340 BIOLOGICAL CHARACTERISTICS AND FUNCTIONAL CAPABILITY OF FELINE ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS

Abstract

Chronic kidney disease is a major cause of mortality in cats (Boyd et al. 2008J. Vet. Intern. Med. 22, 1111–1117). Similarly, the black-footed cat (Felis nigripes; BFC) frequently suffers from kidney failure caused by amyloidosis (Terio et al. 2008 Vet. Pathol. 45, 393–400). Adipose tissue-derived mesenchymal stem cells (AMSC) are a valuable cell source in regenerative medicine for treating certain diseases, including those suffered by endangered species. In the domestic cat (DSH), AMSC have been isolated from subcutaneous (SQ; Quimby et al. 2011 J. Feline Med. Surg. 13, 418–426) and epididymal adipose tissue (Zhang et al. 2014 Stem Cell Rev. Rep. 10, 600–611). Whether AMSC isolated from visceral fat of the abdominal cavity (AB) have similar developmental potential has not been studied. In this study, we (1) compared the biological characteristics of DSH-AMSC isolated from AB and SQ adipose tissue, and (2) evaluated the functional capability of DSH and BFC-AMSC to differentiate into other cell types. The AB and SQ adipose tissues were harvested via laparoscopy or from an incision in the ventral abdomen, respectively. Tissues were digested with collagenase II (1 mg mL–1) at 37°C for 20 to 40 min with shaking at 150 rpm for 20 to 40 min. Cells from the stromal vascular fraction were cultured in DMEM-F12 medium with 12% fetal bovine serum under 5% CO2 in air at 38°C. Results showed that AB biopsies were smaller (1.2 ± 0.2 g) than that of SQ biopsies (3.6 ± 0.7 g). The mean number of nucleated cells per gram from AB biopsies (0.6 to 22 × 106) was similar to that of SQ biopsies (0.4 to 24 ×106). The cell-doubling numbers (days) per passage (P1 to P5) in both cell types remained constant (0.9 to 2.6), but SQ-AMSC at P5 required more cell doublings (4.5 ± 2.1) to reach 50% confluence. The AB-AMSC showed more colony-forming units (CFU; 7.0%) after 8 to 10 days of seeding at 8000 per cm2 than did SQ-AMSC (1.5% CFU). The SQ-AMSC did not form colonies at cell densities below 4000 per cm2. However, AB-AMSC colony formation only substantially decreased when the cell densities were below 1000 per cm2 (0.1%). Flow cytometry analysis revealed higher percentages of CD90+ (92%), CD105+ (80%), and CD146+ (17%) cells in AB-AMSC than in SQ-AMSC (77, 57, and 9%, respectively). Both AB and SQ-AMSC showed negative expressions of CD14–, CD45–, CD73–, and HLA-DR–. Gene expression analysis revealed that pluripotent genes Nanog, KLF4, Oct-4, and proto-oncogene C-Myc were expressed by both cell types, while Sox2 was not expressed in either type of AMSC. Under appropriate stimuli, DSH- and BFC-AMSC demonstrated differentiation potential towards adipogenic, osteogenic, chondrogenic, and neurogenic lineages. The AMSC from both species were less responsive towards osteogenesis than adipogenesis, and BFC cells had more capability to differentiate towards chondrocytes. These results suggest that the defined AMSC population (regardless of site of collection) could potentially be employed as a therapeutic agent for diseased or injured felids, both domestic and endangered.