Despite the importance of ovine as a large animal model for many conditions (i.e., orthopaedic injuries or Transmissible Spongiform Encephalopathies) the characterisation of ovine MSCs (oMSCs) is still limited. During the last decade, there has been an important effort within the scientific community to focus on the characterisation of MSCs obtained from different species, including the sheep. However, most research on MSCs has been performed on cells derived from bone marrow and, to a lesser degree, adipose tissue. The osteogenic and chondrogenic differentiation potential of MSCs in vitro[20–22] and in vivo[23, 24] is currently relatively well understood. Their phenotype for mesenchymal surface cell markers has also been analysed , and their proliferative potential has been shown to be heterogeneous . Although the existence of MSCs in peripheral blood has been demonstrated in many species [17, 18], this work represents the first report describing the isolation of these cells from sheep circulation.
The minimal criteria to define human MSCs proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy are: (1) plastic-adhesion when maintained in standard culture conditions; (2) expression of CD105, CD73 and CD90, and lack expression of the haematopoietic markers CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules and; (3) ability to differentiate to osteoblasts, adipocytes and chondroblasts in vitro. In our study, plastic-adherent cells with a fibroblast-like morphology were obtained from all experimental sheep and were further analysed to determine the expression of mesenchymal markers and their ability to differentiate into adipocytes, osteoblasts and chondrocytes.
In other domestic species  the proportion of MSCs in the peripheral blood is low, which is in agreement with the few colonies of MSCs detected in our original oPB-MSCs cultures. Although the proliferation ability of oPB-MSCs was very different between individuals, the doubling time was generally longer than in other species, such as horse . This difference may be due to the higher percentage of FBS used in the isolation of equine PB-MSCs (30%) and also due to the addition of dexamethasone to the growth media, which has been demonstrated to favour the expansion of MSCs . The variability observed in this work is in accordance with the high heterogeneity in the proliferative potential of oMSCs obtained from bone marrow (oBM-MSCs) .
The absence of a well-defined immunophenotype for PB-MSCs renders the comparison of studies difficult. Moreover, most of the cell surface markers utilized to sort subpopulations of human MSC by flow cytometry have not been validated in sheep . Gene expression-based technologies may be useful for the identification of possible molecules described as MSC markers [31, 32]. In our study, RT-qPCR was performed to quantify the mRNA expression levels of six cell surface antigens considered as either positive (CD29, CD73, CD90 and CD105) or negative (CD34 and CD45) MSC markers in humans.
In accordance with the immunophenotype described for human PB-MSCs [33–35], our expression analysis revealed significant amplification of the typical MSC markers, CD29, CD73 and CD90, and a weak signal for CD105. In contrast, the haematopoietic marker CD45 was not expressed. To our knowledge, there are no published data concerning the gene expression of cell surface markers in oMSCs obtained from other tissues. However, we have observed amplification of CD29, CD73 and CD90 in oBM-MSCs, as well as the lack of CD34 and CD45 expression (unpublished work from our group). Using flow cytometry, the presence of CD29 and CD105 has also been detected in oBM-MSCs [21, 36]. Additionally, oMSCs isolated from adipose tissue (oAT-MSCs) display high expression of CD90 and low immunoreactivity for CD105 . The immunophenotypes of oBM-MSCs  and oAT-MSCs  are negative for the haematopoietic CD34 marker. However, this marker is expressed at low levels in human PB-MSCs  and in equine MSCs derived from adipose tissue  as demonstrated by RT-qPCR. We detected CD34 expression in 5 out of 6 cultures, which may indicate individual variability. Finally, the cells analysed were negative for the haematopoietic marker CD45, as are human MSCs . We have previously found a good correlation between MSC marker gene expression and the immunophenotype detected by flow cytometry in equine MSCs . Although flow cytometry analysis is necessary to validate the immunophenotype of the isolated cells, the gene expression profile observed in this work strongly suggests that the peripheral blood derived fibroblast-like cells obtained as described would fulfill the requirements to be considered as MSCs.
Ovine BM-MSCs can be differentiated into adipocytes, showing lipid droplets in their cytoplasm and the induction of adipogenic markers [20, 36]. Similarly, adipogenic differentiation has been achieved here in all peripheral blood derived cell cultures, although great variability in the size of lipid droplets was observed. The expression of two adipogenic markers was evaluated in the cultures using RT-qPCR. PPARG is considered the master regulator of adipogenesis [40, 41] and is up-regulated in MSCs under adipogenic conditions . SCD is expressed uniquely in adipocytes and catalyzes the rate-limiting step in the synthesis of poly-unsaturated fatty acids, thereby exhibiting a pivotal role in adipocyte metabolism . Inter-individual variability was also noticeable in the expression of these adipogenic markers, which explains the lack of statistically significant differences in PPARG and SCD expression results despite the strong overexpression observed throughout the culture period. We also determined the expression of IL-6, which maintains the proliferative and undifferentiated state of bone marrow-derived MSCs  and is down-regulated during lineage-specific differentiation . In accordance with these reports, a significant decrease was detected in the expression of IL6 in the differentiated cultures. Therefore, using specific staining and gene expression profiles of adipogenic markers, we have confirmed the adipogenic potential of oPB-MSCs.
Similar to the adipogenic analysis, a great individual variation was also observed in the osteogenic potential. Osteogenic mineralization was confirmed on the last day of culture in osteogenic conditions (21 days) by staining calcium deposits with alizarin red. The induction period necessary for visualization of matrix mineralisation in oMSCs varies among different studies. The period reported for oBM-MSC mineralisation ranges from 21 days  to 5 weeks , while 4 weeks are required to differentiate periodontal oMSCs . The weak alizarin red staining observed in some of our experiments could be due to the relatively short period of induction.
Although COL1A1 is considered an early marker of osteoprogenitor cells , we observed either no changes or a strong down-regulation on the 21st day of culture. Besides displaying a rapid mineralisation, oBM-MSCs cultured under osteogenic conditions express increased or declined levels of COL1A1 depending on the differentiation moment . Other authors have reported either no significant increase in COL1A1 mRNA expression levels after osteogenic differentiation in human , porcine  and equine  MSCs, or a down-regulation of this marker in human PB-MSCs during osteogenesis . Therefore, COL1A1 may not be suitable for monitoring osteogenesis in oPB-MSCs. In contrast, BGLAP was upregulated during the differentiation process and was maximally expressed on the last day of culture (day 21), coinciding with the positive alizarin red staining. This is in accordance with the role of BGLAP as a late marker of developing osteoblasts .
The sheep has been used as a large animal model for the studies of chondrogenesis both in vivo and in vitro. The chondrogenic potential of oMSCs has been evaluated mainly in micromass cultures of cells derived from bone marrow [20, 21, 36]. Chondrogenesis was evaluated in our study using a bidimensional culture with a high cell concentration seeding, according to the protocol described by Jäger et al.  for chondrogenic differentiation of ovine umbilical cord blood-derived MSCs. Chondrogenic nodules were observed in both control and chondrogenic media, although the staining was stronger in the induced cultures. The confirmation with molecular markers was not straightforward as the expression of the two components of the extracellular matrix BGN and LUM changed in opposite directions during chondrogenic differentiation. In accordance to our results, the lack of strong BGN overexpression has been reported for chondrogenic induced micropellets of oBM-MSCs . However, further analysis is necessary to fully confirm the ability of oPB-MSCs to differentiate into chondrocytes.
During the last decade, many reports have described the in vitro neural transdifferentiation of MSCs derived from a range of species [2, 54, 55] but, to our knowledge, this has never been investigated in oMSCs. Neurogenic capacity of PB-MSCs would offer exciting possibilities for autologous therapeutic treatments for a variety of neurological disorders. As ovine is a natural model for prion diseases, the transdifferentiation of MSCs into neural cells could provide an excellent in vitro model for the study of these pathologies. Here, we described alterations in the morphology and expression profiles of neurogenic markers (MAP2, NEFM, NELF, NES and TUBB3) that are consistent with neural differentiation. In addition, we detected up-regulation of PRNP, which could also be involved in the morphological changes as the cellular prion protein seems to be necessary for neuritogenesis . The variable success in the ability to transdifferentiate MSCs to a neural phenotype could be influenced by the inter-donor variability of expression of neural-related markers in MSCs prior to differentiation . Nevertheless, our study shows that oPB-MSCs retain the ability to transdifferentiate. Finally, although murine bone marrow stromal cells express the prion protein , this has not been previously shown in species susceptible to prion diseases. In the present work, we have demonstrated the expression of PRNP in oPB-MSCs and its overexpression during neuronal differentiation.