Isolation and characterization of ovine mesenchymal stem cells derived from peripheral blood.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells with capacity to differentiate into several mesenchymal lineages. This quality makes MSCs good candidates for use in cell therapy. MSCs can be isolated from a variety of tissues including bone marrow and adipose tissue, which are the most common sources of these cells. However, MSCs can also be isolated from peripheral blood. Sheep has been proposed as an ideal model for biomedical studies including those of orthopaedics and transmissible spongiform encephalopathies (TSEs). The aim of this work was to advance these studies by investigating the possibility of MSC isolation from ovine peripheral blood (oPB-MSCs) and by subsequently characterizing there in vitro properties. RESULTS: Plastic-adherent fibroblast-like cells were obtained from the mononuclear fraction of blood samples. These cells were analysed for their proliferative and differentiation potential into adipocytes, osteoblasts and chondrocytes, as well as for the gene expression of cell surface markers. The isolated cells expressed transcripts for markers CD29, CD73 and CD90, but failed to express the haematopoietic marker CD45 and expressed only low levels of CD105. The expression of CD34 was variable. The differentiation potential of this cell population was evaluated using specific differentiation media. Although the ability of the cultures derived from different animals to differentiate into adipocytes, osteoblasts and chondrocytes was heterogeneous, we confirmed this feature using specific staining and analysing the gene expression of differentiation markers. Finally, we tested the ability of oPB-MSCs to transdifferentiate into neuronal-like cells. Morphological changes were observed after 24-hour culture in neurogenic media, and the transcript levels of the neurogenic markers increased during the prolonged induction period. Moreover, oPB-MSCs expressed the cellular prion protein gene (PRNP), which was up-regulated during neurogenesis. CONCLUSIONS: This study describes for the first time the isolation and characterization of oPB-MSCs. Albeit some variability was observed between animals, these cells retained their capacity to differentiate into mesenchymal lineages and to transdifferentiate into neuron-like cells in vitro. Therefore, oPB-MSCs could serve as a valuable tool for biomedical research in fields including orthopaedics or prion diseases.

Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue.

BACKGROUND: Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2. RESULTS: At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O2 for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state. CONCLUSIONS: Hypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.

Genome-Wide Methylation Profiling in the Thalamus of Scrapie Sheep.

Scrapie is a neurodegenerative disorder belonging to the group of transmissible spongiform encephalopathy (TSE). Scrapie occurs in sheep and goats, which are considered good natural animal models of these TSE. Changes in DNA methylation occur in the central nervous system (CNS) of patients suffering from prion-like neurodegenerative diseases, such as Alzheimer's disease. Nevertheless, potential DNA methylation alterations have not yet been investigated in the CNS of any prion disease model or naturally infected cases, neither in humans nor in animals. Genome-wide DNA methylation patterns were studied in the thalamus obtained from sheep naturally infected with scrapie at a clinical stage (n = 4) and from controls (n = 4) by performing a whole-genome bisulfite sequencing (WGBS) analysis. Ewes carried the scrapie-susceptible ARQ/ARQ PRNP genotype and were sacrificed at a similar age (4-6 years). Although the average genomic methylation levels were similar between the control and the scrapie animals, we identified 8,907 significant differentially methylated regions (DMRs) and 39 promoters (DMPs). Gene Ontology analysis revealed that hypomethylated DMRs were enriched in genes involved in transmembrane transport and cell adhesion, whereas hypermethylated DMRs were related to intracellular signal transduction genes. Moreover, genes highly expressed in specific types of CNS cells and those previously described to be differentially expressed in scrapie brains contained DMRs. Finally, a quantitative PCR (qPCR) validation indicated differences in the expression of five genes (PCDH19, SNCG, WDR45B, PEX1, and CABIN1) that matched the methylation changes observed in the genomic study. Altogether, these results suggest a potential regulatory role of DNA methylation in prion neuropathology.

Inflammation affects the viability and plasticity of equine mesenchymal stem cells: possible implications in intra-articular treatments.

Mesenchymal stem cells (MSCs) are gaining relevance for treating equine joint injuries because of their ability to limit inflammation and stimulate regeneration. Because inflammation activates MSC immunoregulatory function, proinflammatory priming could improve MSC efficacy. However, inflammatory molecules present in synovial fluid or added to the culture medium might have deleterious effects on MSCs. Therefore, this study was conducted to investigate the effects of inflammatory synovial fluid and proinflammatory cytokines priming on viability and plasticity of equine MSCs. Equine bone marrow derived MSCs (eBM-MSCs) from three animals were cultured for 72 h in media supplemented with: 20% inflammatory synovial fluid (SF); 50 ng/mL IFN-γ and TNF-α (CK50); and 20 ng/mL IFN-γ and TNF-α (CK20). Proliferation assay and expression of proliferation and apoptosis-related genes showed that SF exposed-eBM-MSCs maintained their viability, whereas the viability of CK primed-eBM-MSCs was significantly impaired. Tri-lineage differentiation assay revealed that exposure to inflammatory synovial fluid did not alter eBM-MSCs differentiation potential; however, eBM-MSCs primed with cytokines did not display osteogenic, adipogenic or chondrogenic phenotype. The inflammatory synovial environment is well tolerated by eBM-MSCs, whereas cytokine priming negatively affects the viability and differentiation abilities of eBM-MSCs, which might limit their in vivo efficacy.

Expression of genes involved in immune response and in vitro immunosuppressive effect of equine MSCs.

The immunomodulatory capacities of mesenchymal stem cells (MSCs) have made them the subject of increased clinical interest for tissue regeneration and repair. We have studied the immunomodulatory capacity of equine MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) in cocultures with allogeneic peripheral blood mononuclear cells (PBMCs). Different isoforms and concentrations of phytohaemaglutinin (PHA) were tested to determine the best stimulation conditions for PBMC proliferation and a proliferation assay was performed for 7 days to determine the optimal day of stimulation of PBMCs. The effect of the dose and source of MSCs was evaluated in cocultures of 10(5) PBMCs with different ratios of AT- and BM-MSCs (1:1, 1:10, 1:20 and 1:50). Proliferation rates of the PBMCs were evaluated using BrdU ELISA colorimetric assay. PHA stimulated equine PBMCs reached their peak of growth after 3 days of culture. The immunoassay showed a decrease of the PBMCs growth at high ratio cocultures (1:1 and 1:10). Equine BM-MSCs and AT-MSCs demonstrated an ability to suppress the proliferation of stimulated PBMCs. Although MSCs derived from both sources displayed immunosuppressive effects, AT-MSCs were slightly more potent than BM-MSCs. In addition, the expression of 26 genes coding for different molecules implicated in the immune response was analyzed in cocultures of BM-MSCs and PHA stimulated PBMSCs by reverse transcriptase real time quantitative PCR (RT-qPCR). An upregulation in genes associated with the production of interleukins and cytokines such as TNF-α and TGF-ß1 was observed except for IFN-γ whose expression significantly decreased. The variations of interleukins and cytokine receptors showed no clear patterns. COX-1 and COX-2 showed similar expression patterns while INOs expression significantly decreased in the two cell types present in the coculture. Cyclin D2 and IDO-1 showed an increased expression and CD90, ITG-ß1 and CD44 expression decreased significantly in BM-MSCs cocultured with PHA stimulated PBMCs. On the contrary, CD6 and VCAM1 expression increased in these cells. With regard to the expression of the five genes involved in antigen presentation, an upregulation was observed in both cocultured MSCs and stimulated PBMCs. This study contributes to the knowledge of the immunoregulatory properties of equine MSCs, which are notably important for the treatment of inflammation processes, such as tendinitis and osteoarthritis.

Expansion under hypoxic conditions enhances the chondrogenic potential of equine bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are widely used in regenerative medicine in horses. Most of the molecular characterisations of BM-MSCs have been made at 20% O(2), a higher oxygen level than the one surrounding the cells inside the bone marrow. The present work compares the lifespan and the tri-lineage potential of equine BM-MSCs expanded in normoxia (20% O(2)) and hypoxia (5% O(2)). No significant differences were found in long-term cultures for osteogenesis and adipogenesis between normoxic and hypoxic expanded BM-MSCs. An up-regulation of the chondrogenesis-related genes (COL2A1, ACAN, LUM, BGL, and COMP) and an increase of the extracellular sulphated glycosaminoglycan content were found in cells that were expanded under hypoxia. These results suggest that the expansion of BM-MSCs in hypoxic conditions enhances chondrogenesis in equine BM-MSCs.

Immunophenotype and gene expression profiles of cell surface markers of mesenchymal stem cells derived from equine bone marrow and adipose tissue.

Bone marrow and adipose tissue are the two main sources of mesenchymal stem cell (MSC). The aim of this work was to analyse the immunophenotype of 7 surface markers and the expression of a panel of 13 genes coding for cell surface markers in equine bone marrow and adipose tissue-derived MSCs obtained from 9 horses at third passage. The tri-lineage differentiation was confirmed by specific staining. Equine MSCs from both sources were positive for the MSC markers CD29 and CD90, while were negative for CD44, CD73, CD105, CD45 and CD34. The gene expression of these molecules was also evaluated by reverse transcriptase real-time quantitative PCR along with the expression of 5 other MSC markers. Both populations of cells expressed CD13, CD29, CD44, CD49d, CD73, CD90, CD105, CD106, CD146 and CD166 transcripts. Significant differences in gene expression levels between BM- and AT-MSCs were observed for CD44, CD90, CD29 and CD34. Both cell types were negative for CD45 and CD31. The surface antigens tested revealed a similar phenotypic profile between horse and human MSCs, although specific differences in some surface antigens were noticed.