Nucleotides metabolizing ectoenzymes as possible markers of mesenchymal stem cell osteogenic differentiation

Growing murine mesenchymal stem cells (mMSCs) from mouse bone marrow decreased their rate of proliferation in the presence of benzoylbenzoyl-ATP persistently, but the inhibitory effect of ATP was strong only in a concentration of 50 μmol·L(-1) and lasted for 48 h in culture. These results hinted at ATP hydrolysis by the cell surface enzymes at the lower concentrations and thus it may be not able to inhibit MSCs. By using ATP, ADP, or AMP as substrates, we tested the ectonucleotidase activity on the surface of undifferentiated MSCs and MSC-derived osteoblasts. Here, we report that although nucleoside triphosphate diphosphohydrolase (NTPDase)1 and NTPDase8 are engaged in the metabolism of ATP in MSC-derived osteoblasts, NTPDase3 is responsible for its metabolism in undifferentiated MSCs. In this study, we also realized that osteoblasts effectively metabolize ADP to ATP and AMP. The enzymatic activity of adenylate kinase (AK) is consistent with the high expression level of the AK gene. Therefore, it was tempting to suggest that this enzyme, together with NTPDase1 and NTPDase8, assume the role of specific markers that allowed distinction between differentiated osteoblasts and early undifferentiated MSCs. Additionally, unlike osteoblasts, undifferentiated MSCs demonstrated the activity of 5'-nucleotidase (CD73). However, the expression analysis of CD73 mRNA did not show any differences; CD73 mRNA was expressed in both kinds of cells to the same extent.