Equine Bone Marrow-derived Mesenchymal Stem Research Articles

Effect of platelet lysate on Schwann-like cell differentiation of equine bone marrow-derived mesenchymal stem cells

Currently, treatment for peripheral nerve injuries in horses primarily relies upon physical therapy and anti-inflammatory drugs. In humans, various treatments using mesenchymal stem cells (MSCs) are being attempted. Therefore, in this study, Schwann-like cell differentiation cultures of equine MSCs were prepared using fetal bovine serum (FBS) and equine platelet lysate (ePL). ePL increased the platelet count to 1 × 106/μl, the optimal concentration for culture. In both groups, an elongated morphology at both ends, characteristic of Schwann cells, was observed under the microscope. Real-time PCR analysis of the expression levels of neuronal markers showed that the ePL group tended to express higher levels of Nestin, Musashi1, and Pax3 than the FBS group. p75 was expressed at low levels in both groups. Immunostaining results showed localization of Nestin in both groups of differentiated cells, but the positive cell rate was significantly higher in the ePL group than in the FBS group. Overall, the ePL gro showed good results for Schwann-like cell differentiation, which may be useful for future use in the treatment of equine motor neuron disease. This knowledge could be applied translationaly in the treatment of amyotrophic lateral sclerosis in humans.Overall, the ePL group showed good results for Schwann-like cell differentiation, which may be useful for future use in the treatment of equine motor neuron disease and in the treatment of amyotrophic lateral sclerosis in humans.

Three-Dimensional Culture of Equine Bone Marrow-Derived Mesenchymal Stem Cells Enhances Anti-Inflammatory Properties in a Donor-Dependent Manner.

Three-dimensional (3D) culture of human mesenchymal stem cells (MSCs) as spheroids enhances the production of important regulators of inflammation: prostaglandin E2 (PGE2), interleukin (IL)-6, and tumor necrosis factor-inducible gene 6 (TSG-6). The horse is a model species and suffers from musculoskeletal, ocular, and systemic inflammatory disease. It is unknown if 3D culture promotes enhanced production of immunomodulatory cytokines and regulators in equine MSCs and if there is variation between individual cell donors. We evaluated the feasibility, cell viability, and stem cell marker stability of 3D-cultured equine bone marrow-derived MSCs (eBMSCs) and determined the effect of inflammatory stimulation upon gene expression and secretion of key regulators of inflammation [PGE2, TSG-6, IL-10, IL-6, stromal cell-derived factor 1 (SDF-1)]. Variations in anti-inflammatory phenotype between six donors were investigated, with and without IL-1β stimulation, in either monolayer [two-dimensional (2D)] or 3D culture. Our results showed that eBMSCs self-aggregate in 3D culture while maintaining cell viability and markers of stemness CD90, CD44, CD104, and Oct4. In addition, 3D culture enhances the anti-inflammatory phenotype regardless of inflammatory stimulation by increasing PGE2, IL-6, TSG-6, SDF-1, and IL-10. Finally, anti-inflammatory phenotype was enhanced by IL-1β exposure but showed significant variation between cell lines in the degree of gene upregulation, and what genes were expressed. We conclude that 3D culture of eBMSCs as spheroids alters their anti-inflammatory phenotype, but this effect is influenced by cytokine exposure and cell donor.

TGF-β2 enhances expression of equine bone marrow-derived mesenchymal stem cell paracrine factors with known associations to tendon healing

BackgroundMesenchymal stem cells (MSCs) secrete paracrine factors and extracellular matrix proteins that contribute to their ability to support tissue healing and regeneration. Both the transcriptome and the secretome of MSCs can be altered by treating the cells with cytokines, but neither have been thoroughly investigated following treatment with the specific cytokine transforming growth factor (TGF)-β2.MethodsRNA-sequencing and western blotting were used to compare gene and protein expression between untreated and TGF-β2-treated equine bone marrow-derived MSCs (BM-MSCs). A co-culture system was utilized to compare equine tenocyte migration during co-culture with untreated and TGF-β2-treated BM-MSCs.ResultsTGF-β2 treatment significantly upregulated gene expression of collagens, extracellular matrix molecules, and growth factors. Protein expression of collagen type I and tenascin-C was also confirmed to be upregulated in TGF-β2-treated BM-MSCs compared to untreated BM-MSCs. Both untreated and TGF-β2-treated BM-MSCs increased tenocyte migration in vitro.ConclusionsTreating equine BM-MSCs with TGF-β2 significantly increases production of paracrine factors and extracellular matrix molecules important for tendon healing and promotes the migration of tenocytes in vitro.

Adeno-Associated Virus-Mediated Overexpression of Interleukin-10 Affects the Immunomodulatory Properties of Equine Bone Marrow-Derived Mesenchymal Stem Cells.

Joint injury can cause posttraumatic inflammation, which if severe enough can lead to posttraumatic osteoarthritis (PTOA), a progressive and debilitating condition. Posttraumatic inflammation is characterized by an influx of T lymphocytes and upregulation of inflammatory cytokines and degradative enzymes by activated chondrocytes and synoviocytes. Intra-articular bone marrow-derived mesenchymal stem cell (BM-MSC) injection for the treatment of osteoarthritis (OA) has been of interest due to the immunomodulatory properties of these cells. Interleukin (IL)-10, a potent immunomodulatory cytokine, has also been investigated as an OA therapeutic. Therefore, the objective of this study was to evaluate the combinatorial effects of BM-MSCs and IL-10 in OA using a gene therapy approach. We hypothesized that BM-MSCs overexpressing IL-10 would have superior immunomodulatory effects leading to increased suppression of T cell proliferation and decreased production of proinflammatory cytokines, providing protection of the extracellular matrix (ECM) in a stimulated, co-culture OA model. Treatment groups included the following: untransduced BM-MSC, adeno-associated virus (AAV)-IL10-transduced BM-MSC, and AAV-null transduced BM-MSC, which were unstimulated or stimulated with IL-1β/tumor necrosis factor-α (TNF-α). T cell proliferation was significantly decreased by the presence of BM-MSCs, especially when these BM-MSCs were AAV transduced. There was no significant difference in T cell suppression when cells were cultured with AAV-IL10-transduced or AAV-null transduced BM-MSCs. AAV transduction itself was associated with decreased synthesis of IL-1β, IL-6, and TNF-α. Expression of IL-1β and MMP13 was downregulated in AAV-transduced BM-MSCs and MMP13 expression was downregulated in cartilage explants co-cultured with AAV-transduced BM-MSCs. Despite mitigation of some proinflammatory cascades, rescue of ECM loss, as determined by glycosaminoglycan quantification and histological evaluation, did not occur in either AAV-IL10-transduced or AAV-null transduced co-cultures. Although IL-10 overexpression may enhance BM-MSC-mediated T cell suppression, we did not observe significant modulation of inflammation-driven cartilage degradation in cultures containing AAV-IL10-transduced BM-MSCs. AAV transduction itself does appear to affect paracrine signaling by BM-MSCs, which warrants further investigation.

Effects of continuous passage on the immunomodulatory properties of equine bone marrow-derived mesenchymal stem cells in vitro

BackgroundThe immunomodulatory properties of mesenchymal stem cells (MSCs) have been studied extensively due to their increasing clinical application for tissue regeneration and repair following culture expansion. We have studied the effect of continuous passage on the immunomodulatory capacity of equine bone marrow-derived MSCs (BM-MSCs). Equine BM-MSCs were isolated and culture expanded to passage three, six, and nine (P3, P6, P9). Immunomodulatory properties of each passage were assessed using a T cell proliferation assay and cytokine synthesis following stimulation with interferon gamma (IFN-γ). ResultsEquine BM-MSCs maintained their primary cell morphology and immunophenotype throughout all passages. T cell proliferation was suppressed by all passages of BM-MSCs, compared to peripheral blood mononuclear cells (PBMCs) alone. There was no significant difference in suppression of T cell proliferation between P3, P6, and P9 BM-MSCs. All passages of BM-MSCs significantly increased cytokine synthesis in response to stimulation with IFN-γ. There were no significant differences in production of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) or regulate on activation, normal T cell expressed and secreted (RANTES) following stimulation with IFN-γ between P3, P6, and P9 BM-MSCs. P9 BM-MSCs had significantly increased production of tumor necrosis factor alpha (TNF-α), (IL-1β), and (IL-10) compared to P3 BM-MSCs. Additionally, there was a significant increase in production of (IL-8) in P6 and P9 BM-MSCs in comparison to P3 BM-MSCs. ConclusionsOur findings demonstrate that culture expansion affects some of the immunomodulatory properties of BM-MSCs in vitro, which may suggest that MSCs isolated from a single collection of bone marrow may be culture expanded, but only those from lower passage numbers would be ideal for clinical application.

The effect of equine bone marrow-derived mesenchymal stem cells on the expression of apoptotic genes in neutrophils.

BackgroundBone marrow mesenchymal stem cells (BM‐MSCs), as multipotent cells with self‐renewal and plastic‐adherent properties, have immunomodulatory effects on immune cells, including neutrophils. These cells are in close proximity in bone marrow (BM) sinusoids with non‐multiplicative immature neutrophils. BM‐MSCs exert their immunomodulatory effects on adjacent cells both directly (cell‐to‐cell contact) and indirectly (secretion of soluble factors).ObjectivesThe aim of this study was to evaluate the effect of equine bone marrow mesenchymal stem cells (BM‐MSCs) on the expression of some pro‐ and anti‐apoptotic genes (p53, survivin and Bcl2) in neutrophils co‐cultured with BM‐MSCs.MethodsFor this purpose, peripheral blood neutrophils were isolated and separately co‐cultured for 12 hr with both BM‐MSCs and the BM‐MSCs΄ supernatant. Four groups were included: neutrophils with only culture media (as control), neutrophils co‐cultured with BM‐MScs, neutrophils cultured with BM‐MSCs’ supernatant and neutrophils cultured with lipopolysaccharide (LPS, as positive control). Then, the expression of mentioned genes (p53, survivin and Bcl2) was evaluated by quantitative polymerase chain reaction (qPCR).ResultsCompared with control neutrophils, in neutrophils co‐cultured with both BM‐MSCs and BM‐MSCs’ supernatant, the mRNA expression levels of p53, as pro‐apoptotic gene, and survivin and Bcl2, as anti‐apoptotic genes, were remarkably increased and decreased (p < .05), respectively.ConclusionsThese data revealed the notion that the direct contact of BM‐MSCs is not obligatory for their effects on the apoptotic status of neutrophils and they affect neutrophils via soluble secreted factors, which is promising for clinical implications in equine medicine.

Blood type and breed-associated differences in cell marker expression on equine bone marrow-derived mesenchymal stem cells including major histocompatibility complex class II antigen expression

BackgroundAs the search for an immune privileged allogeneic donor mesenchymal stem cell (MSC) line continues in equine medicine, the characterization of the cells between different sources becomes important. Our research seeks to more clearly define the MSC marker expression of different equine MSC donors.MethodsThe bone marrow-derived MSCs from two equine breeds and different blood donor-types were compared over successive culture passages to determine the differential expression of important antigens. Eighteen Thoroughbreds and 18 Standardbreds, including 8 blood donor (erythrocyte Aa, Ca, and Qa antigen negative) horses, were evaluated. Bone marrow was taken from each horse for isolation and culture of MSCs. Samples from passages 2, 4, 6, and 8 were labelled and evaluated by flow cytometry. The cell surface expression of CD11a/18, CD44, CD90 and MHC class II antigens were assessed. Trilineage assays for differentiation into adipogenic, chondrogenic and osteogenic lines were performed to verify characterization of the cells as MSCs.FindingsThere were significant differences in mesenchymal stem cell marker expression between breeds and blood antigen-type groups over time. Standardbred horses showed a significantly lower expression of MHC class II than did Thoroughbred horses at passages 2, 4 and 6. CD90 was significantly higher in universal blood donor Standardbreds as compared to non-blood donor Standardbreds over all time points. All MSC samples showed high expression of CD44 and low expression of CD11a/18.ConclusionsUniversal blood donor- type Standardbred MSCs from passages 2–4 show the most ideal antigen expression pattern of the horses and passages that we characterized for use as a single treatment of donor bone marrow-derived MSCs. Further work is needed to determine the significance of this differential expression along with the effect of the expression of MHC I on equine bone marrow-derived MSCs.

Enhanced chondrogenic differentiation of equine bone marrow-derived mesenchymal stem cells in zirconia microwell substrata

In human cartilage tissue engineering, three-dimensional zirconia substrata have the potential advantage of producing many uniform cell clusters of controlled size without xenobiotic material, allowing easy clinical application. The objective of this study was to evaluate the possibility of using zirconia porous three-dimensional microwell substrata for chondrogenic differentiation of equine bone marrow-derived mesenchymal stem cells (BMMSCs) in vitro. In regular medium, 8 × 105, 2 × 106, and 5 × 106 equine BMMSCs from five thoroughbred horses were cultured on zirconia microwell substrata for 4 days to allow formation of clusters. The medium was replaced by chondrogenic culture medium. After chondrogenic culture for 7, 14 and 21 days, analysis of collagen type II alpha 1 gene (COL2A1) gene expression and observation of chondrogenic aggregates by scanning electron microscopy (SEM) were performed. SEM showed size-controlled cell clusters and increasing extracellular matrix over time when using 5 × 106 cells. The expression of COL2A1 on day 7 and 14 with 5 × 106 cells was significantly higher than that of conventional pellet culture with 2 × 106 cells. Histological evaluation by immunohistochemical staining for type II collagen (ColII) was performed after chondrogenic culture for 7 days. The clusters showed wide distribution of ColII. The results suggest that the zirconia substrata have the potential to enhance the chondrogenic differentiation of equine BMMSCs, allowing effective equine cartilage tissue engineering without xenobiotic materials.

The Combination of TGF-β3 and BMP-6 Synergistically Promotes the Chondrogenic Differentiation of Equine Bone Marrow-Derived Mesenchymal Stem Cells

Stem cell therapy is a new approach in veterinary medicine for the treatment of complicated disorders such as articular cartilage injuries in horses. Although the equine bone marrow-derived mesenchymal stem cells (eBMSCs) have a unique capacity in chondrogenic differentiation, it needs to be optimized. In the current study, we evaluated the enhancement of chondrogenic differentiation in eBMSCs using transforming growth factor beta-3 (TGF-β3) and bone morphogenic protein 6 (BMP-6) in vitro. The characterized eBMSCs at passage 3 (P3) were cultivated as micro-pellets for 21 days under different conditions: (1) basic culture medium (BCM), (2) chondrogenic differentiation medium (CDM), (3) CDM with BMP-6, (4) CDM with TGF-β3, and (5) CDM with BMP-6 and TGF-β3 to evaluate the chondrogenic differentiation by assessment of pellet size, histological characteristics, and gene expression profile. Pellet size in groups that contained growth factors had a significant difference in comparison with the control group or chondrogenic group without growth factors. The histological assessment showed that the cartilage progression score in groups containing TGF-β3, alone or combined with BMP-6, was significantly higher in comparison with that of the other groups (P < 0.05). Additionally, the aggrecan gene was expressed in all groups except the control group, while collagen type II was just expressed in groups containing TGF-β3, alone or combined with BMP-6. TGF-β3 may have more promising effects on the enhancement of chondrogenic differentiation of eBMSCs in comparison with BMP-6. However, the combination of TGF-β3 and BMP-6 promoted chondrogenic differentiation of eBMSCs in a synergistic manner.

The effect of hypoxia on chondrogenesis of equine synovial membrane-derived and bone marrow-derived mesenchymal stem cells

BackgroundJoint injury is extremely common in equine athletes and post-traumatic osteoarthritis (PTOA), a progressive and debilitating disease, is estimated to affect 60% of horses in the USA. The limited potential for intrinsic healing of articular cartilage has prompted intense efforts to identify a cell-based repair strategy to prevent progression of PTOA. Mesenchymal stem cells (MSCs) have the potential to become an ideal source for cell-based treatment of cartilage lesions; however, full chondrogenic differentiation remains elusive. Due to the relatively low oxygen tension in articular cartilage, hypoxia has been proposed as a method of increasing MSC chondrogenesis. The objective of this study was to investigate the effect of hypoxic culture conditions on chondrogenesis in equine synovial membrane-derived MSCs (SM-MSCs) and bone marrow-derived MSCs (BM-MSCs). MSCs were isolated from synovial membrane and bone marrow collected from 5 horses. Flow cytometric analysis was used to assess cell surface marker expression including CD29, CD44, CD90, CD105, CD45, CD-79α, MHCI and MHCII. MSC pellets were cultured in normoxic (21% O2) or in hypoxic (5% O2) culture conditions for 28 days. Following the culture period, chondrogenesis was assessed by histology, biochemical analyses and gene expression of chondrogenic-related genes including ACAN, COL2b, SOX9, and COL10A1.ResultsBoth cell types expressed markers consistent with stemness including CD29, CD44, CD90, CD105, and MHCI and were negative for exclusion markers (CD45, CD79α, and MHCII). Although the majority of outcome variables of chondrogenic differentiation were not significantly different between cell types or culture conditions, COL10A1 expression, a marker of chondrocyte hypertrophy, was lowest in hypoxic SM-MSCs and was significantly lower in hypoxic SM-MSCs compared to hypoxic BM-MSCs.ConclusionsHypoxic culture conditions do not appear to increase chondrogenesis of equine SM-MSCs or BM-MSCs; however, hypoxia may downregulate the hypertrophic marker COL10A1 in SM-MSCs.

Comparison of the Chondrogenic Differentiation Potential of Equine Synovial Membrane-Derived and Bone Marrow-Derived Mesenchymal Stem Cells.

Focal cartilage injury occurs commonly and often precipitates OA. Mesenchymal stem cells (MSCs) may be useful for repairing cartilage lesions, thereby preventing joint degeneration. Although MSCs isolated from bone marrow have been shown to have chondrogenic potential, synovial membrane-derived MSCs (SM-MSCs) may have superior chondrogenic abilities due to a common progenitor cell between synovium and cartilage. The objective of this study was to directly compare the immunophenotype, proliferative capabilities, and chondrogenic potential of equine SM-MSCs and bone marrow-derived MSCs (BM-MSCs). In order to do this, MSCs were isolated from synovial membrane and bone marrow collected from 6 adult horses. Flow cytometric analysis was used to assess cell surface marker expression including CD29, CD44, CD90, CD105, CD45, CD-79α, MHCI, and MHCII. Proliferation rates and doubling time were quantified in P1 and P2 cells. Trilineage differentiation assays were performed. MSC pellets were cultured in chondrogenic induction media for 28 days. Pellets were stained with toluidine blue to assess proteoglycan deposition. Expression of the chondrogenic-related genes ACAN, COL2b, and SOX9 was quantified using qRT-PCR. The immunophenotypes of BM-MSCs and SM-MSCs were similar with both cell types being positive for expression of stem cell markers (CD29, CD44, CD90, CD105, and MHCI) and negative for exclusion markers (CD45 and CD79α). Although SM-MSCs did not express the exclusion marker, MHCII, expression of MHCII was moderate in BM-MSCs. Overall, chondrogenic differentiation was not significantly between the cell types with histologic parameters, proteoglycan content and gene expression being similar. BM-MSCs showed enhanced osteogenic differentiation compared to SM-MSCs. Synovial membrane is a feasible source of MSCs in the horse, however, superior chondrogenesis in vitro should not be expected under currently described culture conditions.

Equine bone marrow-derived mesenchymal stem cells: optimization of cell density in primary culture.

The primary cell seeding density of bone marrow-derived mononuclear cells (BM-MNCs) affects several cellular behaviors, including attachment to the culture dish, proliferation, and differentiation. The aim of this study was to determine the best density of equine BM-MNCs in primary culture (P0) for obtaining the maximum bone marrow-derived mesenchymal stem cell (BM-MSC) yields at the end of P0. Bone marrow samples of two healthy mares were aspirated. The MNCs were isolated and cultured at different densities (1×105, 2×105, 4×105, 8×105, and 1×106 cells/cm2). Within the 7th and 14th days after seeding, the colonies containing more than 15 cells were counted and the percentage of confluency and the number of cells were calculated on day 21. The lowest density of MNCs was associated with the least number of colonies, number of adherent cells, and confluency percentage, whereas the highest density was associated with the maximum number of colonies and confluency percentage (P<0.05). However, the maximum number of cells at the end of P0 was associated with the intermediate (4×105 cells/cm2) and the highest concentration (P<0.05). The maximum number of MSCs at the end of P0 was obtained at the densities of 1×106 and, especially, at 4×105 cells/cm2.

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity.MethodsPlatelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α.ResultsOur results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed the release of TNF-α when exposed to LPS-stimulated monocytes similar to those cultured in FBS.ConclusionePL has the potential to be used for the expansion of MSCs before clinical application, avoiding the concerns associated with the use of FBS.

In Vitro Effects of High-Intensity Laser Photobiomodulation on Equine Bone Marrow-Derived Mesenchymal Stem Cell Viability and Cytokine Expression.

This study aimed to examine the influence of neodymium-doped yttrium aluminum garnet (Nd:YAG) laser irradiation on equine bone marrow-derived mesenchymal stem cell (MSC) viability, proliferation, and cytokine expression in vitro. Photobiomodulation of cells using monochromatic light is a technique designed to influence cellular processes. Previous studies have shown dose-dependent effects of low-level laser irradiation on cell proliferation and cytokine expression in a range of cell types and species. Evidence for the influence of 1064 nm wavelength near-infrared irradiation on MSCs is sparse, and high-energy doses have shown inhibitory effects. MSC cultures from six horses were exposed to 1064 nm irradiation with an energy density of 9.77 J/cm2 and a mean output power of 13.0 W for 10 sec. MSC viability and proliferation were evaluated through flow cytometry and real-time live cell analysis. Gene expression and cytokine production in the first 24 h after irradiation were analyzed through polymerase chain reaction (PCR), multiplex assay, and enzyme-linked immunosorbent assay. No difference in viability was detected between irradiated and control MSCs. Irradiated cells demonstrated slightly lower proliferation rates, but remained within 3.5% confluence of control cells. Twenty-four hours after irradiation, irradiated MSCs demonstrated a significant increase in expression of interleukin (IL)-10 and vascular endothelial growth factor (VEGF) compared with control MSCs. Under these irradiation parameters, equine MSCs remained viable and expressed increased concentrations of IL-10 and VEGF. IL-10 has an anti-inflammatory action by inhibiting the synthesis of proinflammatory cytokines at the transcriptional level. This response to 1064 nm irradiation shows promise in the photobiomodulation of MSCs to enhance their therapeutic properties.

Transforming Growth Factor-β2 Downregulates Major Histocompatibility Complex (MHC) I and MHC II Surface Expression on Equine Bone Marrow-Derived Mesenchymal Stem Cells Without Altering Other Phenotypic Cell Surface Markers.

Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Effective and safe allogeneic therapy may be hindered, however, by recipient immune recognition and rejection of major histocompatibility complex (MHC)-mismatched MSCs. Development of strategies to prevent immune rejection of MHC-mismatched MSCs in vivo is necessary to enhance cell survival and potentially increase the efficacy and safety of allogeneic MSC therapy. The purposes of this study were to evaluate if transforming growth factor-β2 (TGF-β2) downregulated MHC expression on equine MSCs and to determine if TGF-β2 treatment altered the phenotype of MSCs. Equine bone marrow-derived MSCs from 12 horses were treated with 1, 5, or 10 ng/ml TGF-β2 from initial isolation until MHC expression analysis. TGF-β2-treated MSCs had reduced MHC I and MHC II surface expression compared to untreated controls. TGF-β2 treatment also partially blocked IFN-γ-induced upregulation of MHC I and MHC II. Constitutive and IFN-γ-induced MHC I and MHC II expression on equine MSCs was dynamic and highly variable, and the effect of TGF-β2 was significantly dependent on the donor animal and baseline MHC expression. TGF-β2 treatment did not appear to change morphology, surface marker expression, MSC viability, or secretion of TGF-β1, but did significantly increase the number of cells obtained from culture. These results indicate that TGF-β2 treatment has promise for regulating MHC expression on MSCs to facilitate allogeneic therapy, but further work is needed to maintain MHC stability when exposed to an inflammatory stimulus.

Stemness Signature of Equine Marrow-derived Mesenchymal Stem Cells.

BackgroundApplication of competent cells such as mesenchymal stem cells (MSCs) for treatment of musculoskeletal disorders in equine athletes is increasingly needed. Moreover, similarities of horse and human in size, load and types of joint injuries, make horse as a good model for MSCs therapy studies. This study was designed to isolate and characterize stemness signature of equine bone marrow-derived mesenchymal stem cells (BM-MSCs).MethodsBM of three mares was aspirated and the mononuclear cells (MNCs) were isolated using density gradient. The primary MNCs were cultured and analyzed after tree passages (P3) for growth characteristics, differentiation potentials, and the expression of genes including CD29, CD34, CD44, CD90, CD105, MHC-I, MHC-II and pluripotency related genes (Nanog, Oct-4, Sox-2, SSEA-1, -3, -4) using RT-PCR or immunocytochemistry techniques.ResultsThe isolated cells in P3 were adherent and fibroblast-like in shape with doubling times of 78.15 h. Their clonogenic capacity was 8.67±4% and they were able to differentiate to osteogenic, adipogenic and chondrogenic lineages. Cells showed expression of CD29, CD44, CD90, MHC-I and Sox-2 while no expression for CD34, MHC-II, CD105, and pluripotency stemness markers was detected.ConclusionsIn conclusion, data showed that isolated cells have the basic and minimal criteria for MSCs, however, expressing only one pluripotency gene (sox-2).

Donor-Matched Comparison of Chondrogenic Potential of Equine Bone Marrow- and Synovial Fluid-Derived Mesenchymal Stem Cells: Implications for Cartilage Tissue Regeneration.

Mesenchymal stem cells (MSCs) have been demonstrated to be useful for cartilage tissue regeneration. Bone marrow (BM) and synovial fluid (SF) are promising sources for MSCs to be used in cartilage regeneration. In order to improve the clinical outcomes, it is recommended that prior to clinical use, the cellular properties and, specifically, their chondrogenic potential must be investigated. The purpose of this study is to compare and better understand the in vitro chondrogenic potential of equine bone marrow-derived mesenchymal stem cells (BMMSCs) and synovial fluid-derived mesenchymal stem cells (SFMSCs) populated from the same equine donor. BM- and SF-derived MSCs cultures were generated from five equine donors, and the MSCs were evaluated in vitro for their morphology, proliferation, trilineage differentiation, and immunophenotyping. Differences in their chondrogenic potentials were further evaluated quantitatively using glycosaminoglycan (GAG) content and via immunofluorescence of chondrogenic differentiation protein markers, SRY-type HMG box9, Aggrecan, and collagen II. The BMMSCs and SFMSCs were similar in cellular morphology, viability, and immunophenotype, but, varied in their chondrogenic potential, and expression of the key chondrogenic proteins. The SFMSCs exhibited a significant increase in GAG content compared to the BMMSCs (P < 0.0001) in three donors, suggesting increased levels of chondrogenesis. The expression of the key chondrogenic proteins correlated positively with the GAG content, suggesting that the differentiation process is dependent on the expression of the target proteins in these three donors. Our findings suggest that even though SFMSCs were hypothesized to be more chondrogenic relative to BMMSCs, there was considerable donor-to-donor variation in the primary cultures of MSCs which can significantly affect their downstream application.

Allogeneic major histocompatibility complex-mismatched equine bone marrow-derived mesenchymal stem cells are targeted for death by cytotoxic anti-major histocompatibility complex antibodies.

SummaryBackgroundAllogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Controversy exists, however, over whether major histocompatibility complex (MHC)‐mismatched MSCs are recognised by the recipient immune system and targeted for death by a cytotoxic antibody response.ObjectivesTo determine if cytotoxic anti‐MHC antibodies generated in vivo following MHC‐mismatched MSC injections are capable of initiating complement‐dependent cytotoxicity of MSCs.Study designExperimental controlled study.MethodsAntisera previously collected at Days 0, 7, 14 and 21 post‐injection from 4 horses injected with donor MHC‐mismatched equine leucocyte antigen (ELA)‐A2 haplotype MSCs and one control horse injected with donor MHC‐matched ELA‐A2 MSCs were utilised in this study. Antisera were incubated with ELA‐A2 MSCs before adding complement in microcytotoxicity assays and cell death was analysed via eosin dye exclusion. ELA‐A2 peripheral blood leucocytes (PBLs) were used in the assays as a positive control.ResultsAntisera from all 4 horses injected with MHC‐mismatched MSCs contained antibodies that caused the death of ELA‐A2 haplotype MSCs in the microcytotoxicity assays. In 2 of the 4 horses, antibodies were present as early as Day 7 post‐injection. MSC death was consistently equivalent to that of ELA‐A2 haplotype PBL death at all time points and antisera dilutions. Antisera from the control horse that was injected with MHC‐matched MSCs did not contain cytotoxic ELA‐A2 antibodies at any of the time points examined.Main limitationsThis study examined MSC death in vitro only and utilized antisera from a small number of horses.ConclusionsThe cytotoxic antibody response induced in recipient horses following injection with donor MHC‐mismatched MSCs is capable of killing donor MSCs in vitro. These results suggest that the use of allogeneic MHC‐mismatched MSCs must be cautioned against, not only for potential adverse events, but also for reduced therapeutic efficacy due to targeted MSC death.

Priming Equine Bone Marrow-Derived Mesenchymal Stem Cells with Proinflammatory Cytokines: Implications in Immunomodulation-Immunogenicity Balance, Cell Viability, and Differentiation Potential.

Mesenchymal stem cells (MSCs) have a great potential for treating equine musculoskeletal injuries. Although their mechanisms of action are not completely known, their immunomodulatory properties appear to be key in their functions. The expression of immunoregulatory molecules by MSCs is regulated by proinflammatory cytokines; so inflammatory priming of MSCs might improve their therapeutic potential. However, inflammatory environment could also increase MSC immunogenicity and decrease MSC viability and differentiation capacity. The aim of this study was to assess the effect of cytokine priming on equine bone marrow-derived MSC (eBM-MSC) immunoregulation, immunogenicity, viability, and differentiation potential, to enhance MSC immunoregulatory properties, without impairing their immune-evasive status, viability, and plasticity. Equine BM-MSCs (n = 4) were exposed to 5 ng/mL of TNFα and IFNγ for 12 h (CK5-priming). Subsequently, expression of genes coding for immunomodulatory, immunogenic, and apoptosis-related molecules was analyzed by real-time quantitative polymerase chain reaction. Chromatin integrity and proliferation assays were assessed to evaluate cell viability. Trilineage differentiation was evaluated by specific staining and gene expression. Cells were reseeded in a basal medium for additional 7 days post-CK5 to elucidate if priming-induced changes were maintained along the time. CK5-priming led to an upregulation of immunoregulatory genes IDO, iNOS, IL-6, COX-2, and VCAM-1. MHC-II and CD40 were also upregulated, but no change in other costimulatory molecules was observed. These changes were not maintained 7 days after CK5-priming. Viability and differentiation potential were maintained after CK5-priming. These findings suggest that CK5-priming of eBM-MSCs could improve their in vivo effectiveness without affecting other eBM-MSC properties.

Effects of Dexamethasone Concentration and Timing of Exposure on Chondrogenesis of Equine Bone Marrow-Derived Mesenchymal Stem Cells.

ObjectiveDexamethasone is known to support mesenchymal stem cell (MSC) chondrogenesis, although the effects of dose and timing of exposure are not well understood. The objective of this study was to investigate these variables using a laboratory model of MSC chondrogenesis.DesignEquine MSCs were encapsulated in agarose and cultured in chondrogenic medium with 1 or 100 nM dexamethasone, or without dexamethasone, for 15 days. Samples were analyzed for extracellular matrix (ECM) accumulation, prostaglandin E2 and alkaline phosphatase secretion, and gene expression of selected collagens and catabolic enzymes. Timing of exposure was evaluated by ECM accumulation after dexamethasone was withdrawn over the first 6 days, or withheld for up to 3 or 6 days of culture.ResultsECM accumulation was not significantly different between 1 and 100 nM dexamethasone, but was suppressed ~40% in dexamethasone-free cultures. Prostaglandin E2 secretion, and expression of catabolic enzymes, including matrix metalloproteinase 13, and type X collagen was generally lowest in 100 nM dexamethasone and not significantly different between 1 nM and dexamethasone-free cultures. Dexamethasone could be withheld for at least 2 days without affecting ECM accumulation, while withdrawal studies suggested that dexamethasone supports ECM accumulation beyond day 6.ConclusionOne nanomolar dexamethasone supported robust cartilage-like ECM accumulation despite not having an effect on markers of inflammation, although higher concentrations of dexamethasone may be necessary to suppress undesirable hypertrophic differentiation. While early exposure to dexamethasone was not critical, sustained exposure of at least a week appears to be necessary to maximize ECM accumulation.