Isolation and characterization of olfactory ecto-mesenchymal stem cells from eight mammalian genera

Abstract

Background: Stem cell-based therapies are an attractive option to promote regeneration and repair defective tissues and organs. Thanks to their multipotency, high proliferation rate and the lack of major ethical limitations, "olfactory ecto-mesenchymal stem cells" (OE-MSCs) have been described as a promising candidate to treat a variety of damaged tissues. Easily accessible in the nasal cavity of most mammals, these cells are highly suitable for autologous cell-based therapies and do not face issues associated with other stem cells. However, their clinical use in humans and animals is limited due to a lack of preclinical studies on autologous transplantation and because no well-established methods currently exist to cultivate these cells. Here we evaluated the feasibility of collecting, purifying and amplifying OE-MSCs from different mammalian genera with the goal of promoting their interest in veterinary regenerative medicine. Biopsies of olfactory mucosa from eight mammalian genera (mouse, rat, rabbit, sheep, dog, horse, gray mouse lemur and macaque) were collected, using techniques derived from those previously used in humans and rats. The possibility of amplifying these cells and their stemness features and differentiation capability were then evaluated.

Results: Biopsies were successfully performed on olfactory mucosa without requiring the sacrifice of the donor animal, except mice. Cell populations were rapidly generated from olfactory mucosa explants. These cells displayed similar key features of their human counterparts: a fibroblastic morphology, a robust expression of nestin, an ability to form spheres and similar expression of surface markers (CD44, CD73). Moreover, most of them also exhibited high proliferation rates and clonogenicity with genus-specific properties. Finally, OE-MSCs also showed the ability to differentiate into mesodermal lineages.

Conclusions: This article describes for the first time how millions of OE-MSCs can be quickly and easily obtained from different mammalian genera through protocols that are well-suited for autologous transplantations. Moreover, their multipotency makes them relevant to evaluate therapeutic application in a wide variety of tissue injury models. This study paves the way for the development of new fundamental and clinical studies based on OE-MSCs transplantation and suggests their interest in veterinary medicine.

Keywords: Adult craniofacial stem cells; Dog; Ecto-mesenchymal stem cells; Horse; Non-human primate; Rabbit; Regenerative medicine; Rodent; Sheep; Veterinary medicine.

Fig. 1

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)

Fig. 2

Assessment of proliferative and clonogenic properties of OE-MSCs from different mammalian genera. For each mammalian genus, a clonogenicity efficiency assay was carried out by plating OE-MSCs (passage 7) at low densities and by measuring the number of newly formed colonies after 7 days in culture. All tested genera displayed a high percentage of OE-MSCs capable of forming new colonies (% of clonogenicity, a). The population doubling-time (in hours) was measured for each mammalian genus after 2 months (10 passages) and 3 months (20 passaging) in culture (b). Most of OE-MSC populations display a high proliferation rate but genus specificities are observed. Values reported are the mean (+/− SEM) of three independent experiments carried out in triplicate, on one representative member of each genus

Fig. 3

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