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Papers by Lauren Schnabel

Regenerative Medicine, 2020

Regenerative medicine is commonly used in human and equine athletes. Potential therapies include ... more Regenerative medicine is commonly used in human and equine athletes. Potential therapies include culture expanded stem cells, stromal vascular fraction of adipose tissue, platelet-rich plasma, bone marrow concentrate, or autologous conditioned serum. The purpose of this manuscript is to disseminate findings from a workshop on the development of translational regenerative medicine in the equine field. Five themes emerged: stem cell characterization and tenogenic differentiation; interactions between mesenchymal stem cells, other cells and the environment; scaffolds and cell packaging; blood- and bone marrow-based regenerative medicines; clinical use of regenerative therapies. Evidence gained through the use of regenerative medicine applications in the horse should continue to translate to the human patient, bringing novel regenerative therapies to both humans and horses.

Stem Cell Research & Therapy

Background Mesenchymal stem cells (MSCs) secrete paracrine factors and extracellular matrix prote... more Background Mesenchymal 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. Methods RNA-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. Results TGF-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. Bot...

Frontiers in Veterinary Science, 2017

Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal... more 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.

Stem Cell Research & Therapy

Background: Inflammatory licensed mesenchymal stem cells (MSCs) have the ability to promote funct... more Background: Inflammatory licensed mesenchymal stem cells (MSCs) have the ability to promote functional tissue repair. This study specifically sought to understand how the recipient tissue environment reciprocally affects MSC function. Inflammatory polarized macrophages, modeling an injured tissue environment, were exposed to licensed MSCs, and the resultant effects of MSC immunomodulation and functionality of the MSC secretome on chondrocyte homeostasis were studied. Methods: Inflammatory licensed MSCs were generated through priming with either IFN-γ or polyinosinic: polycytidylic acid (poly I:C). Macrophages were polarized to an inflammatory phenotype using IFN-γ. Licensed MSCs were co-cultured with inflammatory macrophages and immunomodulation of MSCs was assessed in a T-cell proliferation assay. MSC gene expression was analyzed for changes in immunogenicity (MHC-I, MHC-II), immunomodulation (IDO, PTGS2, NOS2, TGF-β1), cytokine (IL-6, IL-8), and chemokine (CCL2, CXCL10) expression. Macrophages were assessed for changes in cytokine (IL-6, IL-10, TNF-α, IFN-γ) and chemokine (CCL2, CXCL10) expression. Conditioned medium representing the secretome from IFN-γ or poly I:C-primed MSCs was applied to IL-1β-stimulated chondrocytes, which were analyzed for catabolic (IL-6, TNF-α, CCL2, CXCL10, MMP-13, PTGS2) and matrix synthesis (ACAN, COL2A1) genes. Results: IFN-γ-primed MSCs had a superior ability to suppress T-cell proliferation compared to naïve MSCs, and this ability was maintained following exposure to proinflammatory macrophages. In naïve and licensed MSCs exposed to inflammatory macrophages, MHC-I and MHC-II gene expression was upregulated. The secretome from licensed MSCs was chondroprotective and downregulated inflammatory gene expression in IL-1β-stimulated chondrocytes. Conclusions: In-vitro inflammatory licensing agents enhanced the immunomodulatory ability of MSCs exposed to inflammatory macrophages, and the resultant secretome was biologically active, protecting chondrocytes from catabolic stimulation. Use of licensing agents produced a more consistent immunomodulatory MSC population compared to exposure to inflammatory macrophages. The clinical implications of this study are that in-vitro licensing prior to therapeutic application could result in a more predictable immunomodulatory and reparative response to MSC therapy compared to in-vivo inflammatory licensing by the recipient environment.

Veterinary Radiology & Ultrasound, 2010

We describe the use of computed tomography (CT) in a 6-year-old mare with upper airway obstructio... more We describe the use of computed tomography (CT) in a 6-year-old mare with upper airway obstruction due to an abnormally thick nasal septum. Upon CT imaging, multifocal, expansile cyst-like lesions were detected in the nasal septum. The histopathologic diagnosis was chronic dissecting chondritis. Resection of the abnormal nasal septum resulted in resolution of the clinical signs.

Cartilage, 2013

The aim of this study was to determine if the noninvasive or minimally invasive and nondestructiv... more The aim of this study was to determine if the noninvasive or minimally invasive and nondestructive imaging techniques of quantitative T2-mapping or multiphoton microscopy (MPM) respectively, could detect differences in cartilage collagen orientation similar to polarized light microscopy (PLM). It was hypothesized that MRI, MPM, and PLM would all detect quantitative differences between repair and normal cartilage tissue. Osteochondral defects in the medial femoral condyle were created and repaired in 5 mature goats. Postmortem, MRI with T2-mapping and histology were performed. T2 maps were generated and a mean T2 value was calculated for each region of interest. Histologic slides were assessed using MPM with measurements of autocorrelation ellipticity, and by PLM with application of a validated scoring method. Collagen orientation using each of the 3 modalities (T2-mapping, MPM, and PLM) was measured in the center of the repair tissue and compared to remote, normal cartilage. MRI, MP...

Stem cell research & therapy, Jan 12, 2015

This study tested the hypothesis that Major Histocompatibility Complex (MHC) incompatible equine ... more This study tested the hypothesis that Major Histocompatibility Complex (MHC) incompatible equine mesenchymal stromal cells (MSCs) would induce cytotoxic antibodies to donor MHC antigens in recipient horses after intradermal injection. No studies to date have explored recipient antibody responses to allogeneic donor MSC transplantation in the horse. This information is critical because the horse is a valuable species for assessing the safety and efficacy of MSC treatment prior to human clinical application. Six MHC heterozygote horses were identified as non-ELA-A2 haplotype by microsatellite typing and used as allogeneic MHC-mismatched MSC recipients. MHC homozygote horses of known ELA-A2 haplotype were used as MSC and peripheral blood leukocyte (PBL) donors. One MHC homozygote horse of the ELA-A2 haplotype was the recipient of ELA-A2 donor MSCs as an MHC-matched control. Donor MSCs, which were previously isolated and immunophenotyped, were thawed and culture expanded to achieve betw...

Stem Cell Research & Therapy, 2012

Introduction: The influence of genetic background on the ability to generate induced pluripotent ... more Introduction: The influence of genetic background on the ability to generate induced pluripotent stem cells (iPSCs) has the potential to impact future applications, but has yet to be examined in detail. The purpose of this study was to determine if genetic background affects the efficiency of generating iPSCs during early reprograming as well as the pluripotent stability of the iPSCs during later stages of reprograming.

Stem Cell Research & Therapy, 2014

The horse is a valuable species to assess the effect of allogeneic mesenchymal stromal cells (MSC... more The horse is a valuable species to assess the effect of allogeneic mesenchymal stromal cells (MSCs) in regenerative treatments. No studies to date have examined recipient response to major histocompatibility complex (MHC)-mismatched equine MSCs. The purposes of this study were to immunophenotype MSCs from horses of known MHC haplotype and to compare the immunogenicity of MSCs with differing MHC class II expression. Methods: MSCs and peripheral blood leukocytes (PBLs) were obtained from Thoroughbred horses (n = 10) of known MHC haplotype (ELA-A2, -A3, and -A9 homozygotes). MSCs were cultured through P8; cells from each passage (P2 to P8) were cryopreserved until used. Immunophenotyping of MHC class I and II, CD44, CD29, CD90, LFA-1, and CD45RB was performed by using flow cytometry. Tri-lineage differentiation assays were performed to confirm MSC multipotency. Recombinant equine IFN-γ was used to stimulate MHC class II negative MSCs in culture, after which expression of MHC class II was re-examined. To assess the ability of MHC class II negative or positive MSCs to stimulate an immune response, modified one-way mixed leukocyte reactions (MLRs) were performed by using MHC-matched and mismatched responder PBLs and stimulator PBLs or MSCs. Proliferation of gated CFSE-labeled CD3+ responder T cells was evaluated via CFSE attenuation by using flow cytometry and reported as the number of cells in the proliferating T-cell gate.

Regenerative Medicine, 2014

Aim: To evaluate the in vitro immunogenic and immunomodulatory properties of induced pluripotent ... more Aim: To evaluate the in vitro immunogenic and immunomodulatory properties of induced pluripotent stem cells (iPSCs) compared with bone marrow-derived mesenchymal stromal cells (MSCs). Materials & Methods: Mouse embryonic fibroblasts (MEFs) were isolated from C3HeB/FeJ and C57BL/6J mice, and reprogrammed to generate iPSCs. Mixed leukocyte reactions were performed using MHCmatched and -mismatched responder leukocytes and stimulator leukocytes, iPSCs or MSCs. To assess immunogenic potential, iPSCs and MSCs were used as stimulator cells for responder leukocytes. To assess immunomodulatory properties, IPSCs and MSCs were cultured in the presence of stimulator and responder leukocytes. MEFs were used as a control. Results: iPSCs had similar immunogenic properties but more potent immunomodulatory effects than MSCs. Co-culture of MHC-mismatched leukocytes with MHC-matched iPSCs resulted in significantly less responder T-cell proliferation than observed for MHC-mismatched leukocytes alone and at more responder leukocyte concentrations than with MSCs. In addition, MHC-mismatched iPSCs significantly reduced responder T-cell proliferation when co-cultured with MHC-mismatched leukocytes, while MHC-mismatched MSCs did not. Conclusion: These results provide important information when considering the use of iPSCs in place of MSCs in both regenerative and transplantation medicine.

Current Pathobiology Reports, 2015

ABSTRACT Mesenchymal stem cells (or multipotent stem cells—MSCs) are multipotent cells that were ... more ABSTRACT Mesenchymal stem cells (or multipotent stem cells—MSCs) are multipotent cells that were initially thought to serve as progenitor cells in tissue remodeling, and are now primarily investigated for their immunomodulatory potential in regenerative medicine approaches. MSCs suppress numerous cell types of the immune system primarily through secretion of paracrine mediators. They have been investigated for their ability to enhance allograft survival, and for the treatment of osteoarthritis and cardiovascular disease. A polarization paradigm where different priming stimuli, reflective of an injured tissue, induce either a pro-inflammatory or immunosuppressive MSC phenotype, provides the potential for manipulating MSCs to obtain more predictable clinical effects. There is a tremendous clinical need and numerous clinical trials are underway using allogeneic MSCs, despite evidence that allogeneic MSCs can be immunogenic. Further laboratory and clinical studies will continue to refine and optimize MSC therapy for a wide variety of regenerative medicine applications.

Veterinary Surgery, 2009

Objective-To report surgical treatment of traumatic lateral patellar luxation using trochlear blo... more Objective-To report surgical treatment of traumatic lateral patellar luxation using trochlear block recession in an alpaca. Study Design-Clinical case report. Animals-Five-year-old female alpaca. Methods-Grade IV/IV lateral, left patella luxation and mild femoropatellar joint effusion was identified by palpation and visual assessment, and confirmed by ultrasonography and radiographs. Trochlear block recession combined with lateral retinacular release and medial imbrication to restore patella function. Results-Progressive improvement in weight bearing occurred during hospitalization (6 days) and at 3.5 weeks, no lameness was observed; radiographically, the patella was in normal anatomic alignment. At 15 months, there were no signs of lameness with unrestricted exercise and the alpaca had given birth to another cria. Conclusions-In this alpaca with traumatic origin of the lateral patellar luxation and normal femoro-tibial alignment, a combination of retinacular imbrication, contralateral release, and trochlear block recession were successful for long-term treatment of lateral patellar luxation. Clinical Relevance-Although trochlear block recession is most commonly performed in small animals, this technique may be useful in treatment of traumatic patellar luxations in camelids. r

Veterinary Surgery, 2013

To (1) evaluate the design and use of a global rating scale assessment instrument in veterinary m... more To (1) evaluate the design and use of a global rating scale assessment instrument in veterinary medical education and; (2) examine the effectiveness of 2 surgical techniques courses for improving the surgical skills of veterinary students. Instrument development; observational; survey-based. Students (n = 16) registered for 2 elective surgical techniques courses were enrolled on a volunteer basis. A 5-point global rating scale instrument was designed for the evaluation of 12 basic surgical skills by faculty evaluators and used to obtain student start and end scores during the courses. Upon conclusion of the courses, students completed a survey from which their opinions on their improvement as well as their desire for feedback were obtained. All authors agreed the instrument was easy to use. As groups, 3rd year students, 4th year students, and all students combined had significantly higher total skill scores at the end of the courses compared to the start of the courses. Individually, 10 students (63%) had significant improvement in surgical skills as a result of their participation in the courses: 4 (100%) 3rd year and 6 (50%) 4th year students. Student survey responses revealed a strong desire for feedback as well as support of formal assessment methods. Only weak agreement was found between student opinions on their improvement and the authors' assessment scores. Assessment instruments are useful for (1) student evaluation and (2) for providing students with feedback on their surgical skills.

The Veterinary Journal, 2013

Stem cells are commonly used in equine practice to treat musculoskeletal disorders including tend... more Stem cells are commonly used in equine practice to treat musculoskeletal disorders including tendonitis, osteoarthritis, and more recently laminitis. As the field of regenerative medicine continues to advance, equine practitioners need contemporary information regarding the choice of stem cell type and recommendations regarding clinical implementation of stem cell therapies. Clinicians must also be aware of the limitation in current knowledge regarding stem cells, and the impending regulatory laws that may limit the use of equine stem cells in clinical patients.

Journal of Veterinary Internal Medicine, 2006

A 23-year-old, 467-kg Palomino mare was examined for evaluation of sudden onset severe ataxia and... more A 23-year-old, 467-kg Palomino mare was examined for evaluation of sudden onset severe ataxia and depression. The mare had been found down in a pasture and was unable to rise. She was observed, by her owner, to be normal 24 hours earlier. This mare had resided ...

Journal of Shoulder and Elbow Surgery, 2012

Repair of rotator cuff tears in experimental models has been significantly improved by the use of... more Repair of rotator cuff tears in experimental models has been significantly improved by the use of enhanced biologic approaches, including platelet-rich plasma, bone marrow aspirate, growth factor supplements, and cell-and gene-modified cell therapy. Despite added complexity, cell-based therapies form an important part of enhanced repair, and combinations of carrier vehicles, growth factors, and implanted cells provide the best opportunity for robust repair. Bone marrow-derived mesenchymal stem cells provide a stimulus for repair in flexor tendons, but application in rotator cuff repair has not shown universally positive results. The use of scaffolds such as platelet-rich plasma, fibrin, and synthetic vehicles and the use of gene priming for stem cell differentiation and local anabolic and anti-inflammatory impact have both provided essential components for enhanced tendon and tendon-to-bone repair in rotator cuff disruption. Application of these research techniques in human rotator cuff injury has generally been limited to autologous platelet-rich plasma, bone marrow concentrate, or bone marrow aspirates combined with scaffold materials. Cultured mesenchymal progenitor therapy and gene-enhanced function have not yet reached clinical trials in humans. Research in several animal species indicates that the concept of gene-primed stem cells, particularly embryonic stem cells, combined with effective culture conditions, transduction with long-term integrating vectors carrying anabolic growth factors, and development of cells conditioned by use of RNA interference gene therapy to resist matrix metalloproteinase degradation, may constitute potential advances in rotator cuff repair. This review summarizes cell-and gene-enhanced cell research for tendon repair and provides future directions for rotator cuff repair using biologic composites. Level of evidence: Review Article.

Journal of Orthopaedic Research, 2009

Tendinitis remains a catastrophic injury among athletes. Mesenchymal stem cells (MSCs) have recen... more Tendinitis remains a catastrophic injury among athletes. Mesenchymal stem cells (MSCs) have recently been investigated for use in the treatment of tendinitis. Previous work has demonstrated the value of insulin-like growth factor-I (IGF-I) to stimulate cellular proliferation and tendon fiber deposition in the core lesion of tendinitis. This study examined the effects of MSCs, as well as IGF-I geneenhanced MSCs (AdIGF-MSCs) on tendon healing in vivo. Collagenase-induced bilateral tendinitis lesions were created in equine flexor digitorum superficialis tendons (SDFT). Tendons were treated with 10 Â 10 6 MSCs or 10 Â 10 6 AdIGF-MSCs. Control limbs were injected with 1 mL of phosphate-buffered saline (PBS). Ultrasound examinations were performed at t ¼ 0, 2, 4, 6, and 8 weeks. Horses were euthanized at 8 weeks and SDFTs were mechanically tested to failure and evaluated for biochemical composition and histologic characteristics. Expression of collagen types I and III, IGF-I, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), and aggrecanase-1 (ADAMTS-4) were similar in MSC and control tendons. Both MSC and AdIGF-MSC injection resulted in significantly improved tendon histological scores. These findings indicate a benefit to the use of MSCs and AdIGF-MSCs for the treatment of tendinitis. ß

Journal of Orthopaedic Research, 2007

Platelet rich plasma (PRP) has recently been investigated for use in tissue regeneration studies ... more Platelet rich plasma (PRP) has recently been investigated for use in tissue regeneration studies that seek to utilize the numerous growth factors released from platelet a-granules. This study examined gene expression patterns, DNA, and collagen content of equine flexor digitorum superficialis tendon (SDFT) explants cultured in media consisting of PRP and other blood products. Blood and bone marrow aspirate (BMA) were collected from horses and processed to obtain plasma, PRP, and platelet poor plasma (PPP). IGF-I, TGF-b1, and PDGF-BB were quantified in all blood products using ELISA. Tendons were cultured in explant fashion with blood, plasma, PRP, PPP, or BMA at concentrations of 100%, 50%, or 10% in serum-free DMEM with amino acids. Quantitative RT-PCR for expression of collagen type I (COL1A1), collagen type III (COL3A1), cartilage oligomeric matrix protein (COMP), decorin, matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-13 (MMP-13) was performed as were DNA and total soluble collagen assays. TGF-b1 and PDGF-BB concentrations were higher in PRP compared to all other blood products tested. Tendons cultured in 100% PRP showed enhanced gene expression of the matrix molecules COL1A1, COL3A1, and COMP with no concomitant increase in the catabolic molecules MMP-3 and MMP-13. These findings support in vivo investigation of PRP as an autogenous, patient-side treatment for tendonitis. ß

The Journal of Bone and Joint Surgery (American), 2010

The purpose of this study was to compare the outcomes of treatment with bone marrow aspirate conc... more The purpose of this study was to compare the outcomes of treatment with bone marrow aspirate concentrate, a simple, one-step, autogenous, and arthroscopically applicable method, with the outcomes of microfracture with regard to the repair of full-thickness cartilage defects in an equine model. Extensive (15-mm-diameter) full-thickness cartilage defects were created on the lateral trochlear ridge of the femur in twelve horses. Bone marrow was aspirated from the sternum and centrifuged to generate the bone marrow concentrate. The defects were treated with bone marrow concentrate and microfracture or with microfracture alone. Second-look arthroscopy was performed at three months, and the horses were killed at eight months. Repair was assessed with use of macroscopic and histological scoring systems as well as with quantitative magnetic resonance imaging. No adverse reactions due to the microfracture or the bone marrow concentrate were observed. At eight months, macroscopic scores (mean and standard error of the mean, 9.4 + or - 1.2 compared with 4.4 + or - 1.2; p = 0.009) and histological scores (11.1 + or - 1.6 compared with 6.4 + or - 1.2; p = 0.02) indicated improvement in the repair tissue in the bone marrow concentrate group compared with that in the microfracture group. All scoring systems and magnetic resonance imaging data indicated that delivery of the bone marrow concentrate resulted in increased fill of the defects and improved integration of repair tissue into surrounding normal cartilage. In addition, there was greater type-II collagen content and improved orientation of the collagen as well as significantly more glycosaminoglycan in the bone marrow concentrate-treated defects than in the microfracture-treated defects. Delivery of bone marrow concentrate can result in healing of acute full-thickness cartilage defects that is superior to that after microfracture alone in an equine model. Delivery of bone marrow concentrate to cartilage defects has the clinical potential to improve cartilage healing, providing a simple, cost-effective, arthroscopically applicable, and clinically effective approach for cartilage repair.

Equine Veterinary Journal, 2014

To determine whether low-dose, low-frequency doxycycline administration is capable of achieving c... more To determine whether low-dose, low-frequency doxycycline administration is capable of achieving chondroprotective concentrations within synovial fluid (SF) while remaining below minimum inhibitory concentration 90 (MIC90) of most equine pathogens and would be an option in the management of osteoarthritis. Objectives: To determine whether low-dose, low-frequency oral administration of doxycycline can attain in vivo SF concentrations capable of chondroprotective effects through reduction of matrix metalloproteinase (MMP)-13 activity, while remaining below MIC90 of most equine pathogens. Study design: Descriptive pharmacokinetic study with crossover design. Methods: Two groups of 6 horses received oral doxycycline. Plasma and SF doxycycline concentrations were measured using high performance liquid chromatography. Group 1 received 5 mg/kg bwt q. 24 h with 21 blood and 8 SF samples collected over 120 h; Group 2 received 5 mg/kg bwt q. 48 h with 27 blood and 11 SF samples collected over 192 h. Cultured synoviocytes were treated with interleukin-1α (1 ng/ml) for 24 h to stimulate MMP synthesis, and then SF was added to the culture medium for 96 h. MMP-13 protein and mRNA were measured in synoviocyte culture medium and synoviocytes, respectively. Results: Mean doxycycline concentration ≥0.043 μg/ml (previously demonstrated to inhibit MMP-13) was achieved in plasma by t = 0.25 h and SF by t = 48 h in Group 1, and in plasma by t = 0.17 h and SF by t = 1 h in Group 2. Synoviocyte culture medium containing doxycycline from Groups 1 and 2 had significantly decreased active MMP-13 protein concentration, and synoviocytes cultured in this medium had significantly decreased MMP-13 gene expression compared to controls. Plasma doxycycline concentration in both groups and SF doxycycline concentration in Group 2 demonstrated a cumulative effect. Conclusions: Low-dose orally administered doxycycline achieves SF concentrations in vivo capable of diminishing MMP-13 expression. This study supports the use of doxycycline as a disease modifying osteoarthritic drug.

Regenerative Medicine, 2020

Regenerative medicine is commonly used in human and equine athletes. Potential therapies include ... more Regenerative medicine is commonly used in human and equine athletes. Potential therapies include culture expanded stem cells, stromal vascular fraction of adipose tissue, platelet-rich plasma, bone marrow concentrate, or autologous conditioned serum. The purpose of this manuscript is to disseminate findings from a workshop on the development of translational regenerative medicine in the equine field. Five themes emerged: stem cell characterization and tenogenic differentiation; interactions between mesenchymal stem cells, other cells and the environment; scaffolds and cell packaging; blood- and bone marrow-based regenerative medicines; clinical use of regenerative therapies. Evidence gained through the use of regenerative medicine applications in the horse should continue to translate to the human patient, bringing novel regenerative therapies to both humans and horses.

Stem Cell Research & Therapy

Background Mesenchymal stem cells (MSCs) secrete paracrine factors and extracellular matrix prote... more Background Mesenchymal 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. Methods RNA-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. Results TGF-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. Bot...

Frontiers in Veterinary Science, 2017

Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal... more 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.

Stem Cell Research & Therapy

Background: Inflammatory licensed mesenchymal stem cells (MSCs) have the ability to promote funct... more Background: Inflammatory licensed mesenchymal stem cells (MSCs) have the ability to promote functional tissue repair. This study specifically sought to understand how the recipient tissue environment reciprocally affects MSC function. Inflammatory polarized macrophages, modeling an injured tissue environment, were exposed to licensed MSCs, and the resultant effects of MSC immunomodulation and functionality of the MSC secretome on chondrocyte homeostasis were studied. Methods: Inflammatory licensed MSCs were generated through priming with either IFN-γ or polyinosinic: polycytidylic acid (poly I:C). Macrophages were polarized to an inflammatory phenotype using IFN-γ. Licensed MSCs were co-cultured with inflammatory macrophages and immunomodulation of MSCs was assessed in a T-cell proliferation assay. MSC gene expression was analyzed for changes in immunogenicity (MHC-I, MHC-II), immunomodulation (IDO, PTGS2, NOS2, TGF-β1), cytokine (IL-6, IL-8), and chemokine (CCL2, CXCL10) expression. Macrophages were assessed for changes in cytokine (IL-6, IL-10, TNF-α, IFN-γ) and chemokine (CCL2, CXCL10) expression. Conditioned medium representing the secretome from IFN-γ or poly I:C-primed MSCs was applied to IL-1β-stimulated chondrocytes, which were analyzed for catabolic (IL-6, TNF-α, CCL2, CXCL10, MMP-13, PTGS2) and matrix synthesis (ACAN, COL2A1) genes. Results: IFN-γ-primed MSCs had a superior ability to suppress T-cell proliferation compared to naïve MSCs, and this ability was maintained following exposure to proinflammatory macrophages. In naïve and licensed MSCs exposed to inflammatory macrophages, MHC-I and MHC-II gene expression was upregulated. The secretome from licensed MSCs was chondroprotective and downregulated inflammatory gene expression in IL-1β-stimulated chondrocytes. Conclusions: In-vitro inflammatory licensing agents enhanced the immunomodulatory ability of MSCs exposed to inflammatory macrophages, and the resultant secretome was biologically active, protecting chondrocytes from catabolic stimulation. Use of licensing agents produced a more consistent immunomodulatory MSC population compared to exposure to inflammatory macrophages. The clinical implications of this study are that in-vitro licensing prior to therapeutic application could result in a more predictable immunomodulatory and reparative response to MSC therapy compared to in-vivo inflammatory licensing by the recipient environment.

Veterinary Radiology & Ultrasound, 2010

We describe the use of computed tomography (CT) in a 6-year-old mare with upper airway obstructio... more We describe the use of computed tomography (CT) in a 6-year-old mare with upper airway obstruction due to an abnormally thick nasal septum. Upon CT imaging, multifocal, expansile cyst-like lesions were detected in the nasal septum. The histopathologic diagnosis was chronic dissecting chondritis. Resection of the abnormal nasal septum resulted in resolution of the clinical signs.

Cartilage, 2013

The aim of this study was to determine if the noninvasive or minimally invasive and nondestructiv... more The aim of this study was to determine if the noninvasive or minimally invasive and nondestructive imaging techniques of quantitative T2-mapping or multiphoton microscopy (MPM) respectively, could detect differences in cartilage collagen orientation similar to polarized light microscopy (PLM). It was hypothesized that MRI, MPM, and PLM would all detect quantitative differences between repair and normal cartilage tissue. Osteochondral defects in the medial femoral condyle were created and repaired in 5 mature goats. Postmortem, MRI with T2-mapping and histology were performed. T2 maps were generated and a mean T2 value was calculated for each region of interest. Histologic slides were assessed using MPM with measurements of autocorrelation ellipticity, and by PLM with application of a validated scoring method. Collagen orientation using each of the 3 modalities (T2-mapping, MPM, and PLM) was measured in the center of the repair tissue and compared to remote, normal cartilage. MRI, MP...

Stem cell research & therapy, Jan 12, 2015

This study tested the hypothesis that Major Histocompatibility Complex (MHC) incompatible equine ... more This study tested the hypothesis that Major Histocompatibility Complex (MHC) incompatible equine mesenchymal stromal cells (MSCs) would induce cytotoxic antibodies to donor MHC antigens in recipient horses after intradermal injection. No studies to date have explored recipient antibody responses to allogeneic donor MSC transplantation in the horse. This information is critical because the horse is a valuable species for assessing the safety and efficacy of MSC treatment prior to human clinical application. Six MHC heterozygote horses were identified as non-ELA-A2 haplotype by microsatellite typing and used as allogeneic MHC-mismatched MSC recipients. MHC homozygote horses of known ELA-A2 haplotype were used as MSC and peripheral blood leukocyte (PBL) donors. One MHC homozygote horse of the ELA-A2 haplotype was the recipient of ELA-A2 donor MSCs as an MHC-matched control. Donor MSCs, which were previously isolated and immunophenotyped, were thawed and culture expanded to achieve betw...

Stem Cell Research & Therapy, 2012

Introduction: The influence of genetic background on the ability to generate induced pluripotent ... more Introduction: The influence of genetic background on the ability to generate induced pluripotent stem cells (iPSCs) has the potential to impact future applications, but has yet to be examined in detail. The purpose of this study was to determine if genetic background affects the efficiency of generating iPSCs during early reprograming as well as the pluripotent stability of the iPSCs during later stages of reprograming.

Stem Cell Research & Therapy, 2014

The horse is a valuable species to assess the effect of allogeneic mesenchymal stromal cells (MSC... more The horse is a valuable species to assess the effect of allogeneic mesenchymal stromal cells (MSCs) in regenerative treatments. No studies to date have examined recipient response to major histocompatibility complex (MHC)-mismatched equine MSCs. The purposes of this study were to immunophenotype MSCs from horses of known MHC haplotype and to compare the immunogenicity of MSCs with differing MHC class II expression. Methods: MSCs and peripheral blood leukocytes (PBLs) were obtained from Thoroughbred horses (n = 10) of known MHC haplotype (ELA-A2, -A3, and -A9 homozygotes). MSCs were cultured through P8; cells from each passage (P2 to P8) were cryopreserved until used. Immunophenotyping of MHC class I and II, CD44, CD29, CD90, LFA-1, and CD45RB was performed by using flow cytometry. Tri-lineage differentiation assays were performed to confirm MSC multipotency. Recombinant equine IFN-γ was used to stimulate MHC class II negative MSCs in culture, after which expression of MHC class II was re-examined. To assess the ability of MHC class II negative or positive MSCs to stimulate an immune response, modified one-way mixed leukocyte reactions (MLRs) were performed by using MHC-matched and mismatched responder PBLs and stimulator PBLs or MSCs. Proliferation of gated CFSE-labeled CD3+ responder T cells was evaluated via CFSE attenuation by using flow cytometry and reported as the number of cells in the proliferating T-cell gate.

Regenerative Medicine, 2014

Aim: To evaluate the in vitro immunogenic and immunomodulatory properties of induced pluripotent ... more Aim: To evaluate the in vitro immunogenic and immunomodulatory properties of induced pluripotent stem cells (iPSCs) compared with bone marrow-derived mesenchymal stromal cells (MSCs). Materials & Methods: Mouse embryonic fibroblasts (MEFs) were isolated from C3HeB/FeJ and C57BL/6J mice, and reprogrammed to generate iPSCs. Mixed leukocyte reactions were performed using MHCmatched and -mismatched responder leukocytes and stimulator leukocytes, iPSCs or MSCs. To assess immunogenic potential, iPSCs and MSCs were used as stimulator cells for responder leukocytes. To assess immunomodulatory properties, IPSCs and MSCs were cultured in the presence of stimulator and responder leukocytes. MEFs were used as a control. Results: iPSCs had similar immunogenic properties but more potent immunomodulatory effects than MSCs. Co-culture of MHC-mismatched leukocytes with MHC-matched iPSCs resulted in significantly less responder T-cell proliferation than observed for MHC-mismatched leukocytes alone and at more responder leukocyte concentrations than with MSCs. In addition, MHC-mismatched iPSCs significantly reduced responder T-cell proliferation when co-cultured with MHC-mismatched leukocytes, while MHC-mismatched MSCs did not. Conclusion: These results provide important information when considering the use of iPSCs in place of MSCs in both regenerative and transplantation medicine.

Current Pathobiology Reports, 2015

ABSTRACT Mesenchymal stem cells (or multipotent stem cells—MSCs) are multipotent cells that were ... more ABSTRACT Mesenchymal stem cells (or multipotent stem cells—MSCs) are multipotent cells that were initially thought to serve as progenitor cells in tissue remodeling, and are now primarily investigated for their immunomodulatory potential in regenerative medicine approaches. MSCs suppress numerous cell types of the immune system primarily through secretion of paracrine mediators. They have been investigated for their ability to enhance allograft survival, and for the treatment of osteoarthritis and cardiovascular disease. A polarization paradigm where different priming stimuli, reflective of an injured tissue, induce either a pro-inflammatory or immunosuppressive MSC phenotype, provides the potential for manipulating MSCs to obtain more predictable clinical effects. There is a tremendous clinical need and numerous clinical trials are underway using allogeneic MSCs, despite evidence that allogeneic MSCs can be immunogenic. Further laboratory and clinical studies will continue to refine and optimize MSC therapy for a wide variety of regenerative medicine applications.

Veterinary Surgery, 2009

Objective-To report surgical treatment of traumatic lateral patellar luxation using trochlear blo... more Objective-To report surgical treatment of traumatic lateral patellar luxation using trochlear block recession in an alpaca. Study Design-Clinical case report. Animals-Five-year-old female alpaca. Methods-Grade IV/IV lateral, left patella luxation and mild femoropatellar joint effusion was identified by palpation and visual assessment, and confirmed by ultrasonography and radiographs. Trochlear block recession combined with lateral retinacular release and medial imbrication to restore patella function. Results-Progressive improvement in weight bearing occurred during hospitalization (6 days) and at 3.5 weeks, no lameness was observed; radiographically, the patella was in normal anatomic alignment. At 15 months, there were no signs of lameness with unrestricted exercise and the alpaca had given birth to another cria. Conclusions-In this alpaca with traumatic origin of the lateral patellar luxation and normal femoro-tibial alignment, a combination of retinacular imbrication, contralateral release, and trochlear block recession were successful for long-term treatment of lateral patellar luxation. Clinical Relevance-Although trochlear block recession is most commonly performed in small animals, this technique may be useful in treatment of traumatic patellar luxations in camelids. r

Veterinary Surgery, 2013

To (1) evaluate the design and use of a global rating scale assessment instrument in veterinary m... more To (1) evaluate the design and use of a global rating scale assessment instrument in veterinary medical education and; (2) examine the effectiveness of 2 surgical techniques courses for improving the surgical skills of veterinary students. Instrument development; observational; survey-based. Students (n = 16) registered for 2 elective surgical techniques courses were enrolled on a volunteer basis. A 5-point global rating scale instrument was designed for the evaluation of 12 basic surgical skills by faculty evaluators and used to obtain student start and end scores during the courses. Upon conclusion of the courses, students completed a survey from which their opinions on their improvement as well as their desire for feedback were obtained. All authors agreed the instrument was easy to use. As groups, 3rd year students, 4th year students, and all students combined had significantly higher total skill scores at the end of the courses compared to the start of the courses. Individually, 10 students (63%) had significant improvement in surgical skills as a result of their participation in the courses: 4 (100%) 3rd year and 6 (50%) 4th year students. Student survey responses revealed a strong desire for feedback as well as support of formal assessment methods. Only weak agreement was found between student opinions on their improvement and the authors' assessment scores. Assessment instruments are useful for (1) student evaluation and (2) for providing students with feedback on their surgical skills.

The Veterinary Journal, 2013

Stem cells are commonly used in equine practice to treat musculoskeletal disorders including tend... more Stem cells are commonly used in equine practice to treat musculoskeletal disorders including tendonitis, osteoarthritis, and more recently laminitis. As the field of regenerative medicine continues to advance, equine practitioners need contemporary information regarding the choice of stem cell type and recommendations regarding clinical implementation of stem cell therapies. Clinicians must also be aware of the limitation in current knowledge regarding stem cells, and the impending regulatory laws that may limit the use of equine stem cells in clinical patients.

Journal of Veterinary Internal Medicine, 2006

A 23-year-old, 467-kg Palomino mare was examined for evaluation of sudden onset severe ataxia and... more A 23-year-old, 467-kg Palomino mare was examined for evaluation of sudden onset severe ataxia and depression. The mare had been found down in a pasture and was unable to rise. She was observed, by her owner, to be normal 24 hours earlier. This mare had resided ...

Journal of Shoulder and Elbow Surgery, 2012

Repair of rotator cuff tears in experimental models has been significantly improved by the use of... more Repair of rotator cuff tears in experimental models has been significantly improved by the use of enhanced biologic approaches, including platelet-rich plasma, bone marrow aspirate, growth factor supplements, and cell-and gene-modified cell therapy. Despite added complexity, cell-based therapies form an important part of enhanced repair, and combinations of carrier vehicles, growth factors, and implanted cells provide the best opportunity for robust repair. Bone marrow-derived mesenchymal stem cells provide a stimulus for repair in flexor tendons, but application in rotator cuff repair has not shown universally positive results. The use of scaffolds such as platelet-rich plasma, fibrin, and synthetic vehicles and the use of gene priming for stem cell differentiation and local anabolic and anti-inflammatory impact have both provided essential components for enhanced tendon and tendon-to-bone repair in rotator cuff disruption. Application of these research techniques in human rotator cuff injury has generally been limited to autologous platelet-rich plasma, bone marrow concentrate, or bone marrow aspirates combined with scaffold materials. Cultured mesenchymal progenitor therapy and gene-enhanced function have not yet reached clinical trials in humans. Research in several animal species indicates that the concept of gene-primed stem cells, particularly embryonic stem cells, combined with effective culture conditions, transduction with long-term integrating vectors carrying anabolic growth factors, and development of cells conditioned by use of RNA interference gene therapy to resist matrix metalloproteinase degradation, may constitute potential advances in rotator cuff repair. This review summarizes cell-and gene-enhanced cell research for tendon repair and provides future directions for rotator cuff repair using biologic composites. Level of evidence: Review Article.

Journal of Orthopaedic Research, 2009

Tendinitis remains a catastrophic injury among athletes. Mesenchymal stem cells (MSCs) have recen... more Tendinitis remains a catastrophic injury among athletes. Mesenchymal stem cells (MSCs) have recently been investigated for use in the treatment of tendinitis. Previous work has demonstrated the value of insulin-like growth factor-I (IGF-I) to stimulate cellular proliferation and tendon fiber deposition in the core lesion of tendinitis. This study examined the effects of MSCs, as well as IGF-I geneenhanced MSCs (AdIGF-MSCs) on tendon healing in vivo. Collagenase-induced bilateral tendinitis lesions were created in equine flexor digitorum superficialis tendons (SDFT). Tendons were treated with 10 Â 10 6 MSCs or 10 Â 10 6 AdIGF-MSCs. Control limbs were injected with 1 mL of phosphate-buffered saline (PBS). Ultrasound examinations were performed at t ¼ 0, 2, 4, 6, and 8 weeks. Horses were euthanized at 8 weeks and SDFTs were mechanically tested to failure and evaluated for biochemical composition and histologic characteristics. Expression of collagen types I and III, IGF-I, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), and aggrecanase-1 (ADAMTS-4) were similar in MSC and control tendons. Both MSC and AdIGF-MSC injection resulted in significantly improved tendon histological scores. These findings indicate a benefit to the use of MSCs and AdIGF-MSCs for the treatment of tendinitis. ß

Journal of Orthopaedic Research, 2007

Platelet rich plasma (PRP) has recently been investigated for use in tissue regeneration studies ... more Platelet rich plasma (PRP) has recently been investigated for use in tissue regeneration studies that seek to utilize the numerous growth factors released from platelet a-granules. This study examined gene expression patterns, DNA, and collagen content of equine flexor digitorum superficialis tendon (SDFT) explants cultured in media consisting of PRP and other blood products. Blood and bone marrow aspirate (BMA) were collected from horses and processed to obtain plasma, PRP, and platelet poor plasma (PPP). IGF-I, TGF-b1, and PDGF-BB were quantified in all blood products using ELISA. Tendons were cultured in explant fashion with blood, plasma, PRP, PPP, or BMA at concentrations of 100%, 50%, or 10% in serum-free DMEM with amino acids. Quantitative RT-PCR for expression of collagen type I (COL1A1), collagen type III (COL3A1), cartilage oligomeric matrix protein (COMP), decorin, matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-13 (MMP-13) was performed as were DNA and total soluble collagen assays. TGF-b1 and PDGF-BB concentrations were higher in PRP compared to all other blood products tested. Tendons cultured in 100% PRP showed enhanced gene expression of the matrix molecules COL1A1, COL3A1, and COMP with no concomitant increase in the catabolic molecules MMP-3 and MMP-13. These findings support in vivo investigation of PRP as an autogenous, patient-side treatment for tendonitis. ß

The Journal of Bone and Joint Surgery (American), 2010

The purpose of this study was to compare the outcomes of treatment with bone marrow aspirate conc... more The purpose of this study was to compare the outcomes of treatment with bone marrow aspirate concentrate, a simple, one-step, autogenous, and arthroscopically applicable method, with the outcomes of microfracture with regard to the repair of full-thickness cartilage defects in an equine model. Extensive (15-mm-diameter) full-thickness cartilage defects were created on the lateral trochlear ridge of the femur in twelve horses. Bone marrow was aspirated from the sternum and centrifuged to generate the bone marrow concentrate. The defects were treated with bone marrow concentrate and microfracture or with microfracture alone. Second-look arthroscopy was performed at three months, and the horses were killed at eight months. Repair was assessed with use of macroscopic and histological scoring systems as well as with quantitative magnetic resonance imaging. No adverse reactions due to the microfracture or the bone marrow concentrate were observed. At eight months, macroscopic scores (mean and standard error of the mean, 9.4 + or - 1.2 compared with 4.4 + or - 1.2; p = 0.009) and histological scores (11.1 + or - 1.6 compared with 6.4 + or - 1.2; p = 0.02) indicated improvement in the repair tissue in the bone marrow concentrate group compared with that in the microfracture group. All scoring systems and magnetic resonance imaging data indicated that delivery of the bone marrow concentrate resulted in increased fill of the defects and improved integration of repair tissue into surrounding normal cartilage. In addition, there was greater type-II collagen content and improved orientation of the collagen as well as significantly more glycosaminoglycan in the bone marrow concentrate-treated defects than in the microfracture-treated defects. Delivery of bone marrow concentrate can result in healing of acute full-thickness cartilage defects that is superior to that after microfracture alone in an equine model. Delivery of bone marrow concentrate to cartilage defects has the clinical potential to improve cartilage healing, providing a simple, cost-effective, arthroscopically applicable, and clinically effective approach for cartilage repair.

Equine Veterinary Journal, 2014

To determine whether low-dose, low-frequency doxycycline administration is capable of achieving c... more To determine whether low-dose, low-frequency doxycycline administration is capable of achieving chondroprotective concentrations within synovial fluid (SF) while remaining below minimum inhibitory concentration 90 (MIC90) of most equine pathogens and would be an option in the management of osteoarthritis. Objectives: To determine whether low-dose, low-frequency oral administration of doxycycline can attain in vivo SF concentrations capable of chondroprotective effects through reduction of matrix metalloproteinase (MMP)-13 activity, while remaining below MIC90 of most equine pathogens. Study design: Descriptive pharmacokinetic study with crossover design. Methods: Two groups of 6 horses received oral doxycycline. Plasma and SF doxycycline concentrations were measured using high performance liquid chromatography. Group 1 received 5 mg/kg bwt q. 24 h with 21 blood and 8 SF samples collected over 120 h; Group 2 received 5 mg/kg bwt q. 48 h with 27 blood and 11 SF samples collected over 192 h. Cultured synoviocytes were treated with interleukin-1α (1 ng/ml) for 24 h to stimulate MMP synthesis, and then SF was added to the culture medium for 96 h. MMP-13 protein and mRNA were measured in synoviocyte culture medium and synoviocytes, respectively. Results: Mean doxycycline concentration ≥0.043 μg/ml (previously demonstrated to inhibit MMP-13) was achieved in plasma by t = 0.25 h and SF by t = 48 h in Group 1, and in plasma by t = 0.17 h and SF by t = 1 h in Group 2. Synoviocyte culture medium containing doxycycline from Groups 1 and 2 had significantly decreased active MMP-13 protein concentration, and synoviocytes cultured in this medium had significantly decreased MMP-13 gene expression compared to controls. Plasma doxycycline concentration in both groups and SF doxycycline concentration in Group 2 demonstrated a cumulative effect. Conclusions: Low-dose orally administered doxycycline achieves SF concentrations in vivo capable of diminishing MMP-13 expression. This study supports the use of doxycycline as a disease modifying osteoarthritic drug.