Basic fibroblast growth factor (bfgf)

The iPSC lines were differentiated into CNCC as previously described^{24 (link)}. In brief, iPSCs were treated with CNCC Derivation media: 1:1 Neurobasal medium/D-MEM F-12 medium (Invitrogen), 0.5× B-27 supplement with Vitamin A (50× stock, Invitrogen), 0.5× N-2 supplement (100× stock, Invitrogen), 20 ng/ml bFGF (Biolegend), 20 ng/ml EGF (Sigma-Aldrich), 5 µg/ml bovine insulin (Sigma-Aldrich) and 1× Glutamax-I supplement (100× stock, Invitrogen). Medium (3 ml) was changed every day. Three days after the appearance of the migratory CNCC, cells were detached using accutase and placed into geltrex-coated plates. The early migratory CNCCs were then transitioned to CNCC early maintenance media: 1:1 Neurobasal medium/D-MEM F-12 medium (Invitrogen), 0.5× B-27 supplement with Vitamin A (50× stock, Invitrogen), 0.5× N-2 supplement (100× stock, Invitrogen), 20 ng/ml bFGF (Biolegend), 20 ng/ml EGF (Sigma-Aldrich), 1 mg/ml bovine serum albumin, serum replacement grade (Gemini Bio-Products # 700-104 P) and 1× Glutamax-I supplement (100× stock, Invitrogen).

For the scratch wound healing assay (n = 3 individual replicates), H5V cells were plated on a 12 well plate and grown until 80% confluence in complete culture medium (DMEM GlutaMAX (Invitrogen, GIBCO, Auckland, New Zealand), 10% heat inactivated fetal bovine serum (PAA), 1% penicillin/streptomycin). Cells were then treated with low serum medium (DMEM GlutaMAX (Invitrogen, GIBCO, Auckland, New Zealand), 0.1% heat inactivated fetal bovine serum (PAA), 1% penicillin/streptomycin) for 24 h. After 24 h, medium was removed and a scratch-wound was introduced across the diameter of each well of a 12 wells plate using a p200 pipette tip. Subsequently, the cells were washed with PBS and medium was replaced by new low serum culture medium containing low serum medium plus 5 ng/ml bFGF (Ref.579606, Biolegend), and low serum medium plus 5 ng/ml bFGF (Ref.579606, Biolegend) with the addition of either 50 or 100 µM K5. Two locations along the scratch-wound were marked per well and scratch-wound closure at these sites was imaged by taking pictures at time 0 h and 18 h after scratch-wound introduction using live phase contrast microscopy (Axiovert 40C, Carl Zeiss Microscopy, White Plains, NY, USA). Average scratch-wound closure after 18 h was objectively calculated per well by measuring difference in cell coverage at 18 h vs 0 h using the wound healing tool macro for ImageJ.

As modified from a published method⁵⁶ , brains were obtained from wild-type or Cav-1^−/− mice at the age of 6–8 weeks with cerebellum and meninges removed. After homogenization, the homogenate of brains was centrifugated at 150 × g for 5 min. The pallet was then resuspended in a 15% dextran solution (mol wt 60,000–76,000, Sigma-Aldrich) and centrifugated at 400 × g to isolate blood vessel fragments. The pallet of fragments was digested in collagenase/dispase (1 mg/ml, Roche) and DNase I (10 μg/ml, Roche) at 37 °C. The dissociated BMECs were washed and seeded onto coated plates in DMEM/F12 media with 20% FBS, 1% PS, 1% endothelial cell growth supplement (SclenCell), 1% l-glutamine, 1% heparin, and 2 ng ml⁻¹ bFGF (Biolegend). In vitro hypoxia was induced by either chronic CoCl₂ (10 μM, Sigma-Aldrich) for 5 consecutive days or OGD treatment for 4 h. Primary mouse OPCs were isolated from pups of postnatal days 0–2 as reported^{57 (link)}. In brief, mixed glial cells were cultured in DMEM/F12 with 10% FBS and 1% PS for 7 days. Then the flasks were shaken on the orbital shaker at 200 × g for 1 h. After washed, the flasks continued to shake for another 18–20 h at 4 × g. OPCs were obtained and plated onto coated plates in DMEM/F12 media containing 1% BSA (Gibco), 20 ng ml⁻¹ ITSS (Roche), 20 ng ml⁻¹ PDGF-BB (Biolegend) and 20 ng ml⁻¹ bFGF (Biolegend).

Cell proliferation (n = 3 individual replicates) was measured using MTT assay. H5V cells were plated at 5.000 cells/well in a 96 well plate and grown until 80–85% confluency in complete culture medium (DMEM GlutaMAX (Invitrogen, GIBCO, Auckland, New Zealand), 10% heat inactivated fetal bovine serum (PAA), 1% penicillin/streptomycin, after which they were incubated with low serum medium (DMEM GlutaMAX (Invitrogen, GIBCO, Auckland, New Zealand), 0.1% heat inactivated fetal bovine serum (PAA), 1% penicillin/streptomycin) for 24 h. The medium was then replaced by treatment mixes consisting of low serum medium plus 5 ng/ml bFGF (Ref.579606, Biolegend), low serum medium plus 5 ng/ml bFGF (Ref.579606, Biolegend) with the addition of either 50 or 100 µM K5. After 24 h incubation, 10µL MTT (Thiazolyl blue tetrazolium bromide, Sigma M5655) was added directly to each well and cells were incubated at 37 °C in a humidified 5% CO₂ environment for 4 h. Subsequently, 75µL medium was removed from each well and 75µL isopropanol/0.1 N HCL was added per well. After incubating the plate for 90 min on a shaker platform, absorbance was read at 570 nm with a Cytation 5 spectrophotometer (BioTek, Vermont, USA).

NSCs were harvested from embryonic C57BL/6J mouse brain (E14) as previously described [14 (link)]. Briefly, cells were resuspended in a DMEM/F12 medium containing B27 (2%, Gibco, USA), EGF (20 ng/ml, BioLegend, USA), bFGF (20 ng/ml, BioLegend, USA), and ITSS (10 μg/ml, Roche, Switzerland) and cultured in a cell incubator. The medium was changed every 3 d, and cells were passaged when the neurospheres grew to 50-100 μm diameter. Cells were plated on poly-D-lysine and laminin precoated coverslips for further experiments.

Sorted myoepithelial cells were centrifuged and resuspended in 1:20 Matrigel (cat. 354234; Corning) and cultured in advanced-DMEM/F12 (cat. 12634010; Life Technologies) supplemented with 10 ng/ml EGF (cat. 585506; Biolegend), 20 ng/ml bFGF (cat. 710304; Biolegend), 4 μg/ml heparin (cat. H3149-10KU; Sigma-Aldrich), 5% newborn calf serum (cat. SH3011803; HyClone), and 5 μM Y-27632.
AT-3 cells, a murine breast cancer cell line derived from MMTV-PyMT tumors in the C57Bl/6 background, were cultured at 7% CO₂ in DMEM high glucose (cat. MT-10-013-CV; Corning) supplemented with 10% FBS premium-select, penicillin–streptomycin (cat. MT30002CI; Corning), 15 mM HEPES (cat. 15630080; Life Technologies), 2 mM l-glutamine (cat. SH3003401; HyClone), NEAA (cat. SH3023801; HyClone), 1 mM sodium pyruvate (cat. 13-115E; Lonza Walkersville), and 1:250,000 2-mercaptoethanol (cat. M6250-100ML; Sigma Aldrich).