Comparison of Canine and Feline Adipose-Derived Mesenchymal Stem Cells/Medicinal Signaling Cells With Regard to Cell Surface Marker Expression, Viability, Proliferation, and Differentiation Potential

Abstract

Remarkable immunomodulatory abilities of mesenchymal stem cells, also called multipotent mesenchymal stromal cells or medicinal signaling cells (MSCs), have entailed significant advances in veterinary regenerative medicine in recent years. Despite positive outcomes from MSC therapies in various diseases in dogs and cats, differences in MSC characteristics between small animal veterinary patients are not well-known. We performed a comparative study of cells' surface marker expression, viability, proliferation, and differentiation capacity of adipose-derived MSCs (ADMSCs) from dogs and domestic cats. The same growth media and methods were used to isolate, characterize, and culture canine and feline ADMSCs. Adipose tissue was collected from 11 dogs and 8 cats of both sexes. The expression of surface markers CD44, CD90, and CD34 was detected by flow cytometry. Viability at passage 3 was measured with the hemocytometer and compared to the viability measured by flow cytometry after 1 day of handling. The proliferation potential of MSCs was measured by calculating cell doubling and cell doubling time from second to eighth passage. Differentiation potential was determined at early and late passages by inducing cells toward adipogenic, osteogenic, and chondrogenic differentiation using commercial media. Our study shows that the percentage of CD44⁺CD90⁺ and CD34^-/- cells is higher in cells from dogs than in cells from cats. The viability of cells measured by two different methods at passage 3 differed between the species, and finally, canine ADMSCs possess greater proliferation and differentiation potential in comparison to the feline ADMSCs.

Keywords: cat; cell surface marker; comparison; differentiation; dog; mesenchymal stem cells; proliferation; viability.

Figure 1

Images of canine (A) and feline (B) ADMSCs. Cells from both species exhibited similar spindle-shaped fibroblast-like morphology.

Figure 2

(C)CD and CDT of canine and feline ADMSCs. Canine ADMSCs (C)CD was statistically significantly higher than feline ADMSCs (C)CD (A, *p < 0.01), and average CDT was statistically significantly lower for canine than for feline ADMSCs (B, *p < 0.05). No sex differences in CD or CDT were observed in either of the species. Interestingly, CDT in canine ADMSCs increased gradually through passages, whereas CDT in feline ADMSCs increased unevenly. In dogs, no statistically significant differences were observed between passages. Interestingly, CDT increased significantly in passage 8 in cats only and was different from all other feline and canine passages (***p < 0.001; C). Results are presented as mean ± SEM.

Figure 3

Expression of canine and feline ADMSC surface markers analyzed by FACS at passage 3. Both canine and feline ADMSCs expressed CD44 and CD90 but did not express CD34.

Figure 4

Percentage of CD440⁺CD90⁺ and CD34^−/− canine and feline cells. Percentage of live ADMSCs expressing CD44 and CD90 was statistically significantly higher in canine than in feline cells (*p < 0.01). The percentage of live CD34 negative ADMSCs was also statistically significantly higher in canine than in feline cells (*p < 0.05). There were no differences in cell marker expression between the sexes of ADMSCs from either of the species. Results are presented as mean ± SEM.

Figure 5

Viability of canine and feline ADMSCs measured by hemocytometer (left) and flow cytometry (right) at passage 3. When measured by hemocytometer, the viability of ADMSCs was similar between both species. When measured by flow cytometry, the viability of feline ADMSCs was statistically significantly lower than that of canine ADMSCs (*p < 0.01). No differences in cell viability between sexes from either species were present, regardless of the method. Results are presented as mean ± SEM.

Figure 6

Multilineage differentiation of cells in passage 2. ADMSCs from cats and dogs successfully underwent osteogenic (A,B), chondrogenic (C,D), and adipogenic differentiation (E,F). In osteogenic differentiation, mineral deposits in the extracellular matrix were stained red by alizarin-red-S (dog A, cat B). Chondrogenic differentiation is indicated by the formation of chondrogenic nodules that stain blue with Alcian blue (dog C, cat D). Red intracellular lipid droplets stained with oil-red-O are indicative of adipogenic differentiation (dog E, cat F). Respective negative controls are shown as inserts in each photomicrograph.

Figure 7

Chondrogenic and osteogenic differentiation at early (P2) and late (P8 for canine cells and P6 for feline cells) passages. Positively stained area in chondrogenic differentiation was statistically significantly more extensive in canine ADMSCs than in feline ADMSCs at passage 2 (*p < 0.05). In osteogenic differentiation, there was a statistically significant difference between the sexes of both species (**p < 0.01) but not between species at passage 2, although there was a statistical trend for difference between species (p = 0.07). In late passages, there was statistically significant difference in osteogenic differentiation between the species, but only in males (a different from b, p < 0.01).