Methods: With the treatment of purmorphamine in combination with the traditional osteogenesis-promoting medium comprising a cock-tail of β-glycerol phosphate, ascorbic acid, dexamethasone and vitamin-D, different time points of purmorphamine treatment was carefully studied in a 2-D in-vitro system starting from day 0 to day 14 in two days interval. Upon 3 weeks differentiation, the cellular proliferation was measured by MTS assay. The osteogenesis was characterized by osteo-lineage specific gene expression through PCR analysis, osteocalcin secretion using a Gla-type osteocalcin EIA kit, alkaline phosphatase secretion through PNPP substrate colorimetric assay, and mineral deposition with alizarin red staining.
Results: Purmorphamine treatment enhanced the cell number increment to over 1.7 folds comparing with control group at day 21. Starting the purmorphamine treatments on day 6 and 8 showed the greatest osteogenic potential than the treatments on other starting time points. At day 21, alkaline phosphatase levels of the cultures with purmorphamine treatment starting at day 6 and 8 in both media and cell lysate are significantly higher than other treatment starting time points. Similarly, in day-21 cell lysate, starting treatment at day 6 and 8 showed higher level of osteocalcin than other time points. Mineralization studies also supported our findings of PCR analysis from transcription level and secretion studies from protein level.
Conclusions: Purmorphamine is able to enhance direct osteogenesis from hESCs.