Human mesenchymal stem cells (hMSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications by virtue of their capacity for self-renewal and multipotent differentiation. Our intent was to characterize the effect of pulsed electromagnetic fields (PEMFs) on the proliferation and osteogenic differentiation of hMSCs in vitro. hMSCs isolated from the bone marrow of adult patients were cultured with osteogenic medium for up to 28 days and exposed to daily PEMF stimulation with single, narrow 300 ms quasi-rectangular pulses with a repetition rate of 7.5 Hz. Relatively greater cell numbers were observed at late stages of osteogenic culture with PEMF exposure. The production of alkaline phosphatase (ALP), an early marker of osteogenesis, was significantly enhanced at day 7 with PEMF treatment in both basal and osteogenic cultures as compared to untreated controls. Furthermore, the expressions of other early osteogenic genes, including Runx2/Cbfa1 and ALP, were also partially modulated by PEMF exposure, indicating that osteogenesis in hMSCs was associated with the specific PEMF stimulation. Based on ALP and alizarin red S staining, the accumulation of ALP protein produced by the hMSCs as well as calcium deposits reached their highest levels at day 28. Our results indicate that extremely low-frequency PEMF stimulation may play a modulating role in hMSC osteogenesis. Taken together, these findings provide insights on the development of PEMF as an effective technology for regenerative medicine. ß

Size-sieved stem (SS) cells isolated from human bone marrow and propagated in vitro are a population of cells with consistent marker typing, and can form bone, fat, and cartilage. In this experiment, we demonstrated that SS cells could be induced to differentiate into neural cells under experimental cell culture conditions. Five hours after exposure to antioxidant agents (β-mercaptoethanol ± retinoic acid) in serum-free conditions, SS cells expressed the protein for nestin, neuron-specific enolase (NSE), neuron-specific nuclear protein (NeuN), and neuron-specific tubulin-1 (TuJ-1), and the mRNA for NSE and Tau

We have previously identified a novel family of secreted, cell-surface proteins expressed in human vascular endothelium. To date, two family members have been described, sharing a characteristic domain structure including an amino-terminal signal peptide followed by multiple copies of epidermal growth factor (EGF)-like repeats, a spacer region, and one CUB domain at the carboxyl terminus. Thus, this family was termed SCUBE for signal peptide-CUB-EGF-like domain containing proteins. Here we described the identification and characterization of one additional member of the SCUBE family named SCUBE3 in humans, sharing an overall 60% protein identity and a similar domain structure with other family members. Real-time quantitative reverse transcriptase-PCR and Northern blot analyses revealed that this gene is highly expressed in primary osteoblasts and the long bones (humerus and femur), followed by a low level of expression in human umbilical vein endothelial cells and in heart. When overexpressed in human embryonic kidney 293T cells, the recombinant hSCUBE3 protein is a secreted glycoprotein that can form oligomers tethered to the cell surface. Interestingly, the secreted hSCUBE3 protein can be further proteolytically processed by a serum-associated protease to release the EGF-like repeats from the CUB domain. The SCUBE3 gene is mapped to human chromosome 6p21.3, a region that has been linked with the locus for a rare form of metabolic bone disease, Paget's disease of bone 1. Together, this novel secreted, cell-surface osteoblast protein may act locally and/or distantly through a proteolytic mechanism, and may play an important role in bone cell biology.