br Materials and methods br Results br

Materials and methods
Results
Discussion Human olfactory stem cells can be grown in neurospheres (Reynolds and Weiss, 1992). Murrell et al. obtained human mucosa by biopsy and generated OSs. They separated olfactory mucosa into lamina propria and epithelium. After separately dissociated, cells of both tissues resuspended into the same culture dish pretreated poly-L-lysine. Their human OSs expressed nestin, GFAP, and Tuj1, and gave rise to neuron and glia under various culture conditions to induce differentiation (Murrell et al., 2005, 2008). When their OSs were dissociated and plated at low density, differentiated cells were observed to express GFAP, O4, Tuj1, and MAP5. The majority of their OSs (50.3%±8.09%) expressed GFAP in culture of serum free medium. Fetal calf serum decreased the proportion of cells expressing GFAP (44.67%±1.76%) and increased the proportions of cells expressing Tuj1 (18.33%±0.88%) and O4 (3.67%±0.88%). CNTF, NGF and retinoic Ro 31-8220 methanesulfonate increased the proportion of cells expressing GFAP (69.00%±3.21%), Tuj1 (25.33%±1.45%), and O4 (50.67%±2.96%), respectively. They also presented that their human OSCs differentiated into non-neural lineages in vitro and in vivo when given the appropriate signals and environments. In this study, we dissociated olfactory mucosa as a whole, and cells of dissociated tissues resuspended in the culture dish pretreated poly-HEMA. The percentage of Tuj1-positive cells was 87.2%±11.7%. These suggest that our human OSs more autonomously differentiate into neurons in our culture condition. Delorme et al. reported that the subtypes of their OSs had the character of mesenchymal stem cells that could help preserve auditory function (Delorme et al., 2010; Feron et al., 2013). These mesenchymal subtypes of their OSs were transplanted into mouse hippocampus, and then they could also induce neurogenesis (Nivet et al., 2011). Mesenchymal OSCs support the survival and differentiation of human hematopoietic stem cells (Diaz-Solano et al., 2012). The lamina propria derived-OSCs have MSC-like properties and secrete factors that enhance both OPC and OEC proliferation (Lindsay et al., 2013). Roisen et al. reported that human olfactory stem cells were harvested from cadavers and patients (Roisen et al., 2001; Winstead et al., 2005). They produced OSs, which contained two subpopulations based on neuronal and glial markers (Roisen et al., 2001). Their OSs consisted of heterogeneous populations in which some cells expressed nestin, A2B5, Tuj1, and peripherin (Zhang et al., 2004). A subset of their A2B5-positive OSCs co-expressed nestin and Tuj1. They also demonstrated that adult human OSCs could be directed toward neuronal lineage restriction by retinoic acid, forskolin, and sonic hedgehog (Zhang et al., 2006). The probability of finding olfactory epithelium in a biopsy specimen is from 30% to 76%, depending on its location (Feron et al., 1998). In this study, the location of surplus olfactory mucosa was the nasal septum in 4 patients and the superior concha in 1 patient. Murrell et al. produced 500 to 2000 spheres from 1 to 4mm2 olfactory mucosa after 7–10days culture. The number of primary OSs ranged from 0 to 2/culture dish in a harvesting area under 15mm2, while the number of primary OSs was over 50/culture dish in a harvesting area over 40mm2. Although generating adequate OSCs seems to require an area of olfactory mucosa over 4mm×10mm, our methods need the improvement of the yield, and scaling down of culture.
Conclusions In this study, adult human OSCs were generated and their characteristics analyzed. Human OSCs were adequately produced from olfactory mucosa with area over 40mm2. Human OSCs autonomously differentiated into neurons. These findings suggest that human OSCs have the potential to be used as a cell source of neural progenitors for their own regenerative grafts, avoiding the need for immunosuppression and ethical controversies.
Acknowledgments