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 (2.0±0.6 days) than those isolated from CB (2.6±1.3 days) and AF (2.3±1.0 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.

A complex feedback of growth factors, secreted by a variety of cell types, is responsible for the mediation of skin healing. Despite the recent advances in wound healing management, this fails up to 50% and skin wounds can still be considered one of the main causes of morbidity, both in human and veterinary medicine. Regenerative medicine, involving mesenchymal stromal cells (MSCs), is nowadays a promising solution for skin wound healing. Indeed, MSCs are involved in the modulation of the inflammatory local response and cell replacing, by a paracrine mode of action. Local application of equine umbilical cord Wharton's jelly MSCs (WJMSCS) was carried out in a 6-months-old filly with a non-healing skin wound. Heterologous WJMSCs were applied four times using a carboxymethylcellulose (CMC) gel, produced dissolving CMC in autologous plasma. At first application the mean wound area was 7.28 ± 0.2 cm². Four days after the last application of WJMSCs, the mean wound area was 1.90 ± 0.03 cm², and the wound regression rate was +74%. No local or systemic side effects were registered after WJMSCs application and no evident exuberant scar was observed after wound healing. At discharge, the mean wound area was 0.38 ± 0.01 cm² and the total regression rate was +80%. Five days later, the wound was completely healed. In the present clinical case report, the use of WJMSCs led to promising clinical results, paving the way for possible future applications in the treatment of chronic wounds in horses.

Both in human and equine species, mesenchymal stem cells (MSCs) from amniotic membrane (AM) and Wharton’s jelly (WJ), may be particularly useful for immediate use or in later stages of life, after cryopreservation in cell bank. The aim of this study was to compare equine AM- and WJ-MSCs in vitro features that may be relevant for their clinical employment. MSCs were more easily isolated from WJ, even if MSCs derived from AM exhibited more rapid proliferation (P < 0.05). Osteogenic and chondrogenic differentiation were more prominent in MSCs derived from WJ. This is also suggested by the lower adhesion of AM cells, demonstrated by the greater volume of spheroids after hanging drop culture (P < 0.05). Data obtained by PCR confirmed the immunosuppressive function of AM and WJ-MSCs and the presence of active genes specific for anti-inflammatory and angiogenic factors (IL-6, IL 8, IL-β1). For the first time, by means of transmission electron microscopy (TEM), we ascertained that equine WJ-MSCs constitutively contain a very impressive number of large vesicular structures, scattered throughout the cytoplasm. Moreover, an abundant extracellular fibrillar matrix was located in the intercellular spaces among WJ-MSCs. Data recorded in this study reveal that MSCs from different fetal tissues have different characteristics that may drive their therapeutic use. These finding could be noteworthy for horses as well as for other mammalian species, including humans.