J Vet Sci.  2018 Jan;19(1):13-20. 10.4142/jvs.2018.19.1.13.

Comparison of the characteristics of canine adipose tissue-derived mesenchymal stem cells extracted from different sites and at different passage numbers

• Yaneselli
• Kuhl CP
• Terraciano PB
• de Oliveira FS
• Pizzato SB
• Pazza K
• Magrisso A
• Torman V
• Rial A
• Moreno M
• Llambí S
• Cirne-Lima E
• Maisonnave J

• KMID: 2402690
• DOI: http://doi.org/10.4142/jvs.2018.19.1.13

Abstract

Mesenchymal stem cells (MSCs) have desirable characteristics for use in therapy in animal models and veterinary medicine, due to their capacity of inducing tissue regeneration and immunomodulation. The objective of this study was to evaluate the differences between canine adipose tissue-derived MSCs (AD-MSCs) extracted from subcutaneous (Sc) and visceral (Vs) sites. Surface antigenic markers, in vitro differentiation, and mineralized matrix quantification of AD-MSCs at different passages (P₄, P₆, and P₈) were studied. Immunophenotypic analysis showed that AD-MSCs from both sites were CD44+, CD90+, and CD45−. Moreover, they were able, in vitro, to differentiate into fat, cartilage, and bone. Sc-AD-MSCs preserve in vitro multipotentiality up to P₈, but Vs-AD-MSCs only tri-differentiated up to P₄. In addition, compared to Vs-AD-MSCs, Sc-AD-MSCs had greater capacity for in vitro mineralized matrix synthesis. In conclusion, Sc-AD-MSCs have advantages over Vs-AD-MSCs, as Sc AD-MSCs preserve multipotentiality during a greater number of passages, have more osteogenic potential, and require less invasive extraction.

Keyword

canine; immunophenotyping; mesenchymal stem cells; multipotent plasticity

MeSH Terms

Antigens, Surface
Cartilage
Immunomodulation
Immunophenotyping
In Vitro Techniques
Mesenchymal Stromal Cells*
Miners
Models, Animal
Regeneration
Veterinary Medicine
Antigens, Surface

Figure

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  Fig. 1 Expressions of CD44, CD90, and CD45 on adipose tissue-derived mesenchymal stem cells of subcutaneous (Sc) or visceral (Vs) origin were assessed by performing flow cytometry (n = 2). Data are representative of analyzed passages.

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  Fig. 2 Tri-differentiation microscopic images of adipose tissue-derived mesenchymal stem cells (AD-MSCs) at P2 in vitro (n = 5). Adipogenic differentiation images at 400× (intracellular lipid vacuoles) for subcutaneous (Sc) and visceral (Vs) (A and G). Chondrogenic differentiation images at 40× (B and H). Osteogenic differentiation images at 40× (C and I). Negative controls are shown in images D–F and J–L.

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  Fig. 3 Evaluation of in vitro differentiation of adipose tissue-derived mesenchymal stem cells from both extraction sites at different passages (n = 5). (A) Adipogenic differentiation. (B) Chondrogenic differentiation. (C) Osteogenic differentiation. The “non-growth” category exhibits an initial gradual increase, becoming abrupt between P6 to P8. The multipotentiality had a negative effect on adipose (p < 0.001) and cartilage (p < 0.01) lineages, but the bone lineage showed a marked tendency with the increase of passages (p = 0.054).

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  Fig. 4 In vitro bone matrix synthesis capacity of adipose tissue-derived mesenchymal stem cells of subcutaneous (Sc) and visceral (Vs) origins at P4 (n = 5). Macroscopic and microscopic (40×) images of Sc (A and B) and Vs (C and D) cells. The Sc cells showed a greater capacity for matrix synthesis than that shown by Vs cells (E). *p < 0.05.

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  Fig. 5 Comparison of in vitro bone matrix synthesis capacity of subcutaneous adipose tissue-derived mesenchymal stem cells at different passages (n = 5). Microscopic images (40×) at P4 (A), P6 (B), and P8 (C). A significant difference is detected between P6 and P8 (D), **p < 0.001.

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