Stemness features in mesenchymal stem cells from equine amniotic fluid, Wharton’s jelly and umbilical cord blood

Foetal adnexa contain a heterogeneous mixture of cells including a stem cell population that express a panel of pluripotency markers, such as Oct4, Nanog, Sox2 and SSEA4 (Pappa and Anagnou, 2009; Dyvia et al, 2012). These extra-embryonic tissues and fluids represent viable, accessible and safe sources. The aim of this work was to evaluate pluripotency markers expression in mesenchymal stem cells isolated from equine amniotic fluid (eAF-MSCs), Wharton’s jelly (eWJ-MSCs) and umbilical cord blood (eUCB-MSCs) as continuation of the characterization of these cells previously performed by our research group (Iacono et al., 2012). Equine AF, WJ and UCB samples were recovered at parturition from 3 mares and (46.7 ± 14.4) ml of AF, (2.4 ± 2.2) g of WJ and (23.3 ± 14.4) ml of UBC were collected at foaling. Isolated cells were isolated and cultured as previously described (Iacono et al. 2012). Briefly, cells were cultured in DMEM-TCM 199 (1:1) plus 10% FBS and incubated in a 5% CO2 humidified atmosphere at 38.5 °C. At passage 3 characterization was performed by immunocytochemistry (ICC) and RT-PCR. ICC was carried out for SSEA4 protein fixing cells with paraformaldehyde (4%), doing an overnight incubation at 4°C with anti-SSEA4 primary antibody and then incubating cells with FITC-labeled secondary antibody for 1 hr at room temperature in the dark. Nuclei were stained with Hoechst and the percentages of positive cells were obtained doing ratio between FITC+ cells and nuclei. Equine skin fibroblasts were used as negative control. RT-PCR was performed to analyze mRNA expression of Nanog and Sox2. Equine blastocysts and equine skin fibroblasts were used respectively as positive and negative controls and GAPDH was employed as reference gene. ICC analysis showed expression of the embryonic stem cells antigen SSEA4 in eAF-MSCs (14.8 ± 9.5 %), eWJ-MSCs (8.9 ± 2.6 %) and eUCB-MSCs (7.1 ± 4.8 %). On the other hand, expression of Nanog and Sox2 was not found in all samples, unlike human AF-MSCs and WJ-MSCs. ICC and RT-PCR analyses for the embryonic marker Oct4 (data not reported) gave unclear results so further studies are necessary. Results obtained in the present study reveal that equine MSCs derived from foetal adnexa are an heterogeneous population and express low level of the embryonic marker SSEA4, but do not express embryonic pluripotent markers like Sox2 and Nanog as described in the literature for other species. So these cells could represent an intermediate stage between pluripotent embryonic stem cells and lineage-restricted adult stem cells, as previously demonstrated for human MSCs (De Coppi et al. 2007; Pappa and Anagnou, 2009). Further work is ongoing to find specific primers and antibodies to assess the expression of OCT4.