Ana Rosa Remacha

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.

Comparative study of equine bone marrow and adipose tissue‐derived mesenchymal stromal cells

REASONS FOR PERFORMING STUDY Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. OBJECTIVES To compare and better understand the behaviour of equine MSCs obtained from bone marrow (BM) and adipose tissue (AT) in culture. METHODS Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expression levels of 7 surface marker genes were quantified using RT-qPCR and CD90 was also analysed by flow cytometry. Differentiation was evaluated using specific staining, measurement of alkaline phosphatase activity and analysis of the mRNA expression. RESULTS High interindividual differences were observed in proliferation in both cell types, particularly during the final days. Statistically significant differences in viability and early apoptosis of cultured AT- and BM-MSCs were found. The highest values of early apoptosis were observed during the first days of culture, while the highest percentage of necrosis and late apoptosis and lowest viability was observed in the last days. Surface marker expression pattern observed is in accordance to other studies in horse and other species. Osteogenic differentiation was evident after 7 days, with an increasing of ALP activity and mRNA expression of osteogenic markers. Adipogenic differentiation was achieved in BM-MSCs from 2 donors with one of the 16 media tested. Chondrogenic differentiation was also observed. CONCLUSIONS Proliferation ability is different in AT-MSCs and BM-MSCs. Differences in viability and early apoptosis were observed between both sources and CD34 was only found in AT-MSCs. Differences in their osteogenic and adipogenic potential were detected by staining and quantification of specific tissue markers. POTENTIAL RELEVANCE To provide data to better understand AT-MSCs and BM-MSCs behaviour in vitro.

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

BackgroundMesenchymal 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.ResultsPlastic-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.ConclusionsThis 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.

BackgroundMesenchymal 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.ResultsAt 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.ConclusionsHypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.

Inflammatory response to the administration of mesenchymal stem cells in an equine experimental model: effect of autologous, and single and repeat doses of pooled allogeneic cells in healthy joints

BackgroundMesenchymal stem cells (MSCs) transplantation has become a promising therapeutic choice for musculoskeletal injuries. Joint-related disorders are highly prevalent in horses. Therefore, these animals are considered as suitable models for testing MSC-based therapies for these diseases. The aim of this study was to investigate the clinical and inflammatory responses to intra-articular single and repeat dose administration of autologous or of pooled allogeneic MSCs in healthy equine healthy joints. Six horses were intra-articularly injected with a single autologous dose of bone marrow derived MSCs (BM-MSCs) and two separate doses of allogeneic BM-MSCs pooled from several donors. All contralateral joints were injected with Lactated Ringer’s Solution (LRS) as the control vehicle. Signs of synovitis and lameness were evaluated at days 0, 1, 2, 3, 5 and 10 after injection. Total protein (TP), white blood cell count (WBC) and neutrophil count (NC) in synovial fluid were also measured at the same time-points.ResultsA mild synovial effusion without associated lameness was observed after all BM-MSCs injections. The second allogeneic injection caused the lowest signs of synovitis. Local temperature slightly increased after all BM-MSCs treatments compared to the controls. TP, WBC and NC in synovial fluids also increased during days 1 to 5 after all BM-MSCs injections. Both, clinical and synovial parameters were progressively normalized and by day 10 post-inoculation appeared indistinguishable from controls.ConclusionsIntra-articular administration of an allogeneic pool of BM-MSCs represents a safe therapeutic strategy to enhance MSCs availability. Importantly, the absence of hypersensitivity response to the second allogeneic BM-MSCs injection validates the use of repeat dose treatments to potentiate the therapeutic benefit of these cells. These results notably contribute to the development of stem cell based therapies for equine and human joint diseases.

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.

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-β1 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-β1 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.

Effect of inflammatory environment on equine bone marrow derived mesenchymal stem cells immunogenicity and immunomodulatory properties.

Mesenchymal stem cells (MSCs) are being investigated for the treatment of equine joint diseases because of their regenerative potential. Recently, the focus mainly has addressed to their immunomodulatory capacities. Inflammation plays a central role in joint pathologies, since the release of proinflammatory mediators to the synovial fluid (SF) leads to the activation of enzymatic degradation of the cartilage. MSCs can modulate the local immune environment through direct or paracrine interaction with immune cells, suppressing their proliferation and re-addressing their functions. Proinflammatory molecules can induce MSC immunoregulatory potential, but they could also increase the expression of immunogenic molecules. Studying the effect of inflammatory environment on MSC immunomodulation and immunogenicity profiles is mandatory to improve cellular therapies. The aim of this study was to analyse the response of equine bone marrow MSCs (eBM-MSCs) to three inflammatory conditions. Equine BM-MSCs from three animals were exposed to: (a) 20% allogeneic inflammatory SF (SF); (b) 50 ng/ml of TNFα and IFNγ (CK50) and (c) 20 ng/ml of TNFα and IFNγ (CK20). After 72 h of exposure, expression of immunogenic and immunomodulation-related molecules, including cell-to-cell contact and paracrine signalling molecules, were analysed by RT-qPCR and flow cytometry. The gene expression of adhesion molecules was upregulated whereas MSC migration-related genes were downregulated by all inflammatory conditions tested. CK culture conditions significantly upregulated the expression of COX-2, iNOS, IDO and IL-6. MHC-I gene expression was upregulated by all conditions, whereas MHC-II was upregulated only after CK priming. The expression of CD40 did not significantly change, whereas the ligand, CD40L, was downregulated in CK conditions. Flow cytometry showed an increase in the percentage of positive cells and mean fluorescence intensity (MFI) of the MHC-I and MHC-II molecules at CK50 conditions, supporting the gene expression results. These outcomes reinforce the change of the immunophenotype of the eBM-MSCs according to the surrounding conditions. Inflammatory synovial environment did not lead to significant changes, so the environment found by eBM-MSCs when they are intraarticular administered may not be enough to activate their immunomodulatory potential. CK priming at tested doses enhances the immunoregulatory profile of eBM-MSCs, which may promote a therapeutic benefit. Even if CK priming induced an upregulation of MHC expression, costimulatory molecule expression however was not upregulated, suggesting that immunogenicity might not be increased. This study provides a better understanding about the behaviour of eBM-MSCs inside the inflamed joint and constitutes a first step to improve MSC-based therapies for equine joint diseases.

Comparison of autologous bone marrow and adipose tissue derived mesenchymal stem cells, and platelet rich plasma, for treating surgically induced lesions of the equine superficial digital flexor tendon

Several therapies have been investigated for equine tendinopathies, but satisfactory long term results have not been achieved consistently and a better understanding of the healing mechanism elicited by regenerative therapies is needed. The aim of this study was to assess the separate effects of autologous bone marrow (BM) and adipose tissue (AT) derived mesenchymal stem cells (MSCs), and platelet rich plasma (PRP), for treating lesions induced in the superficial digital flexor tendon (SDFT) of horses. Lesions were created surgically in both SDFTs of the forelimbs of 12 horses and were treated with BM-MSCs (six tendons), AT-MSCs (six tendons) or PRP (six tendons). The remaining six tendons received lactated Ringers solution as control. Serial ultrasound assessment was performed prior to treatment and at 2, 6, 10, 20 and 45 weeks post-treatment. At 45 weeks, histopathology and gene expression analyses were performed. At week 6, the ultrasound echogenicity score in tendons treated with BM-MSCs suggested earlier improvement, whilst all treatment groups reached the same level at week 10, which was superior to the control group. Collagen orientation scores on histological examination suggested a better outcome in treated tendons. Gene expression was indicative of better tissue regeneration after all treatments, especially for BM-MSCs, as suggested by upregulation of collagen type I, decorin, tenascin and matrix metalloproteinase III mRNA. Considering all findings, a clear beneficial effect was elicited by all treatments compared with the control group. Although differences between treatments were relatively small, BM-MSCs resulted in a better outcome than PRP and AT-MSCs.

Acute phase protein haptoglobin as inflammatory marker in serum and synovial fluid in an equine model of arthritis.

Acute phase proteins are useful inflammatory markers in horses. Haptoglobin (Hp) serum level is increased in horses undergoing different inflammatory processes, including arthritis. However, Hp concentration has not been assessed in inflammatory synovial fluid (SF). The aim of the present study was to investigate the Hp response in serum and SF in horses undergoing experimentally induced arthritis. For this purpose, serum and SF samples were collected from 12 animals before amphotericin B-induced arthritis was created (T0, healthy) and 15days after the lesion induction (T1, joint inflammation) and Hp was determined by single radial immunodiffusion. The Hp increase between T0 and T1 was significant in both serum and SF, and serum Hp concentration at T0 was significantly higher than in SF, but significant differences were not found at T1, indicating a higher Hp increase in SF. A significant positive correlation for Hp concentration between serum and SF samples was found. These results highlight the potential usefulness of Hp as inflammatory marker in horses, showing for the first time the increase of Hp in SF from joint inflammation in the horse.