Construction of a Luciferase Reporter System to Monitor Osteogenic Differentiation of Mesenchymal Stem Cells by Using a Mammalian Artificial Chromosome Vector

In the human body, the skeletal system is one of the largest organs, which undergoes remodeling throughout a person’s lifespan.1 Bone remodeling and fracture healing processes are thought to be mediated via osteoprogenitors and their ancestors, mesenchymal stem cells (MSCs).2 In in vitro studies, a protocol using dexamethasone (DEX) is generally accepted for induction of osteoblastic differentiation of MSCs.3 In a clinical setting, however, the use of DEX is not practical because of its adverse side effects such as glucocorticoid-induced osteoporosis.4 For successful treatment of bone-loss disorders, chemical compounds and bioactive substances that effectively induce bone formation are required. In an effort to develop an evaluation system for bone remodeling, we previously generated transgenic mice that express the luciferase (Luc) reporter gene under the control of human osteocalcin enhancer/promoter sequences.5 Osteocalcin (OC) is the main non-collagenous hydroxyapatite-binding protein, which is synthesized by osteoblasts, odontoblasts and hypertrophic chondrocytes.6 It was recognized as a marker of bone formation6 and has been used for detection of osteoblast maturation and osteogenic activation. 7 Using in vivo bioluminescence imaging in the OC-Luc transgenic mice, Luc activity was induced throughout their skeletons by administration of 1,25-dihydroxyvitamin D3 (VD3), which regulates the expression of a number of genes including OC in bone cells. 8 Furthermore, enhanced bioluminescence was observed at the forced radius fracture site reflecting progression of the fracture repair. Although the OC-Luc transgenic mice would contribute to the final confirmation of drug efficacy selected from hundreds of candidates, an in vitro cell based-assay system is preferable for the first screening of high content candidate compounds. Mammalian artificial chromosome vectors have been utilized for introduction of genes to recipient cells.9 As for the origin of their chromosomes, there are two kinds of artificial chromosomes, i.e. human artificial chromosome (HAC) and mouse artificial chromosome (MAC) that have been constructed by chromosome engineering. Since HAC or MAC is maintained as an independent chromosome in recipient cells, physiological regulation of the loaded gene is expected.10 Previously, feasibility of cell lineage-specific expression of a transgene by a HAC vector was demonstrated in a human MSC line.11 The GFP reporter gene under the regulation of human osteopontin (OPN) promoter loaded on a HAC vector was transferred to a human MSC line. OPN is another marker of bone formation, whose expression is upregulated during maturation of osteoblasts.7 The reporter GFP gene in the MSCs was specifically expressed in response to osteogenic differentiation induction in coordination with the transcription of endogenous OPN. In order to distinguish the osteoblast differentiation process from the subsequently occurred maturation processes accurately, however, reference reporter system, which can trace osteoblast differentiation steps, is required. Our aim is setting up a screening platform to identify compounds that promote or induce differentiation of MSCs towards osteoblasts. To this end, we made a reporter construct composed of the Luc gene under the regulation of OC enhancer/promoter in a MAC vector (OC-Luc/MAC). The MSC lines carrying the OC-Luc/MAC vector were tested for differentiation ability to osteogenic lineage. The induction or restraint of Luc activity responding to the already-known positive stimulus via VD3 or negative stimulus via DEX treatment was also addressed for validation as monitor cells.