The Effects of Transforming Growth Factor-β1 on the Differentiation of Cell Organoids Composed of Gingiva-Derived Stem Cells

Abstract

This study was aimed at evaluating the effects of transforming growth factor-β on the differentiation and mRNA expression of organoids made out of human mesenchymal stem cells. Cell organoids composed of gingiva-derived stem cells were cultured in the presence of transforming growth factor-β1 at concentrations ranging from 0, 1, 10, to 20 ng/ml. Evaluations of the cell morphology of the organoids were performed on days 7, 9, 11, and 14. Quantitative cellular viability was completed on day 14. Alkaline phosphatase activity assays were performed to evaluate the differentiation of stem cells on day 14. Real-time polymerase chain reactions were used to determine the expression levels of TGF-β1, RUNX2, OCN, SOX9, and COL1A1 mRNA on day 14. The stem cells produced well-formed organoids on day 7, and the addition of transforming growth factor-β1 did not result in relevant changes in their shape. The organoids grew in size and became more intact with longer incubation times. On day 14, the diameters were $222.2\pm 9.6$ , $186.1\pm 4.8$ , $197.2\pm 9.6$ , and $211.1\pm 19.2$  m for transforming growth factor-β1 at final concentrations of 0, 1, 10, and 20 ng/ml, respectively. Quantitative cell viability results from day 14 exhibited no significant difference between the groups ( $P>0.05$ ). There was significantly higher alkaline phosphatase activity with the addition of transforming growth factor-β1 with the highest value for the 1 ng/ml group ( $P<0.05$ ). Real-time polymerase chain reaction results demonstrated that the mRNA expression levels of RUNX2, OCN, and SOX were higher in 1 ng/ml but did not reach statistical significance. Treatment with 1 ng/ml of transforming growth factor-β1 significantly increased COL1A1 mRNA expression at day 14. The application of transforming growth factor-β1 increased differentiation, which was confirmed by alkaline phosphatase activity and mRNA expression while maintaining cell viability.