Comparative immunophenotyping of equine multipotent mesenchymal stromal cells: An approach toward a standardized definition

Abstract

Horses are an approved large animal model for therapies of the musculoskeletal system. Especially for tendon disease where cell-based therapy is commonly used in equine patients, the translation of achieved results to human medicine would be a great accomplishment. Immunophenotyping of equine mesenchymal stromal cells (MSCs) remains the last obstacle to meet the criteria of the International Society for Cellular Therapy (ISCT) definition of human MSCs. Therefore, the surface antigen expression of CD 29, CD 44, CD 73, CD 90, CD 105, CD 14, CD 34, CD 45, CD 79a, and MHC II in equine MSCs from adipose tissue, bone marrow, umbilical cord blood, umbilical cord tissue, and tendon tissue was analyzed using flow cytometry. Isolated cells from the different sources and donors varied in their expression pattern of MSC-defining antigens. In particular, CD 90 and 105 showed most heterogeneity. However, cells from all samples were robustly positive for CD 29 and CD 44, while being mostly negative for CD 73 and the exclusion markers CD 14, CD 34, CD 45, CD 79a and MHC II. Furthermore, it was evident that enzymes used for cell detachment after in vitro-culture affected the detection of antigen expression. These results emphasize the need of standardization of MSC isolation, culturing, and harvesting techniques. As the equine MSCs did not meet all criteria the ISCT defined for human MSCs, further investigations for a better characterization of the cell type should be conducted. V C 2014 International Society for Advancement of Cytometry

Equine Bone Marrow and Adipose Tissue Mesenchymal Stem Cells: Cytofluorimetric Characterization, In Vitro Differentiation, and Clinical Application

“…Several sources have been studied for obtaining equine MSCs; however, bone marrow and adipose tissue are the most studied sources of MSCs in this species [17,18,20,21]. With the present work, we confirm what has already been reported in the literature but at the same time, we open Table 4 Flow cytometry analysis of eATMSCs and eBMMSCs at passage 3 of in vitro culture.…”

“…In our article, we took into account laboratory and clinical aspects, to be sure of implanting a cell population as much as possible homogeneous, such as required by the guidelines for the use of cell therapy in veterinary medicine as reported in the Official Gazette of the Italian Republic. However, as recently reported by Paebst et al [18], cell sources used in the present study could reliably meet the ISCT criteria of human MSC definition. In fact, although eBMMSCs and eATMSCs are plastic adherent, they could not meet other minimal criteria which are demanded for human MSCs, and the plastic adherence, as a selection method for equine MSCs, does not lead to uniform populations.…”

“…Hand walk 20 min twice/d 10- 16 Hand walk 30-40 min twice/d [17][18][19][20][21][22][23][24][25] Hand walk 40 min twice/d and trot 5-30 min/d Gradual increase of exercise level eATMSCs (P > .05). No lag phase was observed during the in vitro culture of both cell lines; in fact, no statistically significant differences in the number of CD were registered among different culture passages (P > .05).…”

“…As positive control for CD34 and CD14 equine peripheral blood mononuclear cells (PBMCs) were used, while positive surface markers (according to ISCT guidelines) were evaluated on equine Wharton's Jelly (WJ) MSCs, already tested positive for these markers [14]. It has been previously demonstrated that certain surface markers can be trypsin labile [18]; for this reason, control cells were mechanically detached as described by Paebst et al [18]. In addition, a comparison of the amino acidic sequences of human and horse studied CDs was performed using Blast (Basic Local Alignment Search Tool; Supplementary Figs.…”

“…In horses, cells from equine bone marrow (eBM) and adipose tissue (eAT) have been isolated, and some researchers demonstrated their multilineage differentiation potential by the ability to undergo adipogenic, osteogenic, and chondrogenic differentiation [11,13,16]. However, only few authors determined immunophenotypic characterization of cells from eAT and eBM [17,18] by flow cytometry, as requested for human MSCs by ISCT [15].…”