ). Methyl cellulose 400cP solution was purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan) and DMA was purchased from Sigma Aldrich (St Louis, MO). All experimental mice were purchased from Taconic USA (Hudson, NY) or CLEA Japan, Inc. (Tokyo, Japan). All other reagents were of the highest grade and obtained from standard suppliers.

Pentobarbital (Narcoren) was purchased from Merial (Hallbergmoos, Germany), gelatin from porcine skin from Sigma-Aldrich and low melting point agarose from GERBU (Heidelberg, Germany). ET-1 was purchased from BIOTRENDS (Wangen, Switzerland). Glibenclamide, iberiotoxin, 4-aminopyridine and amlodipine were purchased from Tocris Bioscience (Ellisville, Missouri, USA). Human PDGF-BB was provided by Peprotech (Hamburg, Germany). Imatinib and nilotinib were kindly provided by Novartis (Basel, Switzerland). Standard laboratory chemicals were obtained from Sigma-Aldrich (Steinheim, Germany). The ELISA-kits were acquired from Enzo (Lörrach, Germany).