Robust generation of osteoclast progenitors (OCPs) from hESCs and mCherry hESCs. (A) After 20 days of EB differentiation in the 6-cytokine cocktail, EBs were enzymatically dissociated and analyzed for the expression of CD45 and CD34. Cells were plated back into media containing SCF, FLT3L, and MCSF. Expanded floating cells were removed from the dish and analyzed for expression of CD14. Cultures were continued in tissue culture wells with and without dentine bovine slices along with the addition of RANKL. Between 6 and 10 days after RANKL addition, wells or slices were stained for TRAcP and osteoclasts evaluated as multinucleated TRAcP positive cells. (B) TRAcP⁺ osteoclasts (low and high magnification) grown on tissue culture dishes and (C) on top of dentine slices. (D) Toluidine blue-stained dentine slices after OC removal identifies numerous resorption pits. (E) Zinc finger nuclease-derived ubiquitin-mCherry hESC cell line was evaluated by flow cytometry for mCherry and CD45 after 20 days in EB 6-cytokine cocktail (F) and then switched to osteoclast inducing media which lead to multinucleated mCherry+osteoclasts and mCherry+progenitors.