Characterization and differentiation potential of mesenchymal stem cells isolated from multiple canine adipose tissue sources

Abstract

Background: Mesenchymal stem cells (MSCs) are undifferentiated cells that can give rise to a mesoderm lineage. Adipose-derived MSCs are an easy and accessible source for MSCs isolation, although each source of MSC has its own advantages and disadvantages. Our study identifies a promising source for the isolation and differentiation of canines MSCs. For this purpose, adipose tissue from inguinal subcutaneous (SC), perirenal (PR), omental (OM), and infrapatellar fat pad (IPFP) was isolated and processed for MSCs isolation. In the third passage, MSCs proliferation/metabolism, surface markers expression, in vitro differentiation potential and quantitative reverse transcription PCR (CD73, CD90, CD105, PPARγ, FabP4, FAS, SP7, Osteopontin, and Osteocalcin) were evaluated.

Results: Our results showed that MSCs derived from IPFP have a higher proliferation rate, while OM-derived MSCs have higher cell metabolism. In addition, MSCs from all adipose tissue sources showed positive expression of CD73 (NT5E), CD90 (THY1), CD105 (ENDOGLIN), and very low expression of CD45. The isolated canine MSCs were successfully differentiated into adipogenic and osteogenic lineages. The oil-red-O quantification and adipogenic gene expression (FAS, FabP4, and PPARγ) were higher in OM-derived cells, followed by IPFP-MSCs. Similarly, in osteogenic differentiation, alkaline phosphatase activity and osteogenic gene (SP7 and Osteocalcin) expression were higher in OM-derived MSCs, while osteopontin expression was higher in PR-derived MSCs.

Conclusion: In summary, among all four adipose tissue sources, OM-derived MSCs have better differentiation potential toward adipo- and osteogenic lineages, followed by IPFP-MSCs. Interestingly, among all adipose tissue sources, MSCs derived from IPFP have the maximum proliferation potential. The characterization and differentiation potential of canine MSCs isolated from four different adipose tissue sources are useful to assess their potential for application in regenerative medicine.

Keywords: Adipogenesis; Adipose tissue; Dog; Mesenchymal stem cell; Osteogenesis.

Fig. 1

Tissue collection sites of dogs. A. Inguinal subcutaneous, B. Perirenal, C. Omentum fat, D. Infrapatellar fat pad

Fig. 2

The phenotype of canine MSCs at passage 3 (objective 20x). Panel A. Subcutaneous B. Perirenal C. Omental fat-derived MSCs. All recovered cells have a long spindle-shaped appearence, while, D. Infrapatellar fat pad derived MSCs were relatively smaller in size as compared to panel A, B, and C. Scale bar = 20 μm

Fig. 3

(A) Cell doubling time on days 3, 6, 9, and 12. *** P < 0.001, ** P = 0.004. (B) Cellular Metabolism (MTT assay) at day 3, 6, 9 and 12. The data is shown as mean ± SEM. Significance among days, ***P < 0.001, **P = 0.003. Abbreviations: S/C: Subcutaneous, PR: Perirenal, OM: Omentum, IPFP: Infrapatellar fat pad

Fig. 4

(A) Flow cytometric expression of CD73, CD90, CD105, and CD45 in undifferentiated canine MSCs, derived from different fat sources. (B) Immunostaining of undifferentiated cMSCs derived from different tissue sources. The cells were stained positive with surface antigens CD73, CD90, and CD105 (green) and nuclear staining (DAPI, blue). Scale bar = 25 μm. Abbreviations: S/C: Subcutaneous, PR: Perirenal, OM: Omentum, IPFP: Infrapatellar fat pad

Fig. 5

After 7 days of adipogenesis in the adipogenic media the cells were stained with oil-red-O (A) control and adipocytes (Objective 20×). Scale bar = 20 μm. (B) Quantification of ORO staining between undifferentiated cells (control) and differentiated adipocytes. The data was normalized to the number of cells and expressed as the mean ± SEM. Superscripts letter ^A indicates a level of significance P < 0.001. (C) Immuno-expression of FabP4, cells from all fat sources showed positive expression of FabP4. Scale bar = 25 μm. Abbreviations: S/C: Subcutaneous, PR: Perirenal, OM: Omentum, IPFP: Infrapatellar fat pad

Fig. 6

Alizarin red staining of canine MSCs in osteogenic medium after 21 days. (A) Control and osteogenic cells show deposition of hydroxyapatite mineral stained red with Alizarin red stain (objective 20×). Scale Bar = 20 μm. (B) The ALP activity of differentiated osteocytes compared to undifferentiated control cells. The data was normalized by unit per gram of protein and expressed as mean ± SEM. ^A-CDifferent superscripts letters indicate significant differences (P < 0.05). (C) Immuno-expression of osteopontin in canine MSCs recovered from all four fat sources and kept in osteogenic media showed a positive expression of Osteopontin. Scale bar = 25 μm. Abbreviations: S/C: Subcutaneous, PR: Perirenal, OM: Omentum, IPFP: Infrapatellar fat pad

Fig. 7

At P3, the MSCs from all four fat sources were evaluated for the relative mRNA expression (CNRQ) of CD73 (A), CD90 (B), and CD105 (C) genes. After 7 days of adipogenesis, the differentiated adipocytes were evaluated for expression of FAS (D), FABP4 (E), and PPARγ (F). While, the expression of osteogenic genes SP7 (G), Osteocalcin (H), Osteopontin (I) were evaluated in differentiated cMSC after 21 days in osteogenic media. The data was presented as mean ± SEM. ^A,BDifferent superscripts letter indicates significant differences within a given adipocyte source (P < 0.001). Abbreviations: S/C: Subcutaneous, PR: Perirenal, OM: Omentum, IPFP: Infrapatellar fat pad