Elucidating the Role of RUNX2 in Osteoblastic Differentiation of iPSCs

Objectives: Information is limited regarding the mechanisms involved in the differentiation of induced pluripotent stem cells (iPSCs) into osteoblasts, including the role of Runx2. This study aimed to establish iPSCs from Runx2 homo-deficient mouse (Runx2^-/-) and to elucidate the role of RUNX2 in the differentiation process into osteoblasts.
Methods: Mouse iPSCs (miPSCs) were generated from MEFs of Runx2^-/-, Runx2^+/- and wild type mice by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, using a Sendai virus vector. The miPSCs were cultured in osteoblast basal medium. Osteoblastic differentiation was assessed by ALP activities. Quantitative RT-PCR was performed for Alp, Runx2 and Bsp at 14 and 21 days of osteoblastic differentiation. PCR array was used to compare the mRNA expression profiles in miPSCs from Runx2^-/- and wild type, during osteoblastic differentiation. Genes focused on PCRarray were confirmed in detail by quantitative RT-PCR.
Results: After 14 days of osteoblastic differentiation, ALP-positive cells were observed in all groups. In quantitative RT-PCR analysis, time-dependent increases in Alp mRNA expressions were noted in all groups. At 21 days, the expression level of Bsp in Runx2^-/- miPSCs was significantly lower (approximately by 30%) than that in wild type (p< 0.01). In gene expression array analyses, Rankl was upregulated and Vdr was downregulated in Runx2^-/- relative to wild type. At 14 days, Rankl expression in Runx2^-/- miPSCs was significantly higher (approximately 3-fold) and Vdr expression was significantly lower (by 20%) than that in wild type (p< 0.001).
Conclusions: We succeeded for the first time in the generation of Runx2^-/- miPSCs. Our data suggest that Runx2-independent mechanism of osteogenesis exists in miPSCs. Runx2 may function as a negative regulator of Rankl and positive regulator of Vdr and thereby facilitates osteoblastic differentiation of miPSCs.