[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cell (MSC) therapy is being increasingly used to treat dogs and horses with naturally-occurring diseases. However these animals also serve as critical large animal models for ongoing translation of cell therapy products to the human market. MSC manufacture for clinical use mandates improvement in cell culture systems to meet demands for higher MSC numbers and removal of xeno-proteins (i.e. fetal bovine serum, FBS). While serum-free media (SFM) is commercially available, its affects on MSC phenotype and immunomodulatory functions are not fully known. The objective of this study was to determine if specific MSC culture conditions, MSC expansion in HYPERFlasks® or MSC expansion in a commercially available SFM, would alter MSC proliferation, phenotype or immunomodulatory properties in vitro. MSCs cultured in HYPERFlasks® were similar in phenotype, proliferative capacity and immunomodulatory functions to MSCs grown in standard flasks however MSC yield was markedly increased. HYPERFlasks® therefore provide a viable option to generate greater cell numbers in a streamlined manner. Canine and equine MSCs expanded in SFM displayed similar proliferation, surface phenotype and inhibitory effect on lymphocyte proliferation in vitro. However, MSCs cultured in the absence of FBS secreted significantly less PGE2, and were significantly less able to inhibit IFNγ secretion by activated T-cells. Immunomodulatory functions altered by expansion in SFM were species dependent. Unlike equine MSCs, in canine adipose-derived MSCs, the inhibition of lymphocyte proliferation was not principally modulated by PGE2. The removal of FBS from both canine and equine MSC culture systems resulted in altered immunomodulatory properties in vitro and warrants further investigation prior to moving towards FBS-free culture conditions.
Full-text · Article · Dec 2015 · Stem Cell Reviews and Reports
[Show abstract][Hide abstract] ABSTRACT: Significance:
This study is the first to demonstrate the safety and efficacy of fresh, autologous, adipose-derived stem cell systemic therapy for a naturally occurring, chronic inflammatory disease in cats. The findings demonstrate that this therapy resulted in complete clinical and histological resolution or reduction in clinical disease severity and immune modulation in most cats. This study also identified a potentially useful biomarker that could dictate patient enrollment and shed light on immune modulation mechanism. As a naturally occurring animal model, FCGS also provides a strategic platform for potentially translatable therapy for the treatment of human oral inflammatory disease.
Full-text · Article · Nov 2015 · STEM CELLS TRANSLATIONAL MEDICINE
[Show abstract][Hide abstract] ABSTRACT: Knowledge and resources derived from veterinary medicine represent an underused resource that could serve as a bridge between data obtained from diseases models in laboratory animals and human clinical trials. Naturally occurring disease in companion animals that display the defining attributes of similar, if not identical, diseases in humans hold promise for providing predictive proof of concept in the evaluation of new therapeutics and devices. Here we outline comparative aspects of naturally occurring diseases in companion animals and discuss their current uses in translational medicine, benefits, and shortcomings. Last, we envision how these natural models of disease might ultimately decrease the failure rate in human clinical trials and accelerate the delivery of effective treatments to the human clinical market.
Full-text · Article · Oct 2015 · Science translational medicine
[Show abstract][Hide abstract] ABSTRACT: Significance:
It has been established that mouse and human epidermal neural crest stem cells are somatic multipotent stem cells with proved innovative potential in a mouse model of spinal cord injury (SCI) offering promise of a valid treatment for SCI. Traumatic SCI is a common neurological problem in dogs with marked similarities, clinically and pathologically, to the syndrome in people. For this reason, dogs provide a readily accessible, clinically realistic, spontaneous model for evaluation of epidermal neural crest stem cells therapeutic intervention. The results of this study are expected to give the baseline data for a future clinical trial in dogs with traumatic SCI.
No preview · Article · Aug 2015 · STEM CELLS TRANSLATIONAL MEDICINE
[Show abstract][Hide abstract] ABSTRACT: Abstract
The ability to repair damaged cartilage is a major goal of musculoskeletal tissue engineering. Allogeneic (same species, different individual) or xenogeneic (different species) sources can provide an attractive source of chondrocytes for cartilage tissue engineering, since autologous (same individual) cells are scarce. Immune rejection of non-autologous hyaline articular cartilage has seldom been considered due to the popular notion of “cartilage immunoprivilege”. The objective of this study was to determine the suitability of allogeneic and xenogeneic engineered neocartilage tissue for cartilage repair. To address this, scaffold-free tissue engineered articular cartilage of syngeneic (same genetic background), allogeneic, and xenogeneic origin were implanted into two different locations of the rabbit knee (n = 3 per group/location). Xenogeneic engineered cartilage and control xenogeneic chondral explants provoked profound innate inflammatory and adaptive cellular responses, regardless of transplant location. Cytological quantification of immune cells showed that, while allogeneic neocartilage elicited an immune response in the patella, negligible responses were observed when implanted into the trochlea; instead the responses were comparable to microfracture-treated empty defect controls. Allogeneic neocartilage survived within the trochlea implant site and demonstrated graft integration into the underlying bone. In conclusion, the knee joint cartilage does not represent an immune privileged site, strongly rejecting xenogeneic but not allogeneic chondrocytes in a location-dependent fashion. This difference in location-dependent survival of allogeneic tissue may be associated with proximity to the synovium.
No preview · Article · May 2015 · Acta Biomaterialia
[Show abstract][Hide abstract] ABSTRACT: Intravenous (IV) injection of mesenchymal stem cells (MSCs) is used to treat systemic human diseases and disorders but is not routinely used in equine therapy. In horses, MSCs are primarily isolated from adipose tissue (AT) or bone marrow (BM) and used for treatment of orthopedic injuries through one or more local injections. The objectives of this study were to determine safety and lymphocyte response to multiple allogeneic IV injections of either AT-MSCs or BM-MSCs to healthy horses.
We injected 3 doses of 25 x 10(6) allogeneic MSCs from either AT or BM (a total of 75 x 10(6) MSCs per horse) into 5 and 5, respectively, healthy horses. Horses were followed up for 35 days after the first MSC infusion. We evaluated host inflammatory and immune response including total leukocytes numbers, serum cytokine concentration and splenic lymphocyte subsets.
Repeated injection of allogeneic AT-MSCs or BM-MSCs did not elicit any clinical adverse effects. Repeated BM-MSC injection resulted in increased blood CD8+ T cell numbers. Multiple BM-MSC injections also increased splenic T regulatory cell numbers compared to AT-MSC injected horses but not controls.
These data demonstrate that multiple, IV injections of allogeneic MSCs are well tolerated by healthy horses. No clinical signs or clinico-pathologic measurements of organ toxicity or systemic inflammatory response were recorded. Increased numbers of circulating CD8+ T cell after multiple IV injections of allogeneic BM-MSC may indicate a mild allo-antigen-directed cytotoxic response. Safety and efficacy of allogeneic MSC IV infusions in sick horses remains to be determined.
[Show abstract][Hide abstract] ABSTRACT: Background
Volume reduction and RBC depletion of equine bone marrow specimens are necessary processing steps for the immediate therapeutic use of bone marrow (BM)-derived mesenchymal stem cells (MSC), and for MSC expansion in culture.Objectives
The purpose of the study was to evaluate the ability of the PrepaCyte-CB processing system to reduce volume, deplete RBC, and recover mononuclear cells (MNC) from equine BM specimens.Methods
One hundred and twenty mL of heparinized BM were obtained from each of 90 horses. A CBC was performed on the BM pre- and post-PrepaCyte-CB processing. Volume and RBC reduction, and total nucleated cell (TNC) and MNC recoveries were determined.ResultsBone marrow volume was reduced from 120 mL to 21 mL with a median RBC depletion of 90.1% (range, 62.0–96.7%). The median preprocessing total TNC count was 2.2 × 109 (range, 0.46–7.9 × 109) and the median postprocessing TNC count was 1.7 × 109 (range, 0.3–4.4 × 109; P < .0001), with a median recovery of 73.5% (range, 22.4–216.7%). The median preprocessing total MNC count was 0.9 × 109 (range, 0.1–4.7 × 109) and median postprocessing total MNC count was 0.8 × 109 (range, 0.1–2.7 × 109; P = .06), with a median recovery of 83.7% (range, 15.4–413.9%).Conclusions
The PrepaCyte-CB processing system can be used to deplete both volume and RBC, and recover MNC from equine BM specimens. Further studies assessing the viability of MSC and the efficacy of MSC expansion after using the PrepaCyte-CB processing system are warranted.
No preview · Article · Jan 2015 · Veterinary Clinical Pathology
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells have been proposed to treat liver disease in the dog. The objective of this study was to compare portal, systemic intravenous and splenic injections for administration of mesenchymal stem cells to target the liver in healthy beagle dogs. Four healthy beagle dogs were included in the study. Each dog received mesenchymal stem cells via all three delivery methods in randomized order, 1 week apart. Ten million fat-derived allogeneic mesenchymal stem cells labeled with Technetium-99m (99mTc)-hexamethyl-propylene amine oxime(HMPAO) were used for each injection. Right lateral, left lateral, ventral, and dorsal scintigraphic images were obtained with a gamma camera equipped with a low-energy all-purpose collimator immediately after injection and 1, 6, and 24 h later. Mesenchymal stem cells distribution was assessed subjectively using all four views. Pulmonary, hepatic, and splenic uptake was quantified from the right lateral view, at each time point. Portal injection resulted in diffuse homogeneous high uptake through the liver, whereas the systemic intravenous injection led to mesenchymal stem cell trapping in the lungs. After splenic injection, mild splenic retention and high homogeneous diffuse hepatic uptake were observed. Systemic injection of mesenchymal stem cells may not be a desirable technique for liver therapy due to pulmonary trapping. Splenic injection represents a good alternative to portal injection. Scintigraphic tracking with 99mTc-HMPAO is a valuable technique for assessing mesenchymal stem cells distribution and quantification shortly after administration. Data obtained at 24 h should be interpreted cautiously due to suboptimal labeling persistence.
No preview · Article · Jan 2015 · Veterinary Radiology & Ultrasound
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are a promising therapeutic option for various immune-mediated and inflammatory disorders due to their potent immunomodulatory, and trophic properties. Naturally occurring diseases in large animal species may serve as surrogate animal models of human disease, as they may better reflect the complex genetic, environmental and physiologic variation present in outbred populations. We work with naturally occurring diseases in large animal species to better understand how MSCs work and to facilitate optimal translation of MSC-based therapies. We are investigating the use of MSC therapy for a chronic oral inflammatory disease in cats. During our efforts to expand fat-derived feline MSCs (fMSCs) we observed that ∼50% of the cell lines developed giant foamy multinucleated cells in later passages. These morphologic alterations were associated with proliferation arrest. We hypothesized that the cytopathic effects were caused by infection with a retrovirus, feline foamy virus (FFV). Using transmission electron microscopy (TEM), PCR, and in vitro assays we determined that syncytial cell formation and proliferation arrest in fMSCs were caused by FFV strains that were highly homologous to previously reported FFV strains. We determined that the antiretroviral drug, Tenofovir, may be used to support ex vivo expansion and salvage of FFV-infected fMSC lines. MSC lines derived from specific pathogen free (SPF) cats do not appear to be infected with FFV and may be a source of allogeneic fMSCs for clinical application. FFV infection of fMSC lines may hinder large-scale expansion of autologous MSC for therapeutic use in feline patients.
No preview · Article · Nov 2014 · Stem Cells and Development
[Show abstract][Hide abstract] ABSTRACT: A 9-year-old female spayed English Springer Spaniel was evaluated for a cranial mediastinal mass and lymphocytosis. Flow cytometric immunophenotyping of peripheral blood lymphocytes revealed 97% as CD3 positive, confirming a T-cell lineage. Additionally, T-cell subset assessment showed 53.2% to be double-negative T-lymphocytes, expressing neither CD4 nor CD8 surface markers. The number of double-negative lymphocytes in circulation coincided with the number of T-cell receptor (TCR) γδ-expressing T-cells in circulation. Molecular T-cell clonality analysis of TCR Gamma (TCRG) gene rearrangement showed a polyclonal expansion of T-lymphocytes. Histopathology confirmed the mass to be a thymoma, supporting the diagnosis of thymoma-associated T-cell lymphocytosis. Resolution of the lymphocytosis after removal of the thymoma provided further evidence for this diagnosis. To the authors' knowledge, this case is only the second report of thymoma-associated peripheral lymphocytosis in the veterinary literature, and is the first to report a confirmed thymoma-associated peripheral γδ T-cell lymphocytosis in a dog.
No preview · Article · Oct 2014 · Veterinary Clinical Pathology
[Show abstract][Hide abstract] ABSTRACT: SUMOylation, the covalent attachment of a member of the SUMO (small ubiquitin-like modifier) family of proteins to lysines in target substrates, is an essential post-translational modification in eukaryotes. Microbial manipulation of SUMOylation recently emerged as a key virulence strategy for viruses and facultative intracellular bacteria, the latter of which only have been shown to deploy effectors that negatively regulate SUMOylation. Here, we demonstrate that the obligate intracellular bacterium, Anaplasma phagocytophilum, utilizes an effector, AmpA (A. phagocytophilum post-translationally modified protein A) that becomes SUMOylated in host cells and this is important for the pathogen's survival. We previously discovered that AmpA (formerly APH1387) localizes to the A. phagocytophilum-occupied vacuolar membrane (AVM). Algorithmic prediction analyses denoted AmpA as a candidate for SUMOylation. We verified this phenomenon using a SUMO-affinity matrix to precipitate both native AmpA and ectopically expressed green fluorescent protein (GFP)-tagged AmpA. SUMOylation of AmpA was lysine-dependent, as SUMO-affinity beads failed to precipitate a GFP-AmpA protein when its lysine residues were substituted with arginine. Ectopically expressed and endogenous AmpA were poly-SUMOylated, which was consistent with the observation that AmpA colocalizes with SUMO2/3 at the AVM. Only late during the infection cycle did AmpA colocalize with SUMO1, which terminally caps poly-SUMO2/3 chains. AmpA was also detected in the cytosol of infected host cells, further supporting its secretion and likely participation in interactions that aid pathogen survival. Indeed, whereas siRNA-mediated knockdown of Ubc9 – a necessary enzyme for SUMOylation – slightly bolstered A. phagocytophilum infection, pharmacologically inhibiting SUMOylation in infected cells significantly reduced the bacterial load. Ectopically expressed GFP-AmpA served as a competitive agonist against native AmpA in infected cells, while lysine-deficient GFP-AmpA was less effective, implying that modification of AmpA lysines is important for infection. Collectively, these data show that AmpA becomes directly SUMOylated during infection, representing a novel tactic for A. phagocytophilum survival.
No preview · Article · Oct 2014 · Cellular Microbiology
[Show abstract][Hide abstract] ABSTRACT: The use of cell transplantation for spinal cord injury is a rapidly evolving field in regenerative medicine. Numerous animal models are currently being used. However, translation to human patients is still a challenging step. Dogs are of increasing importance as a translational model for human disease since there is a greater awareness of the need to increase the quality of preclinical data. The use of dogs ultimately brings benefit to both human and veterinary medicine. In this review we analyze experimental and clinical studies using cell transplantation for canine spinal cord injury. Overall, in experimental studies, transplantation groups showed improvement over control groups. Improvements were measured at the functional, electrophysiological, histological, RNA and protein levels. Most clinical studies support beneficial effects of cell transplantation despite the fact that methodological limitations preclude definitive conclusions. However, the mechanisms of action and underlying the behavior of transplanted cells in the injured spinal cord remain unclear. Overall, we conclude here that stem cell interventions are a promising avenue for the treatment of spinal cord injury. Canines are a promising model that may help bridge the gap between translational research and human clinical trials.
Full-text · Article · Aug 2014 · Stem Cell Reviews and Reports
[Show abstract][Hide abstract] ABSTRACT: Veterinary adult stem cell therapy is an emerging area of basic and clinical research. Like their human counterparts, veterinary mesenchymal stem cells (MSCs) offer many potential therapeutic benefits. The characterization of canine-derived MSCs, however, is poorly defined compared to human MSCs. Furthermore, little consensus exists regarding the expression of canine MSC cell surface markers. To address this issue, this study investigated characteristics of cultured canine MSCs derived from both adipose tissue and bone marrow. The canine MSCs were obtained from donors of various breeds and ages. A panel of cell surface markers for canine MSCs was selected based on current human and canine literature and the availability of canine-reactive antibodies. Using flow cytometry, canine MSCs were defined to be CD90(+)CD44(+)MHC I(+)CD14(-)CD29(-)CD34(-)MHC II(-). Canine MSCs were further characterized using real-time RT-PCR as CD105(+)CD73(+)CD14(+)CD29(+)MHC II(+)CD45(-) at the mRNA level. Among these markers, canine MSCs differed from canine peripheral blood mononuclear cells (PBMCs) by the absence of CD45 expression at the mRNA level. A novel high-throughput canine-specific PCR array was developed and used to identify changes in the gene expression profiles of canine MSCs. Genes including PTPRC, TNF, β2M, TGFβ1, and PDGFRβ, were identified as unique to canine MSCs as compared to canine PBMCs. Our findings will facilitate characterization of canine MSCs for use in research and clinical trials. Moreover, the high-throughput PCR array is a novel tool for characterizing canine MSCs isolated from different tissues and potentially from different laboratories.
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are somatic, multipotent stromal cells with potent immunomodulatory and regenerative properties. Although MSCs have pattern recognition receptors and are modulated by Toll-like receptors (TLR) ligands, MSC-microbial interactions are poorly defined. The objectives of this study were to determine the effect of bacterial association on MSC function. We hypothesized that gastrointestinal (GI) bacteria associate with MSCs and alter their immunomodulatory properties. The effect of MSC-microbial interactions on MSC morphology, viability, proliferation, migration, and immunomodulatory functions were investigated. MSCs associated with a remarkable array of enteric pathogens and commensal bacteria. MSC interactions with two model organisms, the pathogen Salmonella typhimurium (ST) and the probiotic Lactobacillus acidophilus (LB) were further investigated. While ST readily invaded MSCs, LB adhered to the MSC plasma membrane. Neither microbe induced MSC death, degeneration or diminished proliferation. Microbial association did not upregulate MHC-II, CD80/86 or CD1 expression. MSC-microbial interaction significantly increased transcription of key immunomodulatory genes including COX2, IL6 and IL8 coupled with significantly increased PGE2, IL6 and IL8 secretion. MSC-ST co-incubation resulted in increased MSC expression of CD54, and significant augmentation of MSC inhibition of mitogen-induced T-cell proliferation. T-cell proliferation was partially restored when PGE2 secretion was blocked from ST-primed MSCs. MSC-microbe interactions have a profound effect on MSC function and may be pivotal in a variety of clinical settings where MSCs are being explored as potential therapeutics in the context of microbial communities such as Crohn's disease, chronic non-healing wounds and sepsis.
Full-text · Article · May 2014 · Stem cells and development
[Show abstract][Hide abstract] ABSTRACT: Anaplasma phagocytophilum, which causes granulocytic anaplasmosis in humans and animals, is a tick-transmitted obligate intracellular bacterium that mediates its own uptake into neutrophils and non-phagocytic cells. Invasins of obligate intracellular pathogens are attractive targets for protecting against or curing infection because blocking the internalization step prevents survival of these organisms. The complement of A. phagocytophilum invasins is incompletely defined. Here, we report the significance of a novel A. phagocytophilum invasion protein, AipA. A. phagocytophilum induced aipA expression during transmission feeding of infected ticks on mice. The bacterium upregulated aipA transcription when it transitioned from its non-infectious reticulate cell morphotype to its infectious dense-cored morphotype during infection of HL-60 cells. AipA localized to the bacterial surface and was expressed during in vivo infection. Of the AipA regions predicted to be surface-exposed, only residues 1 to 87 (AipA1-87 ) were found to be essential for host cell invasion. Recombinant AipA1-87 protein bound to and competitively inhibited A. phagocytophilum infection of mammalian cells. Antiserum specific for AipA1-87 , but not other AipA regions, antagonized infection. Additional blocking experiments using peptide-specific antisera narrowed down the AipA invasion domain to residues 9 to 21. An antisera combination targeting AipA1-87 together with two other A. phagocytophilum invasins, OmpA and Asp14, nearly abolished infection of host cells. This study identifies AipA as an A. phagocytophilum surface protein that is critical for infection, demarcates its invasion domain, and establishes a rationale for targeting multiple invasins to protect against granulocytic anaplasmosis.
Full-text · Article · Feb 2014 · Cellular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Abstract Purpose: To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). Methods: HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. Results: Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. Conclusions: HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities.
Full-text · Article · Jan 2014 · Journal of ocular pharmacology and therapeutics: the official journal of the Association for Ocular Pharmacology and Therapeutics
[Show abstract][Hide abstract] ABSTRACT: 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) all 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 E<sub>2</sub> (PGE<sub>2</sub>) 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 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 TNF-α and IFN-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.
No preview · Article · Jan 2014 · Stem cells and development