through a conventional microscope the cells showing the morphology typical of said cell type the same assay was performed but extending its period to 21 days the medium was changed every three days in the same way as in the previous case osteocalcin fig1 c osteonectin fig1 b and aphl fig1 a since they are genes specific for bone tissue and nanog were determined every week the concentration of osteocalcin osteonectin and aphl increases over the weeks mesodermal differentiation being demonstrated the disappearance of the expression of the gene nanog over three weeks of differentiation is also analyzed human bone cdna ambion was used as the positive control and gadph as the loading control hk the primers used in the amplification were the following for aphl seq id no 13 5 u2032 gaaggtgaaggtcggagtca3 u2032 as the forward primer and seq id no 14 5 u2032 tggactccacgacgtactca3 u2032 as the reverse primer for osteocalcin the same as in the previous case seq id no 7 and seq id no 8 and for osteonectin seq id no 15 5 u2032 aggtatctgtgggagctaatc3 u2032 as the forward primer and seq id no 16 5 u2032 attgctgcacaccttctc3 u2032 as the reverse primer for nanog the forward primer was seq id no 5 and the reverse primer was seq id no 6 the rt pcr was performed in cfx96 bio rad using 50 ng of cdna and sybr green.