Human bone marrow derived mscs

Human bone marrow–derived MSCs (Lonza, Walkersville, MD) were used without further characterization. MSCs were expanded until use at passages 5 and 6 in minimum essential medium–α [α-MEM; with l-glutamine and without ribo/deoxyribonucleosides (Invitrogen, Carlsbad, CA)] supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA) and 1% penicillin (10,000 U ml⁻¹) and streptomycin (10 mg ml⁻¹; Mediatech, Manassas, VA) (P/S). Cell-secreted ECMs were prepared as we described (28 (link), 35 (link), 45 (link), 46 (link)). Briefly, MSCs were seeded at 50,000 cells cm⁻² and cultured in medium supplemented with ascorbate 2-phosphate (50 μg ml⁻¹) for 10 days with medium changes every 2 to 3 days. After culture, monolayers were washed with phosphate-buffered saline (PBS), and cells were removed using a detergent-based solution, followed by deoxyribonuclease (Sigma-Aldrich, St. Louis, MO) treatment (37°C for 1 hour) to remove 99.9% of DNA content from culture after decellularization (45 (link)). Decellularized ECM was washed three times with PBS and mechanically dislodged from culture flasks using a cell scraper. Total protein within the collected ECM was quantified using a bicinchoninic acid protein assay (Thermo Fisher Scientific, Rockford, IL). ECM solutions were frozen in 0.02 N acetic acid at −20°C until use.

Human bone marrow-derived MSCs (Lonza), serially passaged 4 times with or without 50 μg/mL HS8, were sent for karyotyping at the Cytogenetics Laboratory of the Singapore General Hospital. A sample with at least two cells with abnormal chromosomes would be reported as an abnormal karyotype.