Bm mscs

Placental pericytes (Pl-PCs; PromoCell) and bone marrow derived mesenchymal stromal cells (BM-MSCs; PromoCell) were thawed and seeded into untreated/uncoated flasks. Pl-PCs were seeded at a density of 3–4 × 10⁴ cells per cm² into 1 T-150 flask. Pl-PCs were cultured and expanded in Pericyte Growth Medium (PromoCell). BM-MSCs were seeded at a density of 4 × 10⁴ cells per cm² into 1 T-150 flask. BM-MSCs were cultured and expanded in Mesenchymal Stem Cell Growth Medium (PromoCell). Both Pl-PC and BM-MSC cultures were fed once every 2–3 days and cells passaged at 80–90% confluency.

Human bone marrow derived mesenchymal stem cells (bm-MSCs) were purchased from Promocell at Passage 2 and were cultured with MSC Growth Medium 2 (Promocell). Cells were plated in T75 flasks at a density of 4000 cells/cm². Cells were cultured in incubators at 37 °C and 5% CO₂. Cell culture medium was changed every other day. Cells were passaged when 80–90% confluency was reached, using Accutase solution (Promocell) for detachment. Cells of passage 3 to 6 were used for experiments.
Human amnion-derived mesenchymal stem cells (ha-MSC) were provided by Dr. Kenichi Yamahara [40 ]. Their marker expression (positive for CD73 and CD29, negative for CD45 and CD34), as well as their ability to differentiate into adipocytes and osteocytes, at least up to passage 20, was previously established in our lab [9 (link)]. Cells were defrosted from cryopreservation vials at Passage 10 and seeded in T75 flasks at a density of 10,000 cells/cm², and cultured in aMEM (Gibco) with 10% fetal bovine serum (FBS), supplemented with 1% penicillin and streptomycin and 10% of human basic fibroblast growth factor (bFGF, 10 ng/mL). Cells were cultured in incubator at 37 °C and 5% CO₂. Cell culture medium was changed every other day. Cells were passaged when at 80%–90% confluency by detachment using 0.25% Trypsin with 0.2% EDTA (Sigma). Passage 11 to 14 cells were used for experiments.