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In vitro assessment of differentiation abilities of OE-MSCs
Prior to any differentiation, we observed that cells from four studied genera (rat, rabbit, dog and horse) expressed neural proteins GFAP and MAP2 at a basal state. Subsequently, differentiation as-says of these cells into cells of the mesodermal lineage were performed. We found they could be induced to express bio-chemical features specific to osteoblasts, chondroblasts and tenoblasts .
OE-MSCs: Similar characteristics shared by the different genera
Despite some differences between genera, the results indicated that the various mammalian OE-MSCs displayed similar characteristics to those previously described in their human counterparts . Noticeably, these methods were successful in obtaining many highly proliferative cells. Interestingly, though it is not possible to ascertain the absolute purity of stem cells and exclude potential contamination with other cell types, such as the olfactory ensheathing cells, the populations of OE-MSCs were highly homogeneous, when comparing cell morphology, expression of nestin and surface markers
Assessment of neural and mesodermal differentiation abilities of OE-MSCs in vitro. Multipotency was assessed in OE-MSCs from rat, rabbit, dog and horse. Expression of the neural proteins GFAP (a) and MAP2 (b) in red in undifferentiated rat OE-MSCs. Bone differentiation was assessed using Red Alizarin (c) and Von kossa (d) stainings. Dog OE-MSCs were positively labeled in red (c) and in black (d) using these procedures. Chondrogenic differentiation was assessed using Toluidine Blue (e) and Alcian Blue (f) stainings. Horse OE-MSCs were positively labeled in purple (e) and in blue (f) using these procedures. Expression of the tenocytic markers Scleraxis protein (g) and Tenomodulin (h) in red in rabbit OE-MSCs. Each image is representative of multiple independent cultures of each species. Scale bar: 200 μm CD34 and CD44 according to previous studies in human and rat. While the expression of CD34 and CD44 in cells from all genera was similar with the data from human OE-MSCs, we observed higher variability for CD73. First, this marker could not be detected at significant levels in horse OE-MSCs. Although we could not exclude that the antibody used was not able to recognize the horse protein, this observation agrees with other research groups who could not detect CD73, using multiples antibodies, in different horse MSC subtypes that normally express this marker in humans and rodents. While the same technical issues may happen with cells from rabbit, CD73 is not, weakly or highly expressed in rabbit MSCs according to three different studies. However, our results confirm the data of another research team which observed a similar percentage of positive cells (28%). In addition to the expression of these markers, the ability to generate spheres, the strong mitotic activity that remained high in most cases after 20 passages, and the high clonal efficiency rate, serve as indisputable proofs of the stemness features of these cells. For unknown reasons, mouse olfactory stem cells were the most difficult to grow and amplify and were not tested for following evaluations. Accordingly, in addition to the complexity of collecting olfactory mucosa biopsies in living mice, autologous transplantations appear highly difficult in this genus.
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