Isolation, characterization and differentiation of mesenchymal stem cells from amniotic fluid, umbilical cord blood and Wharton's jelly in the horse

Abstract

Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton's Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2 h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically significant differences were found between cell-doubling times (DTs): cells isolated from WJ expanded more rapidly (2.0G0.6 days) than those isolated from CB (2.6G1.3 days) and AF (2.3G1.0 days) (P!0.05). Positive von Kossa and Alizarin Red S staining confirmed osteogenesis. Alcian Blue staining of matrix glycosaminoglycans illustrated chondrogenesis and positive Oil Red O lipid droplets staining suggested adipogenesis. All cell lines isolated were positive for CD90, CD44, CD105; and negative for CD34, CD14 and CD45. These findings suggest that equine MSCs from AF, UCB and WJ appeared to be a readily obtainable and highly proliferative cell lines from a uninvasive source that may represent a good model system for stem cell biology and cellular therapy applications in horses. However, to assess their use as an allogenic cell source, further studies are needed for evaluating the expression of markers related to cell immunogenicity.

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

“…However, as both antigens are expressed by a variety of cells types, they are not part of the ISCT MSC definition. Furthermore, this study confirmed the rare expression of CD 73 on equine MSCs, which had already been stated by several research groups (31,33,39,41,42). Inconsistent findings were reported concerning the expression of CD 105 on equine MSCs.…”

“…This finding was consistently reported in different studies and Original Article for different cell source (33,(40)(41)(42). However, as both antigens are expressed by a variety of cells types, they are not part of the ISCT MSC definition.…”

Phenotypical and functional characteristics of mesenchymal stem cells derived from equine umbilical cord blood

“…Similar results have been reported previously in monolayer cultures (Iacono et al 2012) and in pellet cultures (De Schauwer et al 2011;Koch et al 2007;Schuh et al 2009). The findings were further confirmed by expression of Collagen 2aI gene by RT-PCR as reported previously (Reed and Johnson 2008).…”

“…Mean population doubling time for initial 8 passages in the present study was lower than that reported for UCB-MSCs (Iacono et al 2012) whereas the plating efficiency (2.57 ± 0.16 %) during initial 8 passages was higher than those reported for MSCs isolated from amniotic fluid, umbilical cord matrix and bone marrow (Lovati et al 2011). In human medicine, a lower doubling time (DT) and therefore, a greater proliferative activity has been reported for cells isolated from umbilical cord matrix and from cord blood compared to bone marrow-derived MSCs (Karahuseyinoglu et al 2007).…”

“…The UCB-MSCs have been recognized as important source of MSCs in equines (Iacono et al 2012;Koch et al 2007;Schuh et al 2009). However, MSCs isolated from equine umbilical cord are not fully characterized due to lack of species-specific markers.…”