Nanog promotes osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 by modulating bone morphogenetic protein (BMP) signaling

Abstract

How the pluripotency of stem cells is maintained and the role of transcription factors in this maintenance remain major questions. In the present study, in order to clarify the mechanism underlying the pluripotency of stem cells for the advancement of regenerative medicine, we examined the effect of forced Nanog expression in mesenchymal cells, with a particular focus on osteogenic differentiation. The human mesenchymal stromal cells (hMSCs) or mouse mesenchymal cell line C3H10T1/2 cells were transduced with the Nanog gene or control green fluorescent protein (GFP) gene by using retrovirus vectors. Short-term, forced Nanog gene expression had few effects on the terminal osteogenic differentiation of either hMSCs or C3H10T1/2 cells. To determine its long-term effects, we established C3H10T1/2 cells expressing Nanog constitutively. Constitutive Nanog expression strongly induced osteogenic differentiation of C3H10T1/2 cells. In regard to cell proliferation, constitutive Nanog expression only repressed the proliferation of the cells treated with rhBMP-2. Moreover, Nanog also had the potential to promote the proliferation of C3H10T1/2 cells in the absence of rhBMP-2. Constitutive Nanog expression enhanced phosphorylation of Smad1/5/8 and suppressed Cdk4 and cyclinD1. The promoter activities of both the osteocalcin and Id-1 genes were activated in cells expressing Nanog constitutively. To identify downstream molecules of Nanog involved in the promotion of osteogenic differentiation, we performed a DNA microarray analysis and discovered that NFATc1 was one of the downstream effectors of Nanog. These results indicate that Nanog functions as a modulator of BMP signaling in C3H10T1/2 cells probably through a genome reprogramming process.

Runx1 and Runx3 Are Downstream Effectors of Nanog in Promoting Osteogenic Differentiation of the Mouse Mesenchymal Cell Line C3H10T1/2

“…C3H10T1/ 2 cells constitutively expressing green fluorescent protein (GFP) or Nanog were established as described (Ogasawara et al, 2013). These cells were cultured in high-glucose DMEM containing 10% FBS and 1% penicillin/streptomycin.…”

“…DNA microarray analysis was performed as described (Ogasawara et al, 2013). Briefly, a 250-ng aliquot of total RNA from Nanog-expressing cells or GFP-expressing cells cultured for 7 days with rhBMP-2 (200 ng/mL) was used for the first-and second-strand cDNA synthesis.…”

“…Thus, it is difficult to discuss the physiological role of Nanog in somatic cells. However, there have been several Nanog overexpression experiments reported in somatic cells (Go et al, 2008;Kochupurakkal et al, 2008;Ogasawara et al, 2013;Piestun et al, 2006;Zhang et al, 2005).…”

“…However, MSCs progressively lose their multipotency during long-term culture, resulting in the present limitations of tissue engineering, i.e., the small size and insufficient quality of the engineered tissues. To overcome this problem, the effects of several genes, including Nanog, were investigated (Go et al, 2008;Ogasawara et al, 2013;Simonsen et al, 2002;Terai et al, 2005). In the present study, to clarify the mechanisms underlying the multipotency of MSCs and to develop a novel approach to bone regenerative medicine, we sought to discover the downstream effectors of Nanog in promoting osteogenic differentiation of mesenchymal cells.…”

“…Previously, we reported that the transcription factor Nanog promotes the osteogenic differentiation of the mouse mesenchymal cell line C3H10T1/2 cells through a genome reprogramming process (Ogasawara et al, 2013). Nanog is a homeodomain transcription factor that maintains the selfrenewal of embryonic stem cells (ESCs) (Chambers et al, 2003;Mitsui et al, 2003).…”