Using the combination of a mitogen

Using the combination of a mitogen-activated protein kinase kinase (MEK) inhibitor, PD0325901 (PD03), and a glycogen synthase kinase 3 (GSK3) inhibitor, CHIR99021 (CHIR), which will hereafter be referred to as 2i, mouse ESCs (mESCs) are maintained in a ground state (Ying et al., 2008). CHIR activates canonical Wnt signaling by suppressing the degradation of β-catenin (Bain et al., 2007). Canonical Wnt signaling cues also specify the differentiation of germ layers and multipotent stem KPT-185 into mesodermal cells (Davis and Zur Nieden, 2008). We recently described the gene regulatory networks underlying canonical Wnt signaling-mediated control of mesoderm differentiation and pluripotency in mESCs (Zhang et al., 2013). The formation of osteoblasts is a sequential process. In mesoderm-derived skeletons, cells in the lateral plate mesoderm or the paraxial mesoderm give rise to skeletal progenitors, which then differentiate into bone-forming osteoblasts and cartilage-forming chondrocytes (Akiyama et al., 2005). We and others have shown that hedgehog (Hh) signaling is essential for normal osteoblast development, particularly for the specification of osteo-chondroprogenitors into osteoblast precursors, which express runt-related transcription factor 2 (Runx2), a transcription factor essential for bone formation (Hojo et al., 2012; Long et al., 2004; St-Jacques et al., 1999); the smoothened (SMO) agonist (SAG), a Hh signaling activator, promoted early osteoblast differentiation in perichondrial cells, which consist of osteo-chondroprogenitors (Hojo et al., 2012). This finding suggested that SAG could efficiently differentiate mesodermal cells into osteoblast precursors. Moreover, it also indicated that SAG alone might not be sufficient to direct the differentiation of these precursors into mature osteoblasts. In terms of molecules inducing osteoblast maturation, we identified the helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH) as an osteogenic small molecule that acts on preosteoblasts in a bone morphogenetic protein (BMP)-dependent manner (Ohba et al., 2007b). Furthermore, we recently reported the combinatorial effect of SAG and TH on the differentiation of mesenchymal cells into osteoblasts (Maeda et al., 2013).
Results and Discussion We initially attempted to differentiate 2i-cultured mESCs into mesodermal cells by activating canonical Wnt signaling with CHIR in 2i culture media. CHIR upregulated the mesoderm-related genes T and Mixl1 in a dose-dependent manner relative to day 0 (Figure 1A; Figure S1A available online). The expression of the pluripotency-related genes Nanog, Pou5f1, and Sox2 was suppressed in cells treated with high concentrations of CHIR relative to day 0 (Figures 1A and S1A). In addition, the expression of T was higher on day 5 than on day 7, and higher expression of the mesoderm-related genes was associated with higher expression of osteoblast-related genes in subsequent osteoblast induction (Figure S1B). Therefore, we selected the 5-day treatment protocol using 30 μM CHIR for the mesoderm induction of mESCs. To induce osteoblast differentiation of the mESC-derived mesoderm cells, we then cultured them in 2i culture media containing 1 μM SAG, 1 μM TH, and other osteogenic supplements (Jaiswal et al., 1997). Runx2, Sp7, and Ibsp were upregulated following treatment for 2 weeks with SAG plus TH relative to the control group (Figure 1B). Thus, the stepwise differentiation from mESCs into osteoblasts via mesoderm formation was achieved using the three small molecules CHIR, SAG, and TH. Given the roles of Hh signaling during the development of the CNS (Martí and Bovolenta, 2002), recombinant Hh proteins or SAGs have been used to differentiate pluripotent stem cells into motor neurons (Nizzardo et al., 2010). Moreover, 2i-cultured ESCs preferentially differentiate into ectoderm lineages rather than mesoderm lineages (Marks et al., 2012). These findings led us to examine whether the suppression of Hh signaling during the mesoderm induction would block neuro-ectoderm specification, resulting in enhanced osteoblast differentiation. The combinatorial use of the Hh signaling inhibitor cyclopamine (Cyc) and CHIR during the 5-day mesoderm induction induced the downregulation of Sox1 on day 5 and led to increased osteoblast differentiation (Figure 1C).