Nitric oxide (NO) has been shown to play a crucial role in bone formation in vivo. We sought to determine the temporal effect of NO on murine embryonic stem cells (ESCs) under culture conditions that promote osteogenesis. Expression profiles of NO pathway members and osteoblast-specific markers were analyzed using appropriate assays. We found that NO was supportive of osteogenesis specifically during an early phase of in vitro development (days 3-5). Furthermore, ESCs stably overexpressing the inducible NO synthase showed accelerated and enhanced osteogenesis in vitro and in bone explant cultures. To determine the role of NO in early lineage commitment, a stage in ESC differentiation equivalent to primitive streak formation in vivo, ESCs were transfected with a T-brachyury-GFP reporter. Expression levels of T-brachyury and one of its upstream regulators, beta-catenin, the major effector in the canonical Wnt pathway, were responsive to NO levels in differentiating primitive streak-like cells. Our results indicate that NO may be involved in early differentiation through regulation of beta-catenin and T-brachyury, controlling the specification of primitive-streak-like cells, which may continue through differentiation to later become osteoblasts.
