Omental adipose tissue is a more suitable source of canine Mesenchymal stem cells

Abstract

Background: Mesenchymal Stem Cells (MSCs) are a promising therapeutic tool in veterinary medicine. Currently the subcutaneous adipose tissue is the leading source of MSCs in dogs. MSCs derived from distinct fat depots have shown dissimilarities in their accessibility and therapeutic potential. The aims of our work were to determine the suitability of omental adipose tissue as a source of MSCs, according to sampling success, cell yield and paracrine properties of isolated cells, and compared to subcutaneous adipose tissue.

Results: While sampling success of omental adipose tissue was 100% (14 collections from14 donors) for subcutaneous adipose tissue it was 71% (10 collections from 14 donors). MSCs could be isolated from both sources. Cell yield was significantly higher for omental than for subcutaneous adipose tissue (38 ± 1 vs. 30 ± 1 CFU-F/g tissue, p < 0.0001). No differences were observed between sources regarding cell proliferation potential (73 ± 1 vs. 74 ± 1 CDPL) and cell senescence (at passage 10, both cultures presented enlarged cells with cytoplasmic vacuoles and cellular debris). Omental- and subcutaneous-derived MSCs expressed at the same level bFGF, PDGF, HGF, VEGF, ANG1 and IL-10. Irrespective of the source, isolated MSCs induced proliferation, migration and vascularization of target cells, and inhibited the activation of T lymphocytes.

Conclusion: Compared to subcutaneous adipose tissue, omental adipose tissue is a more suitable source of MSCs in dogs. Since it can be procured from donors with any body condition, its collection procedure is always feasible, its cell yield is high and the MSCs isolated from it have desirable differentiation and paracrine potentials.

Keywords: Adipose tissue; Canine; Dog; Mesenchymal stem cell; Omentum; Source.

Fig. 1

Omental adipose tissue and subcutaneous adipose tissue have MSCs. a Representative photomicrographs of primary culture of subcutaneous- and omental-derived cells. b Flow cytometry analysis after immunostaining with monoclonal antibodies against CD45, CD11b, CD90 and CD44 (green lines) or isotype controls (red lines). c Representative photomicrographs of MSCs stained with Oil Red O (adipogenesis), Safranin O (chondrogenesis) or Alizarin Red (osteogenesis) 3 weeks after differentiation induction. Control undifferentiated cells are also shown. Abbreviations: SC: subcutaneous adipose tissue; OM: omental adipose tissue

Fig. 2

Omental adipose tissue has higher abundance of MSCs than subcutaneous adipose tissue. a Quantitative analysis of CFU-Fs per gram of tissue sampled. b Representative photographs of plates stained with crystal violet 7 days after seeding of nucleated cells from tissue samples. c Quantitative analysis of CPDL up to passage 12. d Representative photomicrographs of S-MSCs and O-MSCs at passages 1 (P1) and 10 (P10). Abbreviations: SC: subcutaneous adipose tissue; OM: omental adipose tissue; CFU-F/g tissue: colony forming units per grams of tissue; S-MSCs: mesenchymal stem cells derived from subcutaneous adipose tissue; O-MSCs: mesenchymal stem cells derived from omental adipose tissue; CDPL: cumulative population doubling level

Fig. 3

MSCs derived from omental and subcutaneous adipose tissues have similar trophic properties. a Quantitative analysis of mRNA levels of trophic factors. b Quantitative analysis of proliferation kinetic of human fibroblast cultivated with S-MSCs CM or O-MSCs CM evaluated during 12 days. c Representative photomicrographs of invaded area at 0 (T0), six (T6) and 12 (T12) hours post exposure to CMs. d Quantitative analysis of invaded area evaluated as the percentage of area free of cells at 6 or 12 h post exposure to CM with respect to 0 h. Abbreviations: S-MSCs: mesenchymal stem cells derived from subcutaneous adipose tissue; O-MSCs: mesenchymal stem cells derived from omental adipose tissue; CM: conditioned medium; bFGF: basic fibroblast growth factor; PDGF: platelet-derived growth factor; HGF: hepatocyte growth factor

Fig. 4

MSCs derived from omental and subcutaneous adipose tissues have similar vasculogenic properties. a Quantitative analysis of mRNAs levels of vasculogenic factors. b Representative photomicrographs of tube assay performed with 3D collagen-embedded human umbilical endothelial cells cultivated 5 h with S-MSCs CM or O-MSCs CM and analyzed by Wimasis software. c Quantitative analysis of total tube length, total branching point and total loops of tube assay among the different conditions. Abbreviations: S-MSCs: mesenchymal stem cells derived from subcutaneous adipose tissue; O-MSCs: mesenchymal stem cells derived from omental adipose tissue; CM: conditioned medium; VEGF: vascular endothelial growth factor; ANG1 angiopoietin 1

Fig. 5

MSCs derived from omental and subcutaneous adipose tissues have similar immunomodulatory properties. a Quantitative analysis of the mRNAs levels of immunomodulatory factors. Each value was normalized to 18S expression. b Quantitative analysis of total CD4+ T-cells stimulated and cultivated with S-MSC CM and O-MSC CM after 5 days. Abbreviations: S-MSCs: mesenchymal stem cells derived from subcutaneous adipose tissue; O-MSCs: mesenchymal stem cells derived from omental adipose tissue; CM: conditioned medium; IDO indoleamine-pyrrole 2,3-dioxygenase; IL-10 interleukin 10