Xenogen-free media provide variable equine mesenchymal stromal cell expansion after a 7-day culture period.

Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation in vivo. The transforming growth factor (TGF)-β, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-β3/Smad signalling in tenogenic differentiation in both human and equine MSC. Primary equine and human MSC isolated from adipose tissue were cultured as monolayers or on tendon-derived decellularized scaffolds to evaluate the influence of the ROCK inhibitor Y-27632 on TGF-β3-induced tenogenic differentiation. The MSC were incubated with and without TGF-β3 (10 ng/ml), Y-27632 (10 μM), or both. On day 1 and day 3, the signalling pathway of TGF-β and the actin cytoskeleton were visualized by Smad 2/3 and phalloidin staining, and gene expression of signalling molecules and tendon markers was assessed. ROCK inhibition was confirmed by disruption of the actin cytoskeleton. Activation of Smad 2/3 with nuclear translocation was evident upon TGF-β3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species (p < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-β3 and ROCK inhibition were combined (p < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-β3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-β3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.

BackgroundThe prevalence of chronic skin wounds in humans is high, and treatment is often complicated by the presence of pathogenic bacteria. Therefore, safe and innovative treatments to reduce the bacterial load in cutaneous wounds are needed. Mesenchymal stromal cells (MSC) are known to provide paracrine signals that act on resident skin cells to promote wound healing, but their potential antibacterial activities are not well described. The present study was designed to examine the antibacterial properties of MSC from horses, as this animal model offers a readily translatable model for MSC therapies in humans. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC on the growth of representative gram-negative and gram-positive bacterial species commonly found in skin wounds and (ii) define the mechanisms by which MSC inhibit bacterial growth.MethodsMSC were isolated from the peripheral blood of healthy horses. Gram-negative E. coli and gram-positive S. aureus were cultured in the presence of MSC and MSC conditioned medium (CM), containing all factors secreted by MSC. Bacterial growth was measured by plating bacteria and counting viable colonies or by reading the absorbance of bacterial cultures. Bacterial membrane damage was detected by incorporation of N-phenyl-1-naphthylamine (NPN). Antimicrobial peptide (AMP) gene and protein expression by equine MSC were determined by RT-PCR and Western blot analysis, respectively. Blocking of AMP activity of MSC CM was achieved using AMP-specific antibodies.ResultsWe found that equine MSC and MSC CM inhibit the growth of E. coli and S. aureus, and that MSC CM depolarizes the cell membranes of these bacteria. In addition, we found that equine MSC CM contains AMPs, and blocking these AMPs with antibodies reduces the effects of MSC CM on bacteria.ConclusionsOur results demonstrate that equine MSC inhibit bacterial growth and secrete factors that compromise the membrane integrity of bacteria commonly found in skin wounds. We also identified four specific AMPs produced by equine MSC. The secretion of AMPs may contribute to the value of MSC as a therapy for cutaneous wounds in both horses and humans.

IntroductionMesenchymal stromal cells (MSCs) have been extensively studied for their promising capabilities in regenerative medicine. Although bone marrow is the best-known source for isolating equine MSCs, non-invasive alternative sources such as umbilical cord blood (UCB), umbilical cord matrix (UCM), and peripheral blood (PB) have also been reported.MethodsEquine MSCs from three non-invasive alternative sources were isolated from six individual mares (PB) and their foals (UCB and UCM) at parturition. To minimize inter-horse variability, the samples from the three sources were matched within the same mare and for UCB and UCM even within the same foal from that specific mare. The following parameters were analyzed: (i) success rate of isolation, (ii) proliferation capacity, (iii) tri-lineage differentiation ability, (iv) immunophenotypical protein, and (v) immunomodulatory mRNA profiles. Linear regression models were fit to determine the association between the source of MSCs (UCB, UCM, PB) and (i) the moment of first observation, (ii) the moment of first passage, (iii) cell proliferation data, (iv) the expression of markers related to cell immunogenicity, and (v) the mRNA profile of immunomodulatory factors, except for hepatocyte growth factor (HGF) as no normal distribution could be obtained for the latter variable. To evaluate the association between the source of MSCs and the mRNA expression of HGF, the non-parametric Kruskal-Wallis test was performed instead.ResultsWhile equine MSCs could be isolated from all the UCB and PB samples, isolation from UCM was successful in only two samples because of contamination issues. Proliferation data showed that equine MSCs from all three sources could be easily expanded, although UCB-derived MSCs appeared significantly faster in culture than PB- or UCM-derived MSCs. Equine MSCs from both UCB and PB could be differentiated toward the osteo-, chondro-, and adipogenic lineage, in contrast to UCM-derived MSCs in which only chondro- and adipogenic differentiation could be confirmed. Regardless of the source, equine MSCs expressed the immunomodulatory genes CD40, CD80, HGF, and transforming growth factor-beta (TGFβ). In contrast, no mRNA expression was found for CD86, indoleamine 2,3-dioxygenase (IDO), and tumor necrosis factor-alpha (TNFα).ConclusionsWhereas UCM seems less feasible because of the high contamination risks and low isolation success rates, UCB seems a promising alternative MSC source, especially when considering allogeneic MSC use.

Simple SummaryRegenerative medicine using platelet-based blood products or adult stem cells offers the prospect of better clinical outcomes with many diseases. In veterinary medicine, most progress has been made with the development and therapeutic use of these regenerative therapeutics in horses, but the clinical need is given in dogs as well. Our aim was to transfer previous advances in the development of horse regenerative therapeutics, specifically the use of platelet lysate for feeding stem cell cultures, to the dog. Here, we describe the scalable production of canine platelet lysate, which could be used in regenerative biological therapies. We also evaluated the canine platelet lysate for its suitability in feeding canine stem cell cultures in comparison to equine platelet lysate used for equine stem cell cultures. Platelet lysate production from canine blood was successful, but the platelet lysate did not support stem cell culture in dogs in the same beneficial way observed with the equine platelet lysate and stem cells. In conclusion, canine platelet lysate can be produced in large scales as described here, but further research is needed to improve the cultivation of canine stem cells.Platelet lysate (PL) is an attractive platelet-based therapeutic tool and has shown promise as xeno-free replacement for fetal bovine serum (FBS) in human and equine mesenchymal stromal cell (MSC) culture. Here, we established a scalable buffy-coat-based protocol for canine PL (cPL) production (n = 12). The cPL was tested in canine adipose MSC (n = 5) culture compared to FBS. For further comparison, equine adipose MSC (n = 5) were cultured with analogous equine PL (ePL) or FBS. During canine blood processing, platelet and transforming growth factor-β1 concentrations increased (p < 0.05 and p < 0.001), while white blood cell concentrations decreased (p < 0.05). However, while equine MSC showed good results when cultured with 10% ePL, canine MSC cultured with 2.5% or 10% cPL changed their morphology and showed decreased metabolic activity (p < 0.05). Apoptosis and necrosis in canine MSC were increased with 2.5% cPL (p < 0.05). Surprisingly, passage 5 canine MSC showed less genetic aberrations after culture with 10% cPL than with FBS. Our data reveal that using analogous canine and equine biologicals does not entail the same results. The buffy-coat-based cPL was not adequate for canine MSC culture, but may still be useful for therapeutic applications.

Mesenchymal stromal cells (MSCs) are a promising cell source for cartilage tissue regeneration in animals and humans but with large inter-donor variation in their in vitro chondrogenic differentiation potential. Underlying molecular mechanisms responsible for culture-expanded MSC heterogeneity remains poorly understood. In this study, we sought to identify variations in miRNA signatures associated with cultured equine MSC chondrogenic differentiation potential from different donors. Neocartilage tissue generated from equine cord blood-derived MSCs (CB-MSCs) was categorized as having either high or low chondrogenic potential based on their histological appearance and quantification of glycosaminoglycan deposition. Using next-generation sequencing, we identified 30 differentially expressed miRNAs amongst undifferentiated MSC cultures that corresponded with their chondrogenic potential. Of note, MSCs with low chondrogenic potential upregulated miR-146a and miR-487b-3p, which was also observed by qRT-PCR. Our findings suggest that miRNA profiling of equine MSC cultures may have prognostic value in selecting MSC donors with regards to their chondrogenic differentiation potential.

BackgroundMesenchymal stromal cells (MSCs) can be used intra-articularly to quell inflammation and promote cartilage healing; however, mechanisms by which MSCs mitigate joint disease remain poorly understood. Galectins, a family of β-galactoside binding proteins, regulate inflammation, adhesion and cell migration in diverse cell types. Galectin-1 and galectin-3 are proposed to be important intra-articular modulators of inflammation in both osteoarthritis and rheumatoid arthritis. Here, we asked whether equine bone marrow-derived MSCs (BMSCs) express higher levels of galectin-1 and -3 relative to synovial fibroblasts and chondrocytes and if an inflammatory environment affects BMSC galectin expression and motility.MethodsEquine galectin-1 and -3 gene expression was quantified using qRT-PCR in cultured BMSCs, synoviocytes and articular chondrocytes, in addition to synovial membrane and articular cartilage tissues. Galectin gene expression, protein expression, and protein secretion were measured in equine BMSCs following exposure to inflammatory cytokines (IL-1β 5 and 10 ng/mL, TNF-α 25 and 50 ng/mL, or LPS 0.1, 1, 10 and 50 μg/mL). BMSC focal adhesion formation was assessed using confocal microscopy, and BMSC motility was quantified in the presence of inflammatory cytokines (IL-1β or TNF-α) and the pan-galectin inhibitor β-lactose (100 and 200 mM).ResultsEquine BMSCs expressed 3-fold higher galectin-1 mRNA levels as compared to cultured synovial fibroblasts (p = 0.0005) and 30-fold higher galectin-1 (p < 0.0001) relative to cultured chondrocytes. BMSC galectin-1 mRNA expression was significantly increased as compared to carpal synovial membrane and articular cartilage tissues (p < 0.0001). IL-1β and TNF-α treatments decreased BMSC galectin gene expression and impaired BMSC motility in dose-dependent fashion but did not alter galectin protein expression. β-lactose abrogated BMSC focal adhesion formation and inhibited BMSC motility.ConclusionsEquine BMSCs constitutively express high levels of galectin-1 mRNA relative to other articular cell types, suggesting a possible mechanism for their intra-articular immunomodulatory properties. BMSC galectin expression and motility are impaired in an inflammatory environment, which may limit tissue repair properties following intra-articular administration. β-lactose-mediated galectin inhibition also impaired BMSC adhesion and motility. Further investigation into the effects of joint inflammation on BMSC function and the potential therapeutic effects of BMSC galectin expression in OA is warranted.

Equine mesenchymal stromal cells (MSCs) have been found to be beneficial for the treatment of many ailments, including orthopedic injuries, due to their superior differentiation potential and immunomodulating properties. Cell therapies require large cell numbers, which are not efficiently generated using conventional static expansion methods. Expansion of equine cord blood-derived MSCs (eCB-MSCs) in bioreactors, using microcarriers as an attachment surface, has the potential to generate large numbers of cells with increased reproducibility and homogeneity compared with static T-flask expansion. This study investigated the development of an expansion process using Vertical-Wheel (VW) bioreactors, a single-use bioreactor technology that incorporates a wheel instead of an impeller. Initially, microcarriers were screened at small scale to assess eCB-MSC attachment and growth and then in bioreactors to assess cell expansion and harvesting. The effect of different donors, serial passaging, and batch versus fed batch were all examined in 0.1 L VW bioreactors. The use of VW bioreactors with an appropriate microcarrier was shown to be able to produce cell densities of up to 1E6 cells/mL, while maintaining cell phenotype and functionality, thus demonstrating great potential for the use of these bioreactors to produce large cell numbers for cell therapies.

Mesenchymal stromal cells (MSCs) are an ideal cell source for allogenic cell therapy due to their immunomodulatory and differentiation properties. Equine MSCs (eMSCs) have been found to be a promising treatment for equine joint injuries including meniscal injuries, cartilage degradation, and osteoarthritis. Although the use of eMSCs has shown efficacy in preliminary studies, challenges associated with biomanufacturing remain. To achieve the required cell numbers for clinical application, bioreactor-based processes are required. Initial studies have shown that eMSCs can be cultivated in microcarrier-based, stirred suspension bioreactor culture at the laboratory 0.1 L scale using a Vertical-Wheel® (VW) bioreactor. However, investigations regarding scale up of these processes to the required biomanufacturing scales are required. This study investigated the scale-up of a equine cord blood MSC (eCB-MSC) bioprocess in VW bioreactors at three scales. This included scale-up from the 0.1–0.5 L bioreactor, scale-up from static culture to the 3 L computer-controlled bioreactor, and scale-up into the 3 L computer-controlled bioreactor using a mock clinical trial process. Results from the various scale-up experiments demonstrated similar cell expansion at the various tested scales. The 3 L computer-controlled system resulted in a final cell densities of 1.5 × 105 cells/cm2 on average, achieving 1.5 × 109 harvested cells. Biological testing of the cells showed that cell phenotype and functionality were maintained after scale-up. These findings demonstrate the scalability of an eCB-MSC bioprocess using microcarriers in VW bioreactors to achieve clinically relevant cell numbers, a critical step to translate MSC treatments from research to clinical applications. This study also represents the first known published study expanding any cell type in the 3 L VW bioreactor.

The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below -80°C and typically below -140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins are used as protectant reagents, but unexpected changes in stem cell fate and downstream toxicity effects have been reported, limiting their wide use in clinical settings. In scientific reviews, there are not much data regarding viability of mesenchymal stromal cells (MSCs) after the freezing/thawing process. During our routine analysis, a poor resistance to cryopreservation of these cells was observed, as well as their weak ability to replicate. This is an important point in the study of MSCs; moreover, it represents a limit for preservation and long-term storage. For this reason, MSCs isolated from equine, ovine, and rodent bone marrow and equine adipose tissue were compared using different cryopreservation solutions for this study of vitality. Our findings showed the best results regarding cell viability using a solution of fetal bovine serum with addition of 10% DMSO. In particular, we noted an increase in survival of equine bone marrow MSCs. This parameter has been evaluated by Trypan blue staining at fixed times (0, 24, and 48 hours post-thaw). This result highlights the fact that equine bone marrow MSCs are the frailest we analyzed. Therefore, it could be useful to delve further into this topic in order to improve the storage possibility for these cells and their potential use in cell-based therapies.

Background: Mesenchymal stromal cells (MSCs) are being investigated as a stroke therapy both in preclinical and clinical trials. MSCs may promote recovery through secretome release and immunomodulation. Approximately one-third of stroke patients are on statins. With cell therapy gaining momentum, we explored the effect of two statins on MSCs. Methods: Clinical grade bone marrow MSCs from 3 healthy donors at passage 2 were thawed and re-suspended in serum free media. Monocytes (Mo) were isolated from peripheral blood of healthy humans and stroke patients. MSCs and Mo were cultured alone as well as in co-culture and exposed to different doses of atorvastatin and simvastatin. At 24 and 48 hours of incubation, viability of MSCs was measured using ATP assay. 5-bromo-2’-deoxyuridine (BrdU) assay was used to measure proliferation. Media from treated MSCs was analyzed for secretomes and treated cells were analyzed for gene expression changes. Data were averaged for 3 donors. Results: Both atorvastatin and simvastatin decreased the viability and proliferation of MSCs (Fig. 1A-B), but at physiologically relevant doses both were maintained above 90%. Clinically relevant doses of both drugs decreased the secretion of TNF-α and IL-6 from MSCs upto two fold (Fig. 1C-D). IL-6 expression decreased and IL-10 expression increased dose-dependently in MSCs (data not shown). When co-cultured with Mo from control vs stroke patients, both statins at physiologically relevant doses increased VEGF release and decreased the secretion of IL-6, IL-8 and MCP-1 (Fig. 1E-H). Conclusion: Exposure of MSCs to statins can alter their immunomodulatory function. Furthermore, these effects are consistent across two commonly prescribed drugs. Our results suggest that stroke trials involving use of intravenous MSCs should consider the impact of statins on MSC function.

The therapeutic potential of mesenchymal stromal cells for cellular therapy has generated increasing interest in human as well as veterinary medicine. Considerable research has been performed on the cryopreservation of expanded mesenchymal stromal cells, but little information is available on the cryopreservation of the original mononuclear cell fraction. The present study describes a protocol to expand equine mesenchymal stromal cells after cryopreserving the mononuclear cells of umbilical cord blood. To this end, mononuclear cells were isolated from 7 umbilical cord blood samples and cryopreserved at a concentration of 1-2 × 10(9) cells/l cold freezing solution. Cells were cryopreserved and kept frozen for at least 6 months before thawing. Frozen cryotubes were thawed in a 37°C water bath. Putative equine mesenchymal stromal cells were immunophenotyped using multicolour flow cytometry based on a selected 9 marker panel. Average cell viability upon thawing was 98.7 ± 0.6%. In 6 out of 7 samples, adherent spindle-shaped cell colonies were observed within 9.0 ± 2.6 days and attained 80% confluency at 12.3 ± 3.9 days. After 3 passages, putative equine mesenchymal stromal cells were successfully immunophenotyped as CD29, CD44 and CD90 positive, and CD45, CD73, CD79α, CD105, MHC II and monocyte-marker negative. Equine mesenchymal stromal cells can be cultured after cryopreservation of the isolated mononuclear cells, a time- as well as cost-efficient approach in equine regenerative medicine.

Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number of clinical applications, e.g., non-healing bone fractures and defects and also non-skeletal degenerative diseases like heart failure. Currently, the numbers of clinical trials that employ MSC are increasing. However, several biological and biotechnological challenges need to be overcome to benefit from the full potential of hMSC. In this current review, we present some of the most important and recent advances in understanding of the biology of hMSC and their current and potential use in therapy.

Mesenchymal stromal cells (MSCs) from various species, such as humans, mice, and horses, were recently found to effectively inhibit the growth of various bacteria associated with chronic infections, such as nonhealing cutaneous wounds, via secretion of antimicrobial peptides. These MSC antimicrobial properties have primarily been studied in the context of the planktonic phenotype, and thus, information on the effects on bacteria in biofilms is largely lacking. The objectives of this study were to evaluate the in vitro efficacy of the MSC secretome against various biofilm‐forming wound pathogens, including the methicillin‐resistant Staphylococcus aureus (MRSA), and to explore the mechanisms that affect bacterial biofilms. To this end, we used equine MSCs, because the horse represents a physiologically relevant model for human wound healing and offers a readily translatable model for MSC therapies in humans. Our salient findings were that the equine MSC secretome inhibits biofilm formation and mature biofilms of various bacteria, such as Pseudomonas aeruginosa, S. aureus, and Staphylococcus epidermidis. Furthermore, we demonstrated that equine MSC secrete cysteine proteases that destabilize MRSA biofilms, thereby increasing the efficacy of antibiotics that were previously tolerated by the biofilms. In light of the rise of antibiotic‐resistant bacterial strains as an increasing global health threat, our results provide the rationale for using the MSC secretome as a complementary treatment for bacterial skin infections in both humans and horses.

Current mesenchymal stromal cell (MSC) delivery methods require infusion/implantation through needles and/or catheters. Little investigation into the effect of delivery via catheter injection has been completed. We hypothesize that injection of rat and human MSCs through various clinically relevant-sized catheters and flow rates will not affect cell viability, characterization, or function. Both rat and human MSCs were injected through 20-, 25-, and 30-gauge needles, as well through an SL-10 microcatheter at rates of 60, 120, 240, and 500 mL/h. MSC viability and apoptotic fraction was measured. MSCs were characterized 24 h after injection with flow cytometric immunophenotyping, and multilineage differentiation was completed. Catheter diameter or flow rate did not affect rat MSC viability. No clinically significant decrease in human MSC viability was observed immediately after injection; however, a delayed decrease in viability was observed at 24 h. No difference in the surface markers CD11b, CD45, CD29, CD49e, CD73, CD90, CD105, and Stro-1 or the capacity for multilineage differentiation (adipogenesis, osteogenesis, and chondrogenesis) was observed for either rat or human MSCs. The injection of human and rat MSCs through various clinically relevant catheters and flow rates did not have a clinically significant effect on viability immediately after injection, indicating compliance with recently published Food and Drug Administration guidelines (viability >70%). Further, no changes in cell characterization or function were observed via measurement of cell surface markers and the capacity for multilineage differentiation, respectively. These results ensure the biocompatibility of MSCs with commonly used delivery methods.

Horses are an approved large animal model for therapies of the musculoskeletal system. Especially for tendon disease where cell-based therapy is commonly used in equine patients, the translation of achieved results to human medicine would be a great accomplishment. Immunophenotyping of equine mesenchymal stromal cells (MSCs) remains the last obstacle to meet the criteria of the International Society for Cellular Therapy (ISCT) definition of human MSCs. Therefore, the surface antigen expression of CD 29, CD 44, CD 73, CD 90, CD 105, CD 14, CD 34, CD 45, CD 79α, and MHC II in equine MSCs from adipose tissue, bone marrow, umbilical cord blood, umbilical cord tissue, and tendon tissue was analyzed using flow cytometry. Isolated cells from the different sources and donors varied in their expression pattern of MSC-defining antigens. In particular, CD 90 and 105 showed most heterogeneity. However, cells from all samples were robustly positive for CD 29 and CD 44, while being mostly negative for CD 73 and the exclusion markers CD 14, CD 34, CD 45, CD 79α and MHC II. Furthermore, it was evident that enzymes used for cell detachment after in vitro-culture affected the detection of antigen expression. These results emphasize the need of standardization of MSC isolation, culturing, and harvesting techniques. As the equine MSCs did not meet all criteria the ISCT defined for human MSCs, further investigations for a better characterization of the cell type should be conducted.