Equine Mesenchymal Stem Cells Research Articles

Donor-derived equine mesenchymal stem cells suppress proliferation of mismatched lymphocytes.

Recently, it has been shown that mesenchymal stem cells (MSCs) do not express the major histocompatibility complex (MHC) II antigen and are able to inhibit proliferation of MHC-mismatched stimulated lymphocytes, enabling their use as in vivo allogeneic transplants. However, prior to clinical application of allo-MSCs, in vitro tests are required to confirm the safety of treatment protocols. To evaluate the immunosuppressive capabilities of equine bone-marrow-derived MSCs (BM-MSCs) on MHC-mismatched lymphocytes. In vitro experiment. Phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) from 3 Thoroughbreds (recipients) were co-cultured with mismatched BM-MSCs from 3 Connemara ponies (donors). Proliferation of lymphocytes was monitored by carboxyfluorescein succinimidyl ester labelling and analysed by flow cytometry. In total, 6 horses were haplotyped using microsatellites to confirm mismatching. Optimisation of the conditions to stimulate Thoroughbred lymphocytes and titration of equine anti-CD4 and anti-CD8 antibodies were performed. Connemara pony and Thoroughbred BM-MSCs were isolated, expanded and characterised by tri-lineage differentiation. Finally, BM-MSCs from both breeds were set up in co-culture at different ratios with stimulated Thoroughbred lymphocytes. Proliferation of CD4(+) and CD8(+) cells was determined by flow cytometry. A high proportion of CD4/CD8 double-positive lymphocytes were found in freshly isolated PBMCs, although this percentage decreased after 4 days of culture. Mismatched BM-MSCs inhibited proliferation of stimulated lymphocytes in a dose-dependent manner, with the greatest suppression occurring at a 1:10 ratio of BM-MSCs to PBMCs. Proliferation of CD4(+) and CD8(+) subpopulations decreased in 1:10 co-culture, with statistical significance in the case of CD8(+) cells, while that of the CD4/CD8 double-positive population was similar to the phytohaemagglutinin control. The results demonstrate dose-dependent immunosuppression of stimulated lymphocytes by mismatched equine BM-MSCs, supporting their future application in allo-MSC clinical treatments.

Equine adipose-derived mesenchymal stem cells: phenotype and growth characteristics, gene expression profile and differentiation potentials.

Because of the therapeutic application of stem cells (SCs), isolation and characterization of different types of SCs, especially mesenchymal stem cells (MSCs), have gained considerable attention in recent studies. Adipose tissue is an abundant and accessible source of MSCs which can be used for tissue engineering and in particular for treatment of musculoskeletal disorders. This study was aimed to isolate and culture equine adipose-derived MSCs (AT-MSCs) from little amounts of fat tissue samples and determine some of their biological characteristics. In this descriptive study, only 3-5 grams of fat tissue were collected from three crossbred mares. Immediately, cells were isolated by mechanical means and enzymatic digestion and were cultured in optimized conditions until passage 3 (P3). The cells at P3 were evaluated for proliferative capacities, expression of specific markers, and osteogenic, chondrogenic and adipogenic differentiation potentials. Results showed that the isolated cells were plastic adherent with a fibroblast-like phenotype. AT-MSCs exhibited expression of mesenchymal cluster of differentiation (CD) markers (CD29, CD44 and CD90) and not major histocompatibility complex II (MHC-II) and CD34 (hematopoietic marker). Cellular differentiation assays demonstrated the chondrogenic, adipogenic and osteogenic potential of the isolated cells. Taken together, our findings reveal that equine MSCs can be obtained easily from little amounts of fat tissue which can be used in the future for regenerative purposes in veterinary medicine.

HORSE SPECIES SYMPOSIUM: Use of mesenchymal stem cells in fracture repair in horses.

Equine bone fractures are often catastrophic, potentially fatal, and costly to repair. Traditional methods of healing fractures have limited success, long recovery periods, and a high rate of reinjury. Current research in the equine industry has demonstrated that stem cell therapy is a promising novel therapy to improve fracture healing and reduce the incidence of reinjury; however, reports of success in horses have been variable and limited. Stem cells can be derived from embryonic, fetal, and adult tissue. Based on the ease of collection, opportunity for autologous cells, and proven success in other models, adipose- or bone marrow-derived mesenchymal stem cells (MSC) are often used in equine therapies. Methods for isolation, proliferation, and differentiation of MSC are well established in rodent and human models but are not well characterized in horses. There is recent evidence that equine bone marrow MSC are able to proliferate in culture for several passages in the presence of autologous and fetal bovine serum, which is important for expansion of cells. Mesenchymal stem cells have the capacity to differentiate into osteoblasts, the bone forming cells, and this complex process is regulated by a number of transcription factors including runt-related transcription factor 2 (Runx2) and osterix (Osx). However, it has not been well established if equine MSC are regulated in a similar manner. The data presented in this review support the view that equine bone marrow MSC are regulated by the same transcription factors that control the differentiation of rodent and human MSC into osteoblasts. Although stem cell therapy is promising in equine bone repair, additional research is needed to identify optimal methods for reintroduction and potential manipulations to improve their ability to form new bone.

Platelet-Rich Plasma Enhances the Cellular Function of Equine Bone Marrow-Derived Mesenchymal Stem Cells

Rationale: Equine mesenchymal stem cells (eMSCs) and platelet-rich plasma (PRP) are cell-based therapies being used clinically to repair damaged tissue of horses. Numerous reports including data from our laboratory show that there are variations in the biological properties of MSCs and platelet – rich plasma, which can impact their biological functions. A single study describes the use of the eMSCs and PRP in tendon healing, with minimal success. The exact mechanism of action using the combination of the two therapies is unknown. This study was performed to evaluate and understand the effects of PRP, if any on the cellular performance of eBMMSCs in culture. Objective: To assess the effects of PRP in vitro on the rate of proliferation, expression of protein markers and osteogenic and chondrogenic differentiation of the primary cultures of eBMMSCs. Methods and Results: A commercially available stall side, portable kit was used to isolate PRP. To investigate the effect of PRP on eBMMSCs, the rate of proliferation of eBMMSCs was measured using the MTS assay, and subsequently the viability and the stemness of eBMMSCs was assessed using fluorescent staining and the expression of CD90. Finally, the osteogenic and chondrogenic differentiation of eBMMSCs was assessed by lineage-specific staining and the expressions of lineage – specific mRNAs. All assays were performed at a concentration of 50 million platelets/mL. Significant increase in proliferation and the differentiation profiles were observed in presence of PRP. Most importantly, the stem cell characteristics of inferior eBMMSCs showed a marked improvement in presence of PRP. Conclusions: The addition of PRP improves the in vitro function of eBMMSCs by enhancing the proliferation and osteo - and chondro - genesis. The presence of an optimal dose of platelets may enhance the in vivo performance of eBMMSCs and may be indicated when using autologous eBMMSCs therapy in the clinic.

Equid herpesvirus 1 (EHV1) infection of equine mesenchymal stem cells induces a pUL56-dependent downregulation of select cell surface markers

Equid herpesvirus 1 (EHV1) is an ubiquitous alphaherpesvirus that can cause respiratory disease, abortion and central nervous disorders. EHV1 is known to infect a variety of different cell types in vitro, but its tropism for cultured primary equine mesenchymal stem cells (MSC) has never been explored. We report that equine MSC were highly permissive for EHV1 and supported lytic replication of the virus in vitro. Interestingly, we observed that an infection of MSC with EHV1 resulted in a consistent downregulation of cell surface molecules CD29 (β1-integrin), CD105 (endoglin), major histocompatibility complex type I (MHCI) and a variable downregulation of CD172a. In contrast, expression of CD44 and CD90 remained unchanged upon wild type infection. In addition, we found that this selective EHV1-mediated downregulation of cell surface proteins was dependent on the viral protein UL56 (pUL56). So far, pUL56-dependent downregulation during EHV1 infection of equine cells has only been described for MHCI, but our present data indicate that pUL56 may have a broader function in downregulating cell surface proteins. Taken together, our results are the first to show that equine MSC are susceptible for EHV1 and that pUL56 induces downregulation of several cell surface molecules on infected cells. These findings provide a basis for future studies to evaluate the mechanisms underlying for this selective pUL56-induced downregulation and to evaluate the potential role of MSC during EHV1 pathogenesis.

Phenotypical and functional characteristics of mesenchymal stem cells derived from equine umbilical cord blood.

Mesenchymal stem cells (MSCs) offer promise as therapeutic aid in the repair of tendon and ligament injuries in race horses. Fetal adnexa is considered as an ideal source of MSCs due to many advantages, including non-invasive nature of isolation procedures and availability of large tissue mass for harvesting the cells. However, MSCs isolated from equine fetal adnexa have not been fully characterized due to lack of species-specific markers. Therefore, this study was carried out to isolate MSCs from equine umbilical cord blood (UCB) and characterize them using cross-reactive markers. The plastic-adherent cells could be isolated from 13 out of 20 (65%) UCB samples. The UCB derived cells proliferated till passage 20 with average cell doubling time of 46.40±2.86h. These cells expressed mesenchymal surface markers but did not express haematopoietic/leucocytic markers by RT-PCR and immunocytochemistry. The phenotypic expression of CD29, CD44, CD73 and CD90 was shown by 96.36±1.28, 93.40±0.70, 73.23±1.29 and 46.75±3.95% cells, respectively in flow cytometry, whereas, reactivity against the haematopoietic antigens CD34 and CD45 was observed only in 2.4±0.20 and 0.1±0.0% of cells, respectively. Osteogenic and chondrogenic differentiation could be achieved using established methods, whereas the optimum adipogenic differentiation was achieved after supplementing media with 15% rabbit serum and 20ng/ml of recombinant human insulin. In this study, we optimized methodology for isolation, cultural characterization, differentiation and immunophenotyping of MSCs from equine UCB. Protocols and markers used in this study can be employed for unequivocal characterization of equine MSCs.

High yield recovery of equine mesenchymal stem cells from umbilical cord matrix/Wharton's jelly using a semi-automated process.

Umbilical cord is an abundant source of perinatal, plastic adherent mesenchymal stem cells (UC-MSCs). UC-MSCs exhibit robust stemness and strong immunosuppressive and regenerative effects in vivo. This protocol describes enzymatic and mechanical dissociation of umbilical cord matrix (Wharton's jelly) that results in efficient isolation of large numbers of fresh nucleated umbilical cord regenerative cells (UC-RCs) that, when cultured on plastic, exhibit similar characteristics of UC-MSCs. This protocol potentially alleviates the need for culture expansion to obtain large numbers of cells required for clinical application. Dissociation is achieved with a blend of collagenase and neutral proteases with agitation at 37 °C in a semi-automatic system. Average expected yield is 1.65 × 10(6) cells/g tissue with 93 % viability. This protocol has been successfully used to isolate an uncultured nucleated regenerative cell population (also referred to as stromal vascular fraction or SVF) from surgically debrided skin and from human, equine, and canine adipose tissue. The procedure requires less than 30 min for tissue dissection and less than 100 min for cell extraction. Quickly obtaining a large number of UC-RCs that have pluripotent differentiation capacity without the complexity and risks of culture expansion could simplify and expand the use of UC-RCs in clinical as well as research applications.

Effects of steroids on the morphology and proliferation of canine and equine mesenchymal stem cells of adipose origin — in vitro research

Disorders of the locomotive system, especially those occurring due to degenerative changes of the joints, are serious problems in daily veterinary medical practice. Steroid injections are the main way of treating these disorders. However, this approach brings usually only temporary effects of pain relief, and may cause many side effects. Alternative therapies focus on regeneration of damaged tissue using adult mesenchymal stem cells (MSCs). Since 2002, the great plasticity and immunomodulatory properties of MSCs isolated from adipose tissue (AdMSCs) have been used successfully in the treatment of degenerative joint diseases (DJD) of both dogs and horses. Possible simultaneous application of steroid therapy and stem cell transplantation could improve the commonly used clinical procedure. In this paper, the influence of the two steroid drugs (betamethasone and methylprednisolone) on AdMSCs was evaluated on the basis of morphology and proliferation rate. Both steroids positively influenced the viability and proliferation state of cells in a concentration of 0.01 mg/ml and 0.1 mg/ml, respectively. However, the concentration of 1 mg/ml had a cytotoxic effect. Moreover, the lower dosage of steroid drugs used in the experiment did not affect the morphology of cells and significantly increased cellular activity. In conclusion, our data demonstrate the stimulating effect of steroid drugs on cell morphology, proliferation rate and cytophysiological activity. These findings may influence the use of stem cells and steroids in applied regenerative veterinary medical practice in the future.

Viability of equine mesenchymal stem cells during transport and implantation.

IntroductionAutologous mesenchymal stem cell (MSC) injection into naturally-occurring equine tendon injuries has been shown to be safe and efficacious and protocols inform translation of the technique into humans. Efficient transfer of cells from the laboratory into tissue requires well-validated transport and implantation techniques.MethodsCell viability in a range of media was determined over 72 hours and after injection through a 19G, 21G or 23G needle. Viability, proliferation and apoptosis were analysed using TrypanBlue, alamarBlue® and AnnexinV assays.ResultsCell viability was similar in all re-suspension media following 24 hour storage, however cell death was most rapid in bone marrow aspirate, platelet-rich plasma and serum after longer storage. Cryogenic media exhibited greatest viability regardless of storage time. Cell proliferation after 24 and 72 hour storage was similar for all media, except after 24 hours in serum wherein proliferation was enhanced. MSC tri-lineage differentiation and viability did not significantly change when extruded through 19G, 21G or 23G needles, but 21G and 23G needles significantly increased apoptotic cells compared to 19G and non-injected controls. All gauges induced a decrease in metabolic activity immediately post-injection but cells recovered by 2 hours.ConclusionsThese data indicate storage and injection influence viability and subsequent cell behaviour and provide recommendations for MSC therapy that implantation of cells should occur within 24 hours of recovery from culture, using larger needle bores.

Multipotency of equine mesenchymal stem cells derived from synovial fluid

Cartilage regeneration with cell therapy following arthroscopic surgery could be used in racehorses with intra-articular fractures (IAF) and osteochondritis dissecans (OCD). The aims of this study were to investigate the origin and multipotency of stromal cells in the synovial fluid (SF) of horses with intra-articular injury and synovitis, and to provide a new strategy for regeneration of lost articular cartilage. Mesenchymal stromal cells were isolated from SF of horses with IAF and OCD. Multipotency was analysed by RT-PCR for specific mRNAs and staining for production of specific extracellular matrices after induction of differentiation. The total number of SF-derived mesenchymal stromal cells reached >1 × 107 by the fourth passage. SF-derived cells were strongly positive (>90% cells positive) for CD44, CD90 and major histocompatibility complex (MHC) class I, and moderately positive (60–80% cells positive) for CD11a/CD18, CD105 and MHC class II by flow cytometry. SF-derived cells were negative for CD34 and CD45. Under specific nutrient conditions, SF-derived cells differentiated into osteogenic, chondrogenic, adipogenic and tenogenic lineages, as indicated by the expression of specific marker genes and by the production of specific extracellular matrices. Chondrogenic induction in culture resulted in a change in cell shape to a ‘stone-wall’ appearance and formation of a gelatinous sheet that was intensely stained with Alcian blue. SF may be a novel source of multipotent mesenchymal stem cells with the ability to regenerate chondrocytes.

Equine mesenchymal stromal cells and embryo-derived stem cells are immune privileged in vitro.

IntroductionAutologous mesenchymal stem cells (MSCs) are an attractive concept in regenerative medicine, but their mechanism of action remains poorly defined. No immune response is reported after in vivo injection of allogeneic equine MSCs or embryo-derived stem cells (ESCs) into the equine tendon, which may be due to the cells’ immune-privileged properties. This study further investigates these properties to determine their potential for clinical application in other tissues.MethodsMitomycin C-treated MSCs, ESCs, or differentiated ESCs (dESCs) were cultured with allogeneic equine peripheral blood mononuclear cells (PBMCs), and their effect on PBMC proliferation, in the presence or absence of interferon-gamma (IFN-γ) was determined. MSCs and super-antigen (sAg)-stimulated PBMCs were co-cultured directly or indirectly in transwells, and PBMC proliferation examined. Media from MSC culture were harvested and used for PBMC culture; subsequent PBMC proliferation and gene expression were evaluated and media assayed for IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin (IL)-10 and IL-6 proteins with enzyme-linked immunosorbent assay (ELISA).ResultsCo-culture of PBMCs with ESCs or dESCs did not affect baseline proliferation, whereas co-culture with MSCs significantly suppressed baseline proliferation. Stimulation of PBMC proliferation by using super-antigens (sAgs) was also suppressed by co-culture with MSCs. Inhibition was greatest with direct contact, but significant inhibition was produced in transwell culture and by using MSC-conditioned media, suggesting that soluble factors play a role in MSC-mediated immune suppression. The MSCs constitutively secrete IL-6, even in the absence of co-culture with PBMCs. MSC-conditioned media also brought about a change in the cytokine-expression profile of sAg-stimulated PBMCs, significantly reducing PBMC expression of IL-6, IFN-γ, and TNF-α.ConclusionsEquine MSCs and ESCs possess a degree of innate immune privilege, and MSCs secrete soluble factors that suppress PBMC proliferation and alter cytokine expression. These properties may make possible the future clinical use of allogeneic stem cells to help standardize and broaden the scope of treatment of tissue injuries.

Clinical follow-up of horses treated with allogeneic equine mesenchymal stem cells derived from umbilical cord blood for different tendon and ligament disorders

Background: Mesenchymal stem cells (MSCs) offer promise as therapeutic aids in the repair of tendon and ligament disorders in sport horses. Equine allogeneic MSCs derived from umbilical cord blood (eUCB-MSCs) can be obtained in a minimally invasive fashion with successful propagation of MSCs.Objective: The objective of this study was to determine the applicability and therapeutic effect of eUCB-MSCs on tendinitis of the superficial digital flexor tendon, desmitis of the suspensory ligament, tendinitis of the deep digital flexor tendon, and desmitis of the inferior check ligament in clinical cases.Methods: A retrospective clinical study was performed. At two equine clinics, 52 warmblood horses were treated with cultured eUCB-MSCs between 2009 and 2012. About 2–10 × 106 cells per lesion were administered. When a lesion was treated twice, the total amount could run up to 20 × 106 cells. Pearson's chi-squared test was used to compare the effect of the injured structure on the success rate, as well as the effect of the age of the horse.Results: Based on repeated examinations, 40 horses (77%) returned to work on the same or a higher level based on information provided by the owner. Neither the injured structure nor the age of the horse had a statistically significant influence on the result.Conclusion: Overall, the results of treatment of some tendon and ligament injuries with eUCB-MSCs in clinical cases are promising.

Equine Mesenchymal Stem Cells Inhibit T Cell Proliferation Through Different Mechanisms Depending on Tissue Source

Mesenchymal stem cells (MSCs) are used in both human clinical trials and veterinary medicine for the treatment of inflammatory and immune-mediated diseases. MSCs modulate inflammation by decreasing the cells and products of the inflammatory response. Stimulated equine MSCs from bone marrow (BM), adipose tissue (AT), cord blood (CB), and umbilical cord tissue (CT) inhibit lymphocyte proliferation and decrease inflammatory cytokine production. We hypothesized that equine MSCs inhibit T cell proliferation through secreted mediators and that MSCs from different tissue sources decrease T cell proliferation through different mechanisms. To test our hypotheses, we inhibited interleukin-6 (IL-6), nitric oxide (NO), and prostaglandin E2 (PGE2) to determine their impact on stimulated T cell proliferation. We also determined how equine MSCs modulate lymphocyte proliferation either via cell cycle arrest or apoptosis. Inhibition of IL-6 or NO did not reverse the immunomodulatory effect of MSCs on activated T cells. In contrast, inhibition of PGE2 restored T cell proliferation, restored the secretion of tumor necrosis factor-α and interferon-γ, and increased IL-10 levels. MSCs from solid-tissue-derived sources, AT and CT, inhibited T cell proliferation through induction of lymphocyte apoptosis while blood-derived MSCs, BM and CB, induced lymphocyte cell cycle arrest. Equine MSCs from different tissue sources modulated immune cell function by both overlapping and unique mechanisms. MSC tissue source may determine immunomodulatory properties of MSCs and may have very practical implications for MSC selection in the application of MSC therapy.

Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential

IntroductionStudies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank.MethodsThe BM-MSCs, AT-MSCs and UC-MSCs were cultured and evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. Additionally, MSCs were assessed for CD105, CD44, CD34, CD90 and MHC-II markers by flow cytometry, and MHC-II was also assessed by immunocytochemistry. To interpret the flow cytometry results, statistical analysis was performed using ANOVA.ResultsThe harvesting and culturing procedures of BM-MSCs, AT-MSCs and UC-MSCs were feasible, with an average cell growth until the third passage of 25 days for BM-MSCs, 15 days for AT-MSCs and 26 days for UC-MSCs. MSCs from all sources were able to differentiate into osteogenic (after 10 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs), adipogenic (after 8 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs) and chondrogenic (after 21 days for BM-MSCs, AT-MSCs and UC-MSCs) lineages. MSCs showed high expression of CD105, CD44 and CD90 and low or negative expression of CD34 and MHC-II. The MHC-II was not detected by immunocytochemistry techniques in any of the MSCs studied.ConclusionsThe BM, AT and UC are feasible sources for harvesting equine MSCs, and their immunophenotypic and multipotency characteristics attained minimal criteria for defining MSCs. Due to the low expression of MHC-II by MSCs, all of the sources could be used in clinical trials involving allogeneic therapy in horses. However, the BM-MSCs and AT-MSCs showed fastest ‘‘in vitro’’ differentiation and AT-MSCs showed highest cell growth until third passage. These findings suggest that BM and AT may be preferable for cell banking purposes.

The influence of static magnetic fields on canine and equine mesenchymal stem cells derived from adipose tissue

The aim of this study was to evaluate the proliferation rate and morphological changes of adipose-derived mesenchymal stem cells of canine and equine origin (Eq- and CaAdMSC). Investigated cells were exposed to a static magnetic field (MF) with the intensity of 0.5 T. Proliferation activity of cells was determined with the Alamar Blue assay. Obtained results, normalized in respect to the control culture, showed that EqAdMSC exposed to MF maintained a high proliferation status, whereas proliferation activity of CaAdMSC cultured in the presence of MF was decreased. Estimations of population doubling time (PDT) also revealed that EqAdMSCs exposed to static MF achieved a twofold increase in the total number of cells in a shorter amount of time than the control culture. The PDT value obtained for investigated CaAdMSCs indicated that MF exposure resulted in the prolongation of population doubling time. Morphology of cells and cellular composition was investigated using a light inverted microscope and a fluorescent microscope. A scanning electron microscope was used for microvesicles (MVs) imaging. Obtained results showed that both cell types maintained fibroblastic morphology and did not reveal signs of apoptosis or necrosis. However, the MF had an influence on the MVs secretion. While EqAdMSCs propagated in the presence of MF were characterized by the abundant MVs presence, CaAdMSCs revealed poor secretory activity. The approach presented provides complex analysis, which enables one to determine changes in equine and canine cytophysiology.

Regenerative Therapies for Equine Degenerative Joint Disease: A Preliminary Study

Degenerative joint disease (DJD) is a major cause of reduced athletic function and retirement in equine performers. For this reason, regenerative therapies for DJD have gained increasing interest. Platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) were isolated from a 6-year-old donor horse. MSCs were either used in their native state or after chondrogenic induction. In an initial study, 20 horses with naturally occurring DJD in the fetlock joint were divided in 4 groups and injected with the following: 1) PRP; 2) MSCs; 3) MSCs and PRP; or 4) chondrogenic induced MSCs and PRP. The horses were then evaluated by means of a clinical scoring system after 6 weeks (T1), 12 weeks (T2), 6 months (T3) and 12 months (T4) post injection. In a second study, 30 horses with the same medical background were randomly assigned to one of the two combination therapies and evaluated at T1. The protein expression profile of native MSCs was found to be negative for major histocompatibility (MHC) II and p63, low in MHC I and positive for Ki67, collagen type II (Col II) and Vimentin. Chondrogenic induction resulted in increased mRNA expression of aggrecan, Col II and cartilage oligomeric matrix protein (COMP) as well as in increased protein expression of p63 and glycosaminoglycan, but in decreased protein expression of Ki67. The combined use of PRP and MSCs significantly improved the functionality and sustainability of damaged joints from 6 weeks until 12 months after treatment, compared to PRP treatment alone. The highest short-term clinical evolution scores were obtained with chondrogenic induced MSCs and PRP. This study reports successful in vitro chondrogenic induction of equine MSCs. In vivo application of (induced) MSCs together with PRP in horses suffering from DJD in the fetlock joint resulted in a significant clinical improvement until 12 months after treatment.

Toepassingen van mesenchymale stamcellen bij het paard: huidige stand van zaken

Mesenchymale stamcellen (MSC) zijn adulte stamcellen met een beperkte differentiatiecapaciteit. Deze cellen kunnen therapeutisch aangewend worden om weefselregeneratie te stimuleren, vorming van littekenweefsel te voorkomen, immuunresponsen te moduleren en ontstekingsprocessen te reguleren. Binnen de diergeneeskunde worden MSC bij het paard voornamelijk gebruikt voor orthopedische toepassingen, hoewel ze ook een rol zouden kunnen spelen in de behandeling van verschillende niet-orthopedische letsels, zoals allerhande immuungemedieerde, ischemische, inflammatoire en neurologische aandoeningen. De efficiëntie van MSC-therapie kan beïnvloed worden door de gebruikte dosis en de manier en het tijdstip van toedienen. Tevens zijn er voor- en nadelen verbonden aan het gebruik van autologe ten opzichte van allogene MSC. Het gebruik van MSC bij paarden biedt immens veel mogelijkheden maar meer fundamenteel onderzoek en goed opgebouwde klinische trials zijn nodig zodat het potentieel van equine MSC ten volle kan geëvalueerd worden en equine MSC-gebaseerde therapieën kunnen geoptimaliseerd worden.

In vitroanalysis of equine, bone marrow‐derived mesenchymal stem cells demonstrates differences within age‐ and gender‐matched horses

Stem cell therapies are used routinely in equine practice. Most published reports characterise stem cells derived from younger horses; however, middle-aged horses are often in athletic performance, and experience degenerative medical conditions. Thus, mesenchymal stem cells (MSCs) from this group should be investigated. To describe differences in in vitro adherence, proliferation and potential for differentiation of equine bone marrow-derived MSCs (equine BMMSCs) harvested from middle-aged (10-13 years old) female donors. Descriptive study of stem cell characteristics. Equine BMMSCs from 6 horses were cultured in vitro and evaluated for viability, proliferation, osteogenesis, chondrogenesis, adipogenesis, cluster-of-differentiation markers and gene expression. Equine BMMSCs from all 6 donors demonstrated fibroblastic, cellular morphology, adherence to plastic and expression of cluster-of-differentiation markers. They varied in their rate of proliferation and trilineage differentiation. The equine BMMSCs of one of 6 donors demonstrated a higher rate of proliferation, enhanced ability for cell passaging and a more robust in vitro differentiation. Comparatively, equine BMMSCs from 2 donors demonstrated a lower rate of proliferation and lack of osteogenic and chondrogenic differentiation. The results of this study confirm that donor-to-donor variation in equine BMMSCs exists and this variation can be documented using in vitro assays. Subjective assessment suggests that the rate of proliferation tends to correlate with differentiation potential.

Development of a cryopreservation protocol for equine mesenchymal stem cells obtained from umbilical cord

In equine medicine, mesenchymal stem cells (MSCs) from bone marrow and adipose tissue are the most common types of adult stem cells (ASC) used for therapeutic approach (VIDAL, M.A. et al. Vet. Surg.,2008; 37:713-24. However, MSC from other sources such as the umbilical cord (UC) have aroused great interest because they share differentiation characteristics between embryonic stem cells and multipotent ASC (CREMONESI, F. et al. Theriogenology,2011; 75:1400-15.The aim of the present work was to evaluate by flow cytometry (FC) the rates of viability and apoptosis in MSC obtained from the umbilical cord Wharton’s jelly matrix (MSC-UC) after cryopreservation with different mediums. For this, samples of UC (n1⁄42) were collected at delivery, processed and cultured in DMEM high glucose supplemented with 20% fetal bovine serum, 1% penicillin/streptomycin, and 1.2% amphotericin B (Invitrogen ) at 37.5 C in a humidified atmosphere, containing 5% CO2 in air. Prior to cryopreservation MSC-UC were characterized showing positive response to adipogenic and osteogenic differentiation, expression of cell surface markers CD44 and intracellular OCT4, absence of MHCII marker, good response to CFU-F assay and normal karyotype (2n1⁄464). MSC were cryopreserved in a controlled freezing system using Mr frosty (-1 C/minute until -80 C -24 hours, Nalgene ) with initial viability of 90 7.42% according to the following protocols: Medium 1: DMEM high glucose, with 20% fetal bovine serum (FBS) and

Combined use of platelet rich plasma and vitamin C positively affects differentiation in vitro to mesodermal lineage of adult adipose equine mesenchymal stem cells

Repair of injured soft and hard tissues in horses can benefit greatly from the use of regenerative therapies with mesenchymal stem cells (MSC). Vitamin-C and platelet-rich-plasma had been used for in vitro differentiation of MSC. This study was aimed to evaluate the effect of vitamin-C, platelet-rich-plasma and their combination on the in vitro differentiation of adipose horse MSC. We isolated MSC from horse fat and differentiated them in vitro into osteogenic and chondrogenic lineages, as demonstrated by specific staining and RT-qPCR of selected genes. Combining vitamin-C and plasma-rich-platelet positively affected the ability of MSC to differentiate in vitro into mesodermal lineages during 14days of culture; this effect was not as marked when differentiation was attempted for 21days. This provides valuable information on the effect of combined use of these molecules in regenerative therapies and their potential application along stem cells for lesions of musculoskeletal tissue in sport horses.