342 QUANTITATIVE IMAGING FLOW CYTOMETRY CHARACTERIZATION OF FUNCTIONALITY AND VIABILITY OF EQUINE ADIPOSE-DERIVED STEM CELLS

Abstract

Equine adipose-derived mesenchymal stem cells (Eq adMSC) are a potential cell source for cell therapy due to their capacity to self-renew and differentiate in specialised cell types. In 2006, the International Society for Cellular Therapy (ISCT) defined human MSC as aplastic-adherent cells showing the capacity of tri-lineage differentiation that express specific surface markers (i.e. CD73, CD90, and CD105). In 2013, under the statements of the ISCT and International Federation of Adipose Therapeutics (IFAT), Bourin et al. added CD44 and CD29 to the list of MSC-specific surface antigens and recommended to include in the validation some markers of functionality and viability. Establishing a specific Eq adMSC panel has, up to now, never been done and is complicated by the nonavailability of equine-specific antibodies. Indeed, to our knowledge, only CD44 and MHCII equine-specific monoclonal antibodies are available commercially. To develop an equine-specific characterisation of Eq adMSC, nonequine antibodies should be tested for cross-reactivity. The aim of the present study was to immunophenotypically characterise Eq adMSC for the presence of membrane and intracellular proteins, as well as for apoptosis and viability. The information would then be used to establish references for equine stem cells. The Eq adMSC were obtained from the subcutaneous fat tissue of 13 adult horses. Briefly, fat tissue was minced, washed in PBS buffer, and digested for 30 min in a solution containing 1 mg mL^–1 of collagenase I. The cells were washed in PBS and resuspended in D-MEM, low glucose-containing 10% FBS, and antibiotics, plated at a density of 2.5 × 10⁴ cells cm^–2, and cultured for 8 days in 5% CO₂ at 37°C. After 24 h, nonadherent cells were washed off and the Eq adMSC were cultured until 90% confluence was obtained. The Eq adMSC were then detached with trypsin, first passaged until 90% confluence, and evaluated. The following markers were evaluated by quantitative imaging flow cytometry (ImageStream MK II, Amnis-Millipore): surface (CD29-RD1, CD44-FITC, and CD105-AF648); haematopoietic (CD34-FITC); intracellular (SOX2-DL488 and OCT3/4-DL488); and apoptosis (Annexin V-FITC and DraQ5). At the same passage, as an additional validation, osteogenic differentiation was induced. After 20 days, Alizarin O Red was used to detect extracellular calcium deposition. Following quantitative imaging flow cytometry analyses, all 13 cell lines were simultaneously positive for CD44, CD29, CD105, SOX2, and OCT3/4 and negative for CD34 (mean ± SD across all cell lines: 95.21 ± 10.4, 99.00 ± 0.93, 97.37 ± 2.90, 79.82 ± 14.99, and 0.37 ± 0.13%, respectively). Low frequencies of apoptotic (1.25 ± 0.5%) and necrotic cells (3.10 ± 0.8%) were found. All the 13 horse cultures differentiated in osteogenic tissue, confirming the efficiency of the purification process. These results demonstrate the presence of the ISCT and IFAT main specific markers in Eq adMSC and validate the efficiency of our purification protocol. This information will be used to further improve our knowledge of Eq adMSC in this species.