J Vet Sci.  2017 Sep;18(3):299-306. 10.4142/jvs.2017.18.3.299.

Extensive characterization of feline intra-abdominal adipose-derived mesenchymal stem cells

• Kim HR
• Lee J
• Byeon JS
• Gu NY
• Lee J
• Cho IS
• Cha SH

• KMID: 2412447
• DOI: http://doi.org/10.4142/jvs.2017.18.3.299

Abstract

Mesenchymal stem cells (MSCs) isolated from various tissues have been well characterized for therapeutic application to clinical diseases. However, in contrast to MSCs from other animal species, the characteristics of feline MSCs have not been fully documented. In this study, we conducted extensive characterization of feline adipose tissue-derived MSCs (fAD-MSCs). Study fAD-MSCs were individually isolated from the intra-abdominal adipose tissues of six felines. The expression levels of cell surface markers and pluripotent markers were evaluated. Next, proliferation capacity was analyzed by performing cumulative population doubling level (CPDL) and doubling time (DT) calculation assays. Differentiation potentials of fAD-MSCs into mesodermal cell lineages were analyzed by examining specific staining and molecular markers. All fAD-MSCs positively expressed cell surface markers such as CD29, CD44, CD90, CD105, CD166, and MHC-I, while CD14, CD34, CD45, and CD73 were negatively expressed. The CPDL of the fAD-MSCs was maintained until passage 5 to 6 (P5 to P6), whereas DT increased after P3 to P4. Also, stem cell-specific pluripotent markers (Oct3/4, Nanog, and SSEA-4) were detected. Importantly, all fAD-MSCs demonstrated mesodermal differentiation capacity. These results suggest that fully characterized fAD-MSCs could be beneficial when considering the use of these cells in feline disease research.

Keyword

adipose tissue-derived mesenchymal stem cells; differentiation; feline; multipotent; proliferation

MeSH Terms

Abdomen
Adipose Tissue/*cytology
Animals
Biomarkers/metabolism
Cats/*anatomy & histology
Cell Differentiation
Cell Proliferation
Flow Cytometry/veterinary
Fluorescent Antibody Technique/veterinary
Mesenchymal Stromal Cells/*cytology/physiology
Multipotent Stem Cells/cytology/physiology
Biomarkers

Figure

• 

  Fig. 1 Expression of MSC surface markers and proliferation ability of fAD-MSCs. (A) Typically, cell morphology was fibroblast-like. (B and C) FACS and RT-PCR analysis: fAD-MSCs positively expressed CD29, CD44, CD90, CD105, CD166, and MHC-I, but CD14, CD34, CD45, CD73, and MHC-II were negatively expressed at P2. (C) GAPDH is shown as a control for RNA sample quality. (D) Cumulative population doubling level (CPDL) linearly increased until P4 or P5, and (E) doubling time (DT) did not increase until P3. CPDL and DT data from three independent experiments are expressed as mean ± SD values. 40× (upper panels in A), 100× (lower panels in A). *p < 0.05, **p < 0.001.

• 

  Fig. 2 Expression of pluripotent markers in fAD-MSCs at passage 2. (A and B) Images from RT-PCR and IFS analyses: fAD-MSCs positively expressed Oct3/4, Nanog, and SSEA-4, but Sox2 was negatively expressed. (A) GAPDH is shown as a control for RNA sample quality. 140×.

• 

  Fig. 3 Multilineage differentiation potential of fAD-MSCs at P2. (A) fAD-MSCs, after being induced in adipogenic differentiation medium, formed lipid droplets. (B) MSCs formed proteoglycans when incubated in chondrogenic differentiation medium. (C) Calcium mineralization was formed after incubation in osteogenic differentiation medium. Oil Red O (A; red), Alcian Blue (B; blue), and Alizarin Red (C; orange red). 200× (A and B), 100× (C).

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