Technical strategies to isolate olfactory ensheathing cells for intraspinal implantation.

Over the past few years, the idea of using intraspinal implantations of olfactory ensheathing cells (OECs) as a therapeutic strategy to enhance recovery after spinal cord injury has quickly moved from experimentation with laboratory mammals to surgical approaches for paralyzed humans. Despite this progression, several important issues have yet to be thoroughly addressed: for instance, which of the many methods currently being used best yields enriched populations of OECs, and how such purity can be empirically tested and validated among different mammalian species, including humans. Here we offer an authoritative review of those methods used to isolate OECs from the olfactory mucosa and/or olfactory bulbs of rats, mice, dogs, pigs, non-human primates, and humans. As well, we assess which biomarkers are currently being utilized to determine the relative proportions of OECs and contaminating cells in these glial cultures. Although there have been numerous review articles regarding OECs in vitro, our review is unique in that it offers a critical assessment of the methods currently being used to generate cultures of mammalian OECs. More specifically, we examine the issue of culture contamination by phenotypically similar Schwann cells. This review is timely because recent clinical usage of OECs has come under intense criticism for a number of reasons, including the reliable identification of cultured human OECs. We believe that once these methodological issues of isolation and characterization of OECs have been resolved, this glial population will offer paralyzed individuals a truly viable cellular strategy for intraspinal therapy.

Doublecortin-expressing cells in the ischemic penumbra of a small-vessel stroke.

A cortical lesion was induced by disrupting the medium-size pial vessels, which led to a cone-shaped cortical lesion and turned into a fluid-filled cavity surrounded by a glial acidic fibrillary protein-positive (GFAP(+)) glia limitans 21 days after injury. Therefore, it mimics conditions of lacunar infarctions, one of the most frequent human stroke pathologies. Doublecortin (DCX)-positive cells were present in the neocortex and corpus callosum at the base of the lesion. The number of DCX-positive cells in the corpus callosum was significantly increased from day 5 to day 14 compared with the control group. In contrast, there were no DCX-positive cells in neocortex of control animals; the DCX-positive cells appeared in the neocortex after lesioning and were maintained until 14 days postlesioning. Some of the DCX-positive cells were also immunoreactive for beta III-tubulin, another marker of immature neurons. They did not stain positively for markers of glia cells. The presence of these DCX-positive cells near the lesion might indicate a migratory pathway for developing neuroblasts from the subventricular zone (SVZ) through the corpus callosum to the lesion. SVZ cells were labeled with a lipophilic molecule, 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) stereotaxical injections. Although rostral migratory stream and olfactory bulb were intensely labeled, no CFSE-containing cells were found in the cortex beneath the lesion. These results do not support the idea that the DCX-positive cells were originating from neural precursors of the SVZ, but they might be generated from local progenitor cells.