presence of the priming medium the expanded and primed stem cells were then dissociated from the mef feeder cells following a brief approximately 3 minutes at 37 u00b0 c exposure to collagenase 200 u ml and nearly intact colonies of the primed stem cells were placed into suspension in the presence of the priming medium mef cm plus 4 ng ml bfgf for a maximum of 3 days the priming medium was replenished daily this step was performed in an effort to re establish and or increase the level of cell cell contacts potentially lost during dissociation but required for mesodermal induction commitment of the primed stem cells to mesodermal progenitor cells occurred with the subsequent exposure of the primed stem cells onto a fibronectin substrate formation of precardiac mesoderm explant mesa factor for murine embryonic stem cell co culture and or derivation of mesoderm conditioned medium at least 1 hour before dissection 15 mm diameter culture wells were incubated with 50 u03bcg ml fibronectin at room temperature the excess of which was replaced with 1 0 ml explant medium dmem 10 fbs 50 u03bcg ml gentamicin prior to the addition of explants explants of lateral plate mesoderm microdissected from hamburger amp hamilton stage 5 chick embryos were used briefly embryos were removed from the yolk and placed in pbs with ventral side up intact sheets of mesoderm were gently teased from underlying ectoderm following a 1 minute digestion in ice cold collagenase dispase 1 mg ml.