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Morphology and stemness features of OE-MSCs from different mammalian genera
After 4 weeks in growth culture medium, olfactory mucosa explants formed homogeneous populations of adherent and highly proliferative cells exhibiting a mesenchymal-like fibroblastic morphology: examples of sheep (a). When grown under appropriate culture conditions, OE-MSCs could generate spheres: examples of rabbit (b). After seven passages, cells express the nestin protein (in green, (c) example of rabbit), a prominent marker of immaturity. OE-MSCs were immunostained with 3 surface markers, quantified using a flow cytometer and expression level compared to isotype: example of macaque (d). Each image is representative of multiple independent cultures of each species. Scale bar: 200 μm (a & b), 100 μm (c) OE-MSCs from different mammalian genera display similar expression of mesenchymal stem cell surface markers.
We analyzed expression of 3 surface markers by flow cytometry. In each genus studied, CD34 expression was extremely low or undetectable with a percentage of cells expressing this marker inferior to 10. CD44 was highly and homogeneously expressed in OE-MSCs from all genera with a percent-age of cells expressing this marker superior to 90 except in horse (68%). Expression of CD73 marker was more variable in the different genera. While percentage of cells % cells expressing markers (mean ± SEM) expressing this marker is superior to 50% in cell population from rat, sheep, dog, gray mouse lemur and macaque, CD73 expression was low in OE-MSCs from rabbit (22%) and extremely low in horse cells (5.5%).
High clonal efficiency and proliferation rate of OE-MSCs
Evaluation of clonal efficiency after seeding of the cells at low density indicated that a high percentage of OE-MSCs from all studied genera could give rise to colonies after 7 days in culture, except mice OE-MSCs which were unable to generate new clonal populations (Fig. 2a). The percentage of OE-MSCs generating new colonies after seven passages ranged between 50% and 70% in rat, dog, horse, sheep, gray mouse lemur and macaque. Rabbit cells displayed a lower clonal efficiency (20%) in comparison to their outstanding proliferation rate (Fig. 2b). Assessment of the population doubling-time in short-term (passage 10, 2 months of culture) and long-term (passage 20, 3 months of culture) passages revealed that most OE-MSCs are quickly dividing cells. As shown in Fig. 2b, cells from all genera, except those derived from gray mouse lemur olfactory mucosa, displayed a doubling-time ranging from 20 and 40 h after 10 passages. Except for the sheep and gray mouse lemur, the doubling-time remained stable after 20 passages and even decreased in horse and macaque cells.
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