Fibroblast growth factor-4 maintains cellular viability while enhancing osteogenic differentiation of stem cell spheroids in part by regulating RUNX2 and BGLAP expression

Abstract

Fibroblast growth factors (FGFs) are growth factors that were initially identified as proteins that stimulate fibroblast proliferation. The aim of the present study was to examine the effects of FGF-4 on the morphology, cellular viability and osteogenic differentiation of stem cell spheroids. Stem cell spheroids were generated using concave microwells in the presence of FGF-4 at concentrations of 0, 50, 100 and 200 ng/ml. Cellular viability was qualitatively assessed by a fluorometric live/dead assay using a microscope and quantitatively determined by using Cell Counting Kit-8. Furthermore, alkaline phosphatase activity and calcium deposition were determined to assess osteogenic differentiation. Reverse transcription-quantitative PCR (RT-qPCR) was performed to evaluate the mRNA expression levels of Runt-related transcription factor 2 (RUNX2) and bone γ-carboxyglutamate protein (BGLAP). Spheroidal shapes were achieved in the microwells on day 1 and a significant increase in the spheroid diameter was observed in the 200 ng/ml FGF-4 group compared with the control group on day 1 (P<0.05). The results regarding viability using Cell Counting Kit-8 in the presence of FGF-4 at 50, 100 and 200 ng/ml at day 1 were 98.0±2.5, 106.2±17.6 and 99.5±6.0%, respectively, when normalized to the control group (P>0.05). Furthermore, the alkaline phosphatase activity was significantly elevated in the 200 ng/ml group, when compared with the control group. The RT-qPCR results demonstrated that the mRNA expression levels of RUNX2 and BGLAP were significantly increased at 200 ng/ml. Therefore, the present results suggested that the application of FGF-4 maintained cellular viability while enhancing the osteogenic differentiation of stem cell spheroids, at least partially by regulating RUNX2 and BGLAP expression levels.

NELL-1 Increased the Osteogenic Differentiation and mRNA Expression of Spheroids Composed of Stem Cells

“…Several studies have explored the underlying mechanisms modulated by NELL-1 [5,25,39]. NELL-1 is involved in upregulating runt-related transcription factor 2, a key transcription factor associated with osteoblast differentiation [20,40]. NELL-1 is activated through the mitogen-activated protein kinase-extracellular signal-regulated kinase signaling pathway in pre-osteoblasts on titanium surfaces [25].…”

“…Two-color-based assays (Live/Dead Kit assay, Molecular Probes, Eugene, OR, USA) based on esterase activity and integrity of the plasma membrane were used for qualitative cell viability. The spheroids were incubated for 30 min at room temperature after adding 2 µL of 50 mM calcein acetoxymethyl ester working solution and 4 µL of 2 mM ethidium homodimer-1 [20]. Three spheroids were analyzed for each group at each time point.…”

Gingiva-Derived Mesenchymal Stem Cells: Potential Application in Tissue Engineering and Regenerative Medicine - A Comprehensive Review

“…Similarly, 3D-spheroid GMSCs generated under different culture conditions also exhibit enhanced stemness, trilineage and neural differentiation potentials, and cytokine secretion (152,156). Several studies show that GMSCs cultured in patterned microwells can aggregate into 3D-spheoids with enhanced osteogenic potentials, which are further augmented by the addition of lovastatin or FGF-4 (153)(154)(155).…”

Effects of Connective Tissue Growth Factor on the Cell Viability, Proliferation, Osteogenic Capacity and mRNA Expression of Stem Cell Spheroids

“…A previous report showed that bone morphogenetic protein-4 promoted osteogenic differentiation through upregulation of collagen I and Sp7 expression [1]. Fibroblast growth factors were shown to be involved in osteogenic differentiation of stem cell spheroids while maintaining cell viability [4]. Optimal concentrations of growth factors for proliferation or differentiation activity have been studied previously [1,4,5].…”

“…Fibroblast growth factors were shown to be involved in osteogenic differentiation of stem cell spheroids while maintaining cell viability [4]. Optimal concentrations of growth factors for proliferation or differentiation activity have been studied previously [1,4,5]. Increases in osteogenic differentiation were achieved in a dose-dependent manner at up to 200 ng/mL of fibroblast growth factor-4 [4].…”