Heparin sodium salt in pbs

The protocol of iPSCs differentiation into iNSCs involving EB formation was based on the method presented by Yuan et al. [17 (link)]. The whole procedure can be divided into several distinct steps. At approximately 80% confluency iPSCs colonies were firstly transferred from 4 wells of 6-well cell culture plate (Nunc, Thermo Fisher Scientific) coated with Geltrex to a 6 cm non-adhesive culture dish/T25 non-adhesive culture bottle (Sarstedt, Germany) and grown in suspension culture in Embryoid Body Formation medium (DMEM/F-12 with GlutaMAX-I (1X), KnockOut Serum Replacement 20%, 2-Mercaptoethanol 100 μM, Non Essential Amino Acids 1%) for 4 days. During the next 4 days, 5 × 10⁻⁷ M retinoic acid (Sigma-Aldrich) was added daily to the suspension culture. Next, 5 to 10 EBs were plated on Poly-L-Ornithine (20 μg/ml)/Laminin (10 μg/ml) (both from Sigma Aldrich)–coated 6-well adherent culture dishes (Nunc) and grown in Neural Stem Cell Induction medium (DMEM/F12 1X, B-27 Supplement 2% both from Life Technologies), supplemented with 10 ng/ml hLIF (Merck Milipore), 2 μg/ml heparin sodium salt in PBS (STEMCELL Technologies, Canada), 20 ng/ml EGF (Sigma Aldrich), 10 ng/ml bFGF for the following 7 days. After reaching 85% confluency, 0.5–1 × 10⁶ cells were seeded on Geltrex-coated dishes with ReNcell medium (Merck Millipore) supplemented with 20 ng/ml EGF and 20 ng/ml bFGF.

Human GBM samples were obtained from consenting patients, as approved by the Hamilton Health Sciences/McMaster Health Sciences Research Ethics Board. Brain tumor samples were dissociated as previously described [17 (link)] and cultured as neurospheres in Neurocult complete (NCC) media, a chemically defined serum-free neural stem cell medium (STEMCELL Technologies, Vancouver, BC, Canada), supplemented with human recombinant epidermal growth factor (20 ng/mL: STEMCELL Technologies, Vancouver, Canada), basic fibroblast growth factor (20 ng/mL; STEMCELL Technologies, Vancouver, Canada), heparin (2 μg/mL 0.2% Heparin Sodium Salt in PBS; STEMCELL Technologies, Vancouver, Canada), antibiotic-antimycotic (10 mg/mL; Wisent Bioproducts, Saint Bruno, QC, Canada) in ultra-low attachment plates (Corning, New York, NY, USA). Primary GBM cells (BT 428, BT 458 and BT 624) were cultured in NSC complete media and flow-sorted for CD133+ and CD133− populations as described previously [18 (link),19 (link)]. Transfections were carried out by Lipofectamine 2000 as per the manufacturer’s instructions.