L ascorbic acid 2 phosphate aa

Murine-induced pluripotent
stem cells (iPSCs) (clone TαP4) were cultured on six-well plates
coated with 0.2% gelatin in a growth medium at 37 °C and 5% CO₂. The growth medium was replenished every other day. When
the confluency reached 60–70%, cells were trypsinized and seeded
onto the sterile scaffolds. IPSCs seeded on the scaffolds were cultured
with the growth medium. When cells reached confluency, the medium
was changed to a differentiation medium consisting of IMDM supplemented
with 20% FBS, 1% NEAA, 1% P/S, 0.1 mM β-ME, and 50 μg/mL l-ascorbic acid-2-phosphate (AA) (Sigma) to initiate cardiac
differentiation.^{30 (link),38 (link)} The differentiation medium was
replenished every day. Once beating cells were observed, the FBS content
of the differentiation medium was lowered to 5%, and AA was removed
from the differentiation medium. Cells were cultured with this medium
until the 14th day of culture. The medium was changed every day.^{39 (link)}

The osteogenic media was prepared by adding 10 mM β-glycerophosphate (β-GP, Sigma-Aldrich, Australia), 50 μg/mL L-ascorbic acid 2-phosphate (AA, Sigma-Aldrich, Australia), 10 nM dexamethasone (DEX, Sigma Aldrich, NSW, Australia), 10% (v/v) FBS, 1% (v/v) P/S into DMEM. hBMSCs were seeded in 24-well plates at a density of 2.5×10⁴/cm². After the cell was attached to the plate, culture media was refreshed with the mixture of conditioned media from BG/PBG treated macrophages and fresh osteogenic media at a ratio of 1:1. Media were refreshed every three days.