Porcine heparin sodium salt

Purified recombinant EhSWP1 (20 μg) or 20 μg of bovine serum albumin (Sigma-Aldrich, Massachusetts, USA) were mixed with 50 μl of pre-equilibrated heparin-sepharose beads (50% slurry) with 1× PBS (GE Healthcare, Buckinghamshire, UK) at 4 °C for 1 h with radial rotation. For the heparin competition assay, various concentrations (0.1, 1, 10 and 100 mg/ml) of porcine heparin sodium salt (Sigma-Aldrich, Massachusetts, USA) were mixed with recombinant EhSWP1 prior to incubation with heparin-sepharose beads. The beads were then washed three times with 1× PBS (5 min incubation in each washing step). Proteins were eluted with elution buffer (2 M NaCl in 1× PBS). All protein fractions were visualized by 12.5% SDS-PAGE with Coomassie blue staining. To quantify the level of heparin binding, the intensity of the protein band was quantified using Scion Image software (Version 4.0). Level of heparin binding in the group without competitor (0 mg/ml heparin group) was used for normalization.

BMSC were isolated from bone marrow aspirates collected from a consenting healthy male (BMSC Donor 1) and female (BMSC Donor 2), both volunteers in their twenties. The collection was performed with ethical approval from the Mater Health Services Human Research Ethics Committee and Queensland University of Technology Human Ethics Committee (Ethics No.: 1300000833). BMSCs were enriched from the donors’ bone marrow by a density gradient separation using Ficoll-Paque (GE Healthcare). Mononuclear cells were suspended in low glucose Dulbecco’s Modified Eagle Medium (LG-DMEM) supplemented with 10% FBS, 100 U/mL penicillin/streptomycin (Pen/Strep; Gibco), 10 ng/mL fibroblast growth factor-1 (FGF-1; Peprotech) and 5 μg/mL porcine heparin sodium salt (Sigma-Aldrich). Cells were cultured overnight in a 20% O₂ and 5% CO₂ atmosphere at 37°C as described previously.^{26 (link),34 (link)} The next day, the medium and non-adherent cells were aspirated from cultures, and fresh medium added, and the cultures were incubated in a 2% O₂ and 5% CO₂ atmosphere at 37°C. When the cells reached ~80% confluency, they were passaged using 0.25% (V/V) Trypsin-EDTA in PBS (Thermo Fisher) and reseeded into new T175 flasks at a density of approximately 1500 cells/cm². BMSCs from the third passage were used in the subsequent studies.