Osteogenic nodule formation from single embryonic stem cell-derived progenitors

The process of bone formation can be approximated in vitro in the form of a mineralized nodule. Osteoprogenitors and mesenchymal stem cells (MSCs), the immediate precursors of the osteoprogenitor, proliferate and differentiate into osteoblasts when placed into culture. These osteoblasts secrete and mineralize a matrix during a period of 3-4 weeks. The differentiation potential of embryonic stem (ES) cells suggests that ES cells should also have the ability to form osteogenic nodules in vitro. ES cells were allowed to form embryoid bodies (EBs) and were cultured in suspension for 2 days; EBs were disrupted and plated as single cells at concentrations as low as 25 cells/cm². We provide five lines of evidence for osteogenesis in these ES cell-derived cultures: (1) cell and colony morphology as revealed by phase-contrast microscopy, (2) mineralization of extracellular matrix as revealed by von Kossa staining, (3) quantitative real-time PCR (QRT-PCR) analysis of cDNA from entire plates and individual colonies revealing expression of genes characteristic of, and specific for, osteoblasts, (4) confocal microscopy of nodules from osteocalcin-green fluorescent protein (GFP) ES cell lines demonstrating the appropriate stage and position of osteoblasts expressing the reporter, and (5) immunostaining of nodules with a type I collagen antibody. Our method of initiating osteogenesis from ES cell-derived cultures is the only described method that allows for the observation and manipulation of the commitment stage of mesengenesis from single embryonic progenitors.