Human recombinant basic fibroblast growth factor

To evaluate the ability of hCSCs to differentiate into ECs, hCSCs were cultured in a DMEM high glucose medium supplemented with 20% FBS, 1% P/S, and 30 ng/mL recombinant human basic fibroblast growth factor (R&D Systems) for 7 days. To differentiate hCSCs into smooth muscle cells (SMCs), hCSCs were cultured in Medium 231 supplemented with 5% FBS, 1% P/S, and 1× smooth muscle differentiation supplement (Gibco) for 7 days. The medium was changed every 2 days. To induce cellular differentiation into CM, hCSCs were cultured in a MEM/EBSS medium (HyClone) supplemented with 2% FBS, 1% P/S, and 10 nM dexamethasone (Sigma-Aldrich) for 28 days. The medium was changed every day. The ability of cells to differentiate was analyzed by qRT-PCR and immunofluorescence.

Mouse neurofibroma/DRG-derived sphere culture was performed as described (26 (link)). Specifically, we plated trypan blue–negative cells at 1 × 10⁴ cells per 24-well low-binding plates in 1 ml of medium containing Dulbecco’s modified Eagle’s medium (DMEM):F-12 (3:1) and recombinant human epidermal growth factor (20 ng/ml; R&D Systems), 20 ng/ml recombinant human basic fibroblast growth factor (R&D Systems), 1% B-27 (Invitrogen), and heparin (2 μg/ml; Sigma-Aldrich). We maintained cultures at 37°C and 5% CO₂. To passage, we centrifuged sphere cultures, dissociated and replated at 1 × 10⁴ cells/ml in fresh sphere medium as described (6 (link)).

Sub-cutaneous adipose tissue was obtained by outpatient tumescence liposuction under local anesthesia with patient consent. ASCs were isolated according to Wolbank et al. [27 (link)] and cultured in DMEM-low glucose/HAM's F-12 (GE-Healthcare) supplemented with 4mM L-glutamine (Sigma), 10% fetal calf serum (Sigma) and 1ng/mL recombinant human basic fibroblast growth factor (R&D Systems) at 37°C, 5% CO₂ and 95% air humidity. Cells were passaged once a week at a split ratio of 1:2 to 1:6 according to the growth characteristics.

Freshly dissociated melanoma cells were cultured in ultralow attachment plates (Corning) with ‘melanoma medium' containing 50% low-glucose Dulbecco's modified Eagle's medium, 30% Neurobasal (Invitrogen), 1% penicillin/streptomycin, 1% nonessential amino acids (Gibco), 50 mM 2-mercaptoethanol (Sigma), 1% N2 supplement, 2% B27 supplement (Gibco), recombinant human basic fibroblast growth factor (20 ng ml⁻¹) and insulin-like growth factor 1 (20 ng ml⁻¹) (R&D Systems).

The control pluripotent stem cell lines used in this study were H7 (hESC, WiCell), UTA.00112.hFF [fetal wild-type from ATCC (WT)-hiPSC], and UTA.01006.WT (WT-hiPSC derived from a healthy adult). For LQT1, 4 patient-specific hiPSC lines were used: UTA.00208.LQT1, UTA.00211.LQT1, UTA.00313.LQT1 and UTA.00303.LQT1. For LQT2 2 patient-specific hiPSC lines were used: UTA.00514.LQT2 and UTA.00525.LQT2. The pluripotent stem cells were cultured on a mouse embryonic fibroblast (MEF, Millipore) feeder cell layer in KSR medium consisting of KnockOut Dulbecco’s Modified Eagle’s Medium (KO-DMEM, Invitrogen) supplemented with 20 % KnockOut serum replacement (KO-SR, Invitrogen), 1 % non-essential amino acids (NEAA, Lonza), 1 % Glutamax (Invitrogen), 50 U/mL penicillin/streptomycin (Lonza), and recombinant human basic fibroblast growth factor (R&D Systems). The cells were passaged once a week by treating the pluripotent stem cell colonies with collagenase IV (Gibco) and seeding them onto a fresh MEF feeder layer.

Primary gliostoma cells were dissociated from cell culture dishes and resuspended in PBS with 0.5% BSA and 2mM EDTA and incubated with CD133/1 microbeads (Miltenyi Biotech) followed by positive magnetic cell separation using several MACS columns. The purified CD133⁺ cells were stained with anti- CD133/2-PE (Miltenyi Biotech) and analyzed on a BD FACSCalibur. Then, neurosphere cultures were obtained by growing CD133⁺ tumor cells in stem cell-permissive DMEM/F12 medium supplemented with 20ng/ml each of human recombinant epidermal growth factor and human recombinant basic fibroblast growth factor (R and D Systems), and human leukemia inhibitory factor (Chemicon) and 2% B27 (Life Technologies). These culture conditions enabled tumor cells to retain the molecular characteristics of the primary tumor, with only minor changes in differentiation, expression pattern, and genetic mutation profile.
Cultured primary astrocytes were generated from a slightly injured brain tissue fragment obtained after consent from a patient with cerebral trauma. The grey matter was dissected and dispersed repeatedly after washing in PBS. Primary astrocytes were cultured according to the protocol described by Darling et al [32].