Insulin-like growth factor 2-enhanced osteogenic differentiation of stem cell spheroids by regulation of Runx2 and Col1 expression.

Insulin-like growth factor 2 (IGF-2) is a growth factor that is involved in various functions of cells, including stem cells. The effects of IGF-2 on the cellular viability and osteogenic differentiation of stem cell spheroids were investigated in the present study. Stem cell spheroids were formed using concave microwells in the presence of IGF-2 at final concentrations of 0, 10 and 100 ng/ml. Cellular viability was measured qualitatively using a microscope and quantitatively using an assay kit based on water-soluble tetrazolium salt. The level of alkaline phosphatase activity, and an anthraquinone dye assay for calcium deposit evaluation, were used to assess osteogenic differentiation. A quantitative PCR analysis was conducted to evaluate the expression of Runx2 and Col1. Spheroid formation was noticed on day 1 in the microwells, and the spheroidal shape was maintained up to day 7. The cell viability assay values for IGF-2 at 0, 10 and 100 ng/ml at day 1 were 0.193±0.002, 0.191±0.002 and 0.201±0.006, respectively (P>0.05). The absorbance values at 405 nm for the alkaline phosphatase activity assays on day 21 were 0.221±0.006, 0.375±0.010 and 0.280±0.015 for IGF-2 at 0, 10 and 100 ng/ml, respectively. There were significantly higher values for IGF-2 in the 10 and 100 ng/ml groups when compared with the control (P<0.05). Significantly higher Alizarin red staining was noted for IGF-2 in the 10 ng/ml group when compared with the unloaded control at day 21 (P<0.05). Quantitative PCR revealed that mRNA levels of Runx2 and Col1 were significantly higher at 100 ng/ml on day 7. Conclusively, the present study demonstrated that the application of IGF-2 increased alkaline phosphatase activity, Alizarin red staining, and Runx2 and Col1 expression of stem cell spheroids.