Gelatin

To examine different substrates’ effects on NPCs transdifferentiation of tdASC, the treated cell culture dishes (Corning) were coated with the following substrates (all obtained from Sigma-Aldrich except if otherwise mentioned): gelatin, poly-d-lysine, human laminin, type I collagen, and Matrigel (Corning). In brief, tdASC was dissociated with Accutase (Life Technologies) and plated on different matrix tissue culture plates (Corning Inc.) Matrix detail as follows: 0.1% gelatin was prepared with sterile ddH₂O and was used at 200 μL/cm². Type 1 collagen was used at 0.25~1 mg/cm². Poly-d-lysine was dissolved with sterile ddH₂O. The poly-d-lysine solution was added to plates at a concentration of 0.1–1 mg/mL and left at room temperature overnight. Laminin was prepared at 10–100 μg/mL in PBS, and laminin was diluted to 1 µg/mL in DPBS, and then 1 mL of solution added to 6 well-plates and keep at 37 °C for 2 h or at 4 °C for 24 h.
For laminin/poly-d-lysine coating as described above, dishes were pretreated with poly-d-lysine at room temperature overnight and then laminin was added at 1 μg/mL in DPBS at 37 °C for 2 h, then the excess substrate was removed and dishes were rinsed with PBS.

The migration or invasion Transwell assays were conducted using 24-well Gelatin- or Matrigel-coated Transwell chambers (8-μm pore size, Corning Coster). At 24 h after transfection, the cells were seeded into the upper chamber and cultured with OPTI-MEM (Gibco) without FBS in a humidified incubator at 37 °C. For the migration assay, the seeding density was 1 × 10⁵ cells/well. For invasion assay, the seeding density was 0.75 × 10⁵ cells/well. The bottom chamber contained the complete medium containing 20% FBS. After migration for 12 h or invasion for 24 h, the filters were rinsed with PBS and fixed in the Hema3^® fixative (Fisher Scientific) for 30 min. The non-invaded/non-migrated cells in the upper chamber were removed gently, and the filters were stained with the Hema3^® solution I and II for 30 min in accordance with the manufacturer’s instructions. Lastly, the filters were mounted onto the slides with gridded coverslips and counted under an optical microscope (OLYMPUS IX51).

HUVECs were seeded on a 100 mm TC-Treated Cell Culture Dish (Cat. No. 353003; Corning, NY, USA) coated with 0.1% gelatin (Cat. No. G1393; Sigma) and cultured to confluence in EGM-2 medium (Cat. No. cc3162; Lonza, Basel, Switzerland). Cell dishes were set up into a cone plate flow system that sheared at 15 dynes/cm² for laminar flow or ±5 dynes/cm² and 1 Hz frequency for oscillating flow for 24 h. The flow system was kept at 37 °C temperature and 95% humidified air with 5% CO₂. Cells were then washed in PBS and further processed for Western blot, RNA extraction, or flow cytometry analysis.