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Among the potential stem cell candidates for regenerative therapy, adult nasal stem cells present in the olfactory mucosa are a promising candidate both for human and veterinary medicine. This easily accessible peripheral tissue contains highly proliferative stem cells that do not face ethical or technical issues associated with other stem cells types. Characterized as a member of the mesenchymal stem cell (MSC) superfamily, these cells are known as “olfactory ecto-mesenchymal stem cells” (OE-MSCs). OE-MSCs are located in a permanently self-renewing nervous tissue with an ectodermal embryonic origin and assumed to be derived from neural crest cells, like other adult stem cells of the craniofacial area . They are multipotent, providing a potential source of stem cells for treating numerous types of tissue damages . Moreover, human OE-MSCs can be quickly and easily propagated in sufficient numbers to meet the requirements for cell transplantation without showing tumorigenicity risks. Although the therapeutic potential of OE-MSCs has not yet been assessed in human or veterinary medicine, their therapeutic effect has been evaluated in various rodent models of tissue damage such as myocardial infarct, spinal cord trauma, cochlear damage, Parkinson’s disease, and ischemic/hypoxic injury of the hippo-campus. Despite the promising results reported by these studies, their clinical use in humans and animals is limited by the lack of well-established methods for the collection of these cells from living animals, and for their purification and amplification. To overcome this problem, the authors of the present study recently developed an efficient and minimally invasive procedure for autologous transplantation of OE-MSCs in rats. According to this method, each animal is the donor as well as the receiver of its own cells, thereby excluding complications and side effects associated with other grafting methods. The present study evaluated the feasibility of collecting, purifying and amplifying OE-MSCs from living animals belonging to eight genera of mammals, relevant for basic research or clinical veterinary practice. In the prospect of future autologous transplantation therapies, we also assessed OE-MSCs stemness features.
Collection of olfactory mucosa from different mammalian genera
Biopsies of nasal olfactory mucosa were obtained as previously described in humans and rats with some modifi-cations. According to the morphology and accessibility of the nasal cavity, three protocols were used to access the olfactory mucosa. In mice, muzzle was dissected as previously described in euthanized rats [20]. For rats and gray mouse lemurs, a less invasive method, requiring nasal bone perforation at the junction with the frontal bone, was used to access the olfactory mucosa, as previously described in living rats [34]. In all other gen-era, biopsies were directly obtained by nasal cavity exploration with pliers, as previously described in humans [20, 35]. Details for each genus and the strategies used for biopsies are summarized. For all genera, the fragments of olfactory mucosa were placed, immediately after excision, in culture medium [Dulbecco’s Modified Eagle’s Medium/Ham’s F12 (DMEM/F12) supplemented with 10% Fetal Bovine Serum (FBS), 200 units/mL of penicillin, 200 μg/mL of streptomycin (Life Technologies), 0.25 μg/ml of Amphotericin B (Fungizone, Sigma/Aldrich) and 12.5 μg/ml of Plasmocin Treatment (InvioGen)]. For horse cells, the culture medium was supplemented with 1 μg/ml of Ketocona-zole (Sigma/Aldrich) to prevent fungal contamination due to stabling. Then, the pieces of olfactory mucosa placed in the culture medium were stored in a refrigerated container until processing. Anesthesia and surgical procedures were performed according to the European law on Animal Care Guide-lines and the Animal Care Committee of Aix-Marseille University and Ethic Committee of IRSEA (C2EA125) approved our protocols.
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