Cell Culture and FACS
Undifferentiated hESCs, H1 (WA-01, XY, passages 40–65) and H9 (WA-09, XX, passages 35–45), were cultured on mitotically inactivated mouse embryonic fibroblasts (Specialty Media, Phillipsburg, New Jersey, United States) and maintained under growth conditions and passaging techniques described previously [3]. OP9 cells were maintained in alpha MEM medium containing 20% fetal bovine serum (FBS) and 2 mM L-glutamine. Mesenchymal differentiation was induced by plating 10 × 103 to 25 × 103 cells/cm2 on a monolayer of OP9 cells in the presence of 20% heat-inactivated FBS in alpha MEM medium. Flow-activated cell sorting (FACS) (CD73-PE; PharMingen, San Diego, California, United States) was performed on a MoFlo (Cytomation, Fort Collins, Colorado, United States). All human ES cell–derived mesenchymal precursor cell (hESMPC) lines in this study are of polyclonal origin. Primary human bone marrow–derived MSCs and primary human foreskin fibroblasts (both from Poietics, Cambrex, East Rutherford, New Jersey, United States) were grown in alpha MEM medium containing 10% FBS and 2 mM L-glutamine.