Laminin A

ES cells and neural differentiation are detailed elsewhere [38 (link)]. The LC1 and other ES cell–derived NS cells were routinely generated by re-plating d 7 adherent neural differentiation cultures (typically 2–3 × 10⁶ cells into a T75 flask) on uncoated plastic in NS-A medium (Euroclone, Milan, Italy) supplemented with modified N2 [36 (link)] and 10 ng/ml of both EGF and FGF-2 (NS expansion medium). Over 3–5 d, cells formed aggregates that, after harvesting and sedimentation to remove debris, subsequently attached to fresh plastic and outgrew NS cells. After addition of 0.5 μg/ml of puromycin to differentiating adherent cultures at d 7, 46C-NS cells were generated. Cells were re-plated 3 d later into an uncoated T75 flask in N2B27 media with 10 ng/ml of both EGF and FGF-2 (Peprotech, Rocky Hill, New Jersey, United States) in the absence of puromycin. To derive clonal lines, including NS-5, single cells were plated into 96-well microwell plates (Nalge Nunc International, Rochester, New York, United States) by limiting dilution. and the presence of one cell per well was scored 1 h after plating.
For derivation directly from foetal CNS, primary cultures were generated using standard protocols from cortex or striatum of E16.5 mouse embryos and subsequently allowed to attach on flasks treated with 0.1% gelatin. Outgrowing cells were then expanded on gelatin using NS expansion medium. Clonal derivatives of the cortical line Cor-1 were established by plating at very low density (1,000 cells per 9-cm plate) and expanding individual colonies.
For derivation from established neurospheres, derived from E14 foetal brain and maintained for 40 passages in EGF plus FGF-2, cultures were dissociated to single cells using Accutase (Sigma, St. Louis, Missouri, United States) and plated at 10⁴ cells/ml on gelatin-coated culture flasks in NS expansion medium.
For passaging established NS cell lines, we routinely used trypsin/EDTA or PBS and split cells 1:3 to 1:5 every 2–3 d. For astrocyte differentiations, NS cells were re-plated onto 4-well plates at 1 × 10⁵ cells/well in NS-A medium supplemented with 1% fetal calf serum or 10 ng/ml BMP4 (R&D Systems, Minneapolis, Minnesota, United States). For neuronal differentiation, 5 × 10⁴ NS cells were plated into poly-ornithine/laminin treated wells in NS-A supplemented with FGF-2 alone. After 7 d, the media was switched to NS-A supplemented with B27 (GIBCO, San Diego, California, United States) without growth factor. Half of the medium was exchanged every 2–3 d during the differentiation. For clonal differentiation, 1,000 cells from NS-5 or Cor-1, cultures were plated in 10-cm plates pre-treated with laminin, expanded for 12 d in EGF/FGF-2, and differentiated in situ as above. For electrophysiological studies, 1.5 × 10⁵ NS cells were plated into poly-L-ornithine-treated 35-mm dishes in NS-A medium supplemented with N2 and B27 (both at 0.5%) and FGF-2 (5 ng/ml). After 7 d, the medium was switched to the mix NS-A:Neurobasal (1:1), supplemented with B27 (GIBCO) without growth factors. To sustain neuronal maturation, after a further 7 d, the medium was switched to the mix NS-A:Neurobasal (1:3) supplemented with B27 (GIBCO) and brain derived neurotrophic factor (20 ng/ml) and nerve growth factor (R&D Systems; 50 ng/ml). Throughout neuronal differentiation, half of the medium was replaced every 2–3 d. Further details of NS cell derivation, propagation, and differentiation are provided in Protocol S1.

For preparation of the laminin‐coated plate, flat‐bottom 96‐well microplates (Corning) were incubated with highly purified and refined laminin‐511 E8 fragments (Easy imatrix‐511, Matrixome Inc) at 0.5 μg/cm². After overnight incubation at 4°C, the excessive laminin was aspirated. For the laminin‐uncoated plate, the plate was similarly incubated with PBS only. Following the instructions provided by the respective manufacturer, each cell line was preincubated with 25 μg/mL of anti‐CD49f (#313614, BioLegend),
³⁰ (link) 12.5 μg/mL of anti‐CD29 (#921304, BioLegend),
³¹ (link) or 10 μg/mL of anti‐CD104 (#325‐820, Ancell) neutralizing antibodies or isotype control rat IgG2α,κ (#400502, BioLegend) (CD49f and CD29 blocking) or mouse IgG (#6602398, Beckman Coulter) (CD104 blocking) for 30 min. To verify the effect of imatinib on laminin adhesion, parental KOPN55bi cells or T315I‐acquired KOPN55biR cells
²⁸ (link) were preincubated with 0.5 μM of imatinib for 7 days. After washing with PBS, the cells were resuspended in the medium at 0.5 × 10⁶/mL, and 100 μL of the cells was plated into each well of the laminin‐coated 96‐well plate in triplicate and allowed to attach to laminin for 3 h. After 3‐hour incubation of the cells on the plate with or without laminin coating, the plate was mechanically shaken using a plate mixer (Mikura Orbis Personal Single Plate Shaker, Mikura, West Sussex, UK) at 1300 rpm for 15 s. Then, the numbers of floating cells in the supernatant were evaluated after Trypan blue exclusion staining under an optical microscope. Meanwhile, the cell nuclei attached to the plate were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries) for 20 min and stained by 1 μg/mL DAPI (#DM037, Dojindo Laboratories) for 20 min. In each cell nuclei photograph taken under a fluorescence microscope at 100‐fold magnification, three independent areas of 200 μm in diameter were evaluated by GIMP software (Free Software Foundation Inc.), and the numbers of attached cell nuclei were analyzed with ImageJ software (free software).

Immunohistochemistry was performed using a standard protocol. Briefly, brain sections were fixed in 4% PFA for 15 min at room temperature. After extensive washes, brain sections were blocked in blocking buffer (5% normal donkey serum, 3% BSA, 0.3% Triton X-100 in PBS) for 1 h and incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: anti-Laminin-α1 (1:200, R&D, MAB4656), anti-Laminin-α2 (1:300, Sigma, L0663), anti-Laminin-α4 (1:300, R&D, AF3837), anti-Laminin α5 (1:800, a generous gift from Dr. Jeffrey Miner), anti-Nidogen (1:200, Invitrogen, MA1-06501), anti-Collagen IV (1:400, Novus, NB120-6586), anti-Claudin-5 (1:400, Invitrogen, 34-1600), anti-Claudin-5 (1:200, Invitrogen 35-2500), anti-ZO-1 (1:400, Invitrogen, 61-7300), anti-AQP-4 (1:500, Millipore, AB3594), anti-PDGFRβ (1:400, Cell Signaling, 3169 S), anti-Hemoglobin (1:500, Cloud-Clone, PAB409Mu01), anti-Caveolin-1(1:400, Cell Signaling, 323AS), rabbit anti-MFSD2A (1:200, a generous gift from Dr. Chenghua Gu), and anti-Podocalyxin (1:400, R&D Systems, AF1556). Brain sections were then washed in PBS and incubated with appropriate fluorescent secondary antibodies (Invitrogen) for 1 h at room temperature. After extensive wash, the sections were mounted with DAPI Mount. All fluorescent images were captured using Nikon Eclipse Ti and/or LSM 710 confocal microscopes. Imaging processing was performed using ImageJ and Adobe Photoshop.

The Laminin-α5^flox/flox mice [24 (link)] and platelet-derived growth factor receptor beta (PDGFRβ)-Cre⁺ mice [25 (link)] were generous gifts from Drs. Jeffrey Miner and Volkhard Lindner, respectively. The Tie2-Cre⁺ (Jax:008863) mice were obtained from the Jackson Laboratory. The α5-TPKO (Laminin-α5^flox/flox;PDGFRβ-Cre⁺;Tie2-Cre⁺) mice were generated by crossing the above three mouse lines. Since PDGFRβ-Cre⁺;Tie2-Cre⁺ mice failed to show obvious defects and are indistinguishable from Laminin-α5^flox/flox mice, both were used as controls for α5-TP-KO mice. All mice were maintained on a C57Bl6 background. Mice (both sexes) at the age of 4∼6 months were used in this study. Mice were housed in the animal facility of the University of South Florida and provided with ad libitum access to food and water. The experimental protocols and procedures of this study were approved by the Institutional Animal Care and Use Committee at the University of South Florida and were conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.