Methods: To test for Spry2 expression, commercially obtained human MSCs were cultured in osteogenic medium. Spry2 expression was then knocked-down using RNA interference (RNAi). These cells were treated with osteogenic differentiation media for 2h, 24h, 2 days, 4 days and 6 days respectively. To show the effectiveness of the knock-down and to test the effects of Spry2 on osteogenic differentiation, RNA was extracted and qRT-PCR was performed to measure the relative gene expression levels of Spry2 and the osteoblastic marker genes Runx-2 and ALP.
Results: When MSCs were cultured in osteogenic medium, Spry2 expression was induced within 2h. After RNAi treatment Spry2 expression was reduced by 93 ± 4% and remained low for up to 5 days (55±37%) although by 7 days no consistent pattern of knock-down was seen. Culture of MSCs in osteogenic medium resulted in upregulated expression of Runx-2 and ALP. Simultaneous knock down of Spry2 resulted in 3 fold increase in Runx-2 expression compared with controls within 2 hours (311 ± 201 %) and by 143 ± 101% by 24 hours. No consistent pattern of effect of Spry2 knock-down was seen on ALP expression.
Conclusion: The results demonstrate that Spry2 knock-down stimulates Runx-2 expression significantly. These data suggest that Spry2 may be an important negative regulator of osteoblastic differentiation of MSCs.