Scale bar is 10?m

Scale bar is 10?m. Canine ensheathing cells of the bulb modulate neurite outgrowth of developing human neurons To analyze potential interactions between the isolated olfactory ensheathing and Schwann cells with neurons, we measured how canine glial cells influence parameters of neurite outgrowth. the effects on neurite growth. OB-OECs and Schwann cells migrated faster than OM-OECs in a scratch wound assay. Glial cell migration was not modulated by cGMP and cAMP signaling, but activating protein kinase C enhanced motility. All three glial cell types displayed phagocytic activity in a microbead assay. In co-cultures with of human model (NT2) neurons neurite growth was maximal on OB-OECs. Conclusions These data provide evidence that OB- and OM-OECs AX20017 display distinct migratory behavior and conversation with neurites. OB-OECs migrate faster and enhance neurite growth of human model neurons better than Schwann cells, suggesting distinct and inherent properties of these closely-related cell types. Future studies will have to address whether, and how, these cellular properties correlate with the behavior Vegfb after transplantation. co-culture system. Another important feature of this study is the establishment of a Schwann cell-free preparation as reported [18]. The olfactory mucosa contains OECs and myelinating Schwann cells from trigeminal afferents and other non-myelinating cells. Moreover, the close phenotypic resemblance of OECs and Schwann cells and the expression of marker molecules such as the neurotrophin receptor p75 (p75NTR) and glial protein S100 represent obstacles for the selective identification and purification of pure OEC preparations that are free of Schwann cells. Using magnetic activated cell sorting, it has recently been shown that contaminating Schwann cells can be depleted from canine OEC preparations allowing further characterization of purified OECs from olfactory bulb (OB-OECs), olfactory mucosa (OM-OECs), and Schwann cells from fibular nerve [18]. To advance our understanding how these various groups of glial cells may facilitate axonal regeneration in the damaged CNS various assays were performed. Since a permissive environment created by transplants of migratory glial cells contributes to axonal outgrowth in the injured CNS, initially we investigated the cellular motility of the purified three glial types. To compare cell motility, a scratch migration assay which measures cell migration during the closure of a wound AX20017 that is scratched into a confluent cell monolayer was used. In addition, it was investigated whether motility could be up-regulated by chemical manipulation of intracellular signaling cascades. So far, we found no evidence that glial migration is usually influenced by application of cGMP or cAMP signaling molecules [19,20], but activating PKC enhances motility. Glial cells may aid repair processes in the CNS by clearing cellular debris via phagocytosis. Using a phagocytosis assay, we exhibited internalization of fluorescent microspheres into all three glial cell types. Finally, glial cells were analyzed for their potential to improve neurite outgrowth in a co-culture system with human NT2 model neurons. These neurons were derived from the Ntera2/D1 clone of a well characterized teratocarcinoma cell line, which can be induced to differentiate into fully functional post mitotic neurons by retinoic acid treatment. NT2 cells resemble human embryonic stem cells [21] and the differentiation of NT2 cells into neurons has been suggested to mimic aspects of vertebrate neurogenesis [22-25]. The co-culture assays using OECs and SCs represent a needed prerequisite to evaluate the potential therapeutic impact of the three glial cell types for repair of spinal cord injuries in a large animal translational model and their future clinical application. Results Scratch migration assay One therapeutic aspect of OEC cell transplantation for treatment of SCI is related to the glial ability to migrate within the injury AX20017 site and to accompany regenerating neurites. To compare the motility of the purified canine glial cells, we used a scratch migration assay which tracks cell migration during the closure of a wound that is scratched into a confluent cell monolayer (Physique?1A) [26,27]. Immunocytochemical staining of purified cultures confirmed p75 neurotrophin receptor (p75NTR) expression in all types of glial cells (Physique?1B-D). High magnification images depicted a patchy appearance of immunoreactivity around the glial cell surface, indicative of selective membrane incorporation under cell culture conditions. We seeded the cells into 24-well-plates and performed a scratch wound to the confluent cell monolayer using a pipette tip (Physique?1A). Figures?1E and F show how a scratch wound in a confluent layer of purified OECs from the olfactory bulb induces glial migratory behaviour. The closure of the gap was monitored by following the advancement of the cell front over 8?h. Because this time interval is usually too short for significant cell proliferation, presence of cells in the gap largely reflects migration (Physique?1F)..