Characterization of mesenchymal stem cells derived from adipose tissue of a cougar (Puma concolor)

Abstract

Adipose derived mesenchymal stem cells (AMSCs) have been isolated from domestic and wild cats. For wild cats, the isolation of AMSCs has been reported in the black-footed cats (Felis nigripes) and guigna (Leopardus guigna). Stromal vascular fraction (SVF) isolated from cougar adipose tissue have been used to restore elbow functionality in the cougar (Puma concolor) but multipotent characteristics of these cells have not been described. The present study describes for the first time the isolation and characterization of mesenchymal stem cells derived from adipose tissue of cougar. AMSCs and fibroblasts from six months female cougar were isolated and cultured in DMEM/F12, supplemented with FBS 10% + 1% Antibiotic/Antifungal + 2.4 mM L-Glutamine + 2.4 mM pyruvate up to passage 5. Expression of pluripotent and surface marker genes was evaluated at mRNA level. Mesodermal differentiation (adipogenic, osteogenic and chondrogenic) was described. AMSCs expressed mRNA of pluripotent genes Oct4, Nanog, Sox2 and Klf4 and surface markers Cd44, Cd90, Cd105 and MHCII. Fibroblasts showed similar mRNA expression with the exception of Sox2. AMSCs obtained from cougar exhibit multipotency features similar to domestic cats MSC, nevertheless, other analyses are required. AMSCs from cougar could be a source of interest for treatment of individuals that remain in captivity or arrive to wildlife rehabilitation centers.

Keywords: Puma concolor; cell therapy; feline; stem cell; wild cat.

Figure 1. Cougar AMSC and fibroblasts cultured in vitro. (a) AMSC from cougar at P1; (b) Fibroblasts from cougar at P1.

Figure 2. Multilineage differentiation of cougar AMSC. Cells showed differentiation capacity towards adipogenic, chondrogenic and osteogenic lineages. (a) Control without differentiation; (b) In adipogenic differentiation cells showed an increased granularity and larger intracellular oil droplets stained red by Oil Red; (c) Osteogenic induction was evidenced by calcium phosphate deposit stained red by Alizarin Red; (d) Aggregates with proteoglycan content after 28 days of chondrogenic induction culture showed intense Alcian Blue staining.

Figure 3. Agarose gel electrophoresis of the comparative expression of selected transcripts from AMSC and dermal fibroblasts from Puma concolor . Pluripotency genes: Oct4, Nanog, Klf4 and Sox2. Surface markers: CD44, CD90 and Major histocompatibility complex MHCII . Sdha was employed as a housekeeping. Molecular weight ladder is not shown, the length of the amplicons for each transcript is indicated in Table 2.

Figure 4. Detection of OCT4 and SOX2 by immunocytochemistry in AMSC from Puma concolor. (a and c) negative controls of expression for OCT4 and SOX2 respectively; the cells were fixed, primary antibody was omitted and the cells were incubated with the secondary anti-rabbit HRP sheep conjugated antibody and revealed with DAB; (b) Strong detection of OCT4 in cytoplasm and nuclei and (d) SOX2 immunodetection in the cytoplasm of some cells. Antibodies, catalogue numbers and specificity are described in materials and methods.