From Blood to Lesioned Brain: An In Vitro Study on Migration Mechanisms of Human Nasal Olfactory Stem Cells

Figure 5

Among selected overexpressed cytokines, only CCL2 and SPP1 stimulate OE-MSC migration. Effects of human recombinant cytokines (i.e., hC3A, hCCL2, hSPP1, and hCXCL10) on human OE-MSC migration were assessed using modified Boyden chambers (a–e) and agarose spot assay (f–j). (a–c) Representative images of membrane filter fields from modified Boyden chambers display Hoechst-stained nuclei of migrating human OE-MSCs, 12 hours after the addition of buffer or cytokines (1,000 ng/mL) in the lower chamber. Under the 40x objective, the migrating stem cells attached to the lower side of the membrane (white arrows) were easily distinguishable from nonmigrating cells (unfocused nuclei). Scale bar: 50 μm. Mean percentage of migrating OE-MSCs per field in response to cytokines at increasing concentrations (d) or to a cocktail of the two active cytokines at the concentration showing maximum efficacy (1,000 ng/mL) (e). (f–h) Representative images of an area of jellified agarose drop (upper left zone) containing buffer or cytokines at 10 μg/mL. Twenty-four hours after plating, OE-MSCs were observed around the drop and human OE-MSCs penetrated into the drop in the presence of active cytokines (hCCL2, hSPP1). Scale bar: 200 μm. Mean number of migrating human OE-MSCs in the drop area in response to buffer or cytokines at 10 μg/mL (i) or a cocktail of the two active cytokines used at the same concentration (j). Values reported are the mean (±SEM) of at least three independent experiments performed in triplicate for modified Boyden chambers and in ten replicate drops for agarose spot assays. ( and , using Student’s t-test.)

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