Calcein

calcein injection method was conducted for the MAR measurement. calcein is a fluorescent dye that binds to and labels newly mineralized bone, indicating mineral apposition between the 2 calcein injection time points (Castro et al., 2019 (link); Tompkins et al., 2023a (link)). Briefly, one bird from each pen was injected subcutaneously with 20 mg/Kg of calcein (Sigma Aldrich, St. Louis, MO) solution based on body weight at 3 DPI (d 17) and 7 DPI (d 21). At 10 DPI (d 24), the birds were euthanized, and the right tibias were removed, cleaned from the adherent tissue, and fixed in 70% ethanol. For analysis, a bone section of approximately 0.5 mm was cut from the mid-diaphysis using a circular saw (Ryobi, Anderson, SC). The slides were then examined under a compound microscope (BZ-Z800, Keyence Inc., Itasca, IL), and images were taken at 4× magnification using a fluorescent light (BZ-Z800, Keyence Inc., Itasca, IL). The distance between the 2 calcein labels was measured using BZ-800 Analyzer (BZ-Z800, Keyence Inc., Itasca, IL).

The iPSCs were dissociated and plated on Matrigel (human embryonic stem cell-qualified Matrigel, BD, USA) coated 96-well plates at 20,000 cells per well in 100 μl of the Nutristem medium containing 10 μM Y-27632 (Nacalai Tesque). On day 1, an additional 100 μl of the Nutristem medium with the drug(s) was added to each well. On day 2, 100 μl of the Nutristem medium with 4 μg/ml calcein-AM (Dojindo, Japan) was added to each well, and the plate was incubated for 30 min at 37 °C. The cells stained with calcein were imaged by a fluorescence microscope (BZ-X800, KEYENCE, Japan). After thresholding the images, the stained cell areas were calculated by using Image J (https://imagej.nih.gov/ij/index.html, NIH, USA). 2-Deoxy-d-glucose (2DG, Sigma), JX 06 (Tocris, USA), sodium dichloroacetate (DCA, Sigma), 2, 2-dichloroacetophenone (DAP, Sigma), and taurine (Sigma) were added to the culture medium when needed.