Jennifer M. Cassano

Identification of equine major histocompatibility complex haplotypes using polymorphic microsatellites

A system for identifying equine major histocompatibility complex (MHC) haplotypes was developed based on five polymorphic microsatellites located within the MHC region on ECA 20. Molecular signatures for 50 microsatellite haplotypes were recognized from typing 353 horses. Of these, 23 microsatellite haplotypes were associated with 12 established equine leucocyte antigen (ELA) haplotypes in Thoroughbreds and Standardbreds. Five ELA serotypes were associated with multiple microsatellite subhaplotypes, expanding the estimates of diversity in the equine MHC. The strong correlations between serological and microsatellite typing demonstrated a linkage to known MHC class I protein polymorphisms and validated this assay as a useful supplement to ELA serotyping, and in some applications, a feasible alternative method for MHC genotyping in horse families and in population studies.

Induced pluripotent stem cells have similar immunogenic and more potent immunomodulatory properties compared with bone marrow-derived stromal cells in vitro

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 MHC-matched 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.

Host genetic influence on papillomavirus-induced tumors in the horse.

The common equine skin tumors known as sarcoids have been causally associated with infection by bovine papillomavirus (BPV). Additionally, there is evidence for host genetic susceptibility to sarcoids. We investigated the genetic basis of susceptibility to sarcoid tumors on a cohort of 82 affected horses and 270 controls genotyped on a genome‐wide platform and two custom panels. A Genome Wide Association Study (GWAS) identified candidate regions on six chromosomes. Bayesian probability analysis of the same dataset verified only the regions on equine chromosomes (ECA) 20 and 22. Fine mapping using custom‐produced SNP arrays for ECA20 and ECA22 regions identified two marker loci with high levels of significance: SNP BIEC2‐530826 (map position 32,787,619) on ECA20 in an intron of the DQA1 gene in the Major Histocompatibility Complex (MHC) class II region (p = 4.6e‐06), and SNP BIEC2‐589604 (map position 25,951,536) on ECA22 in a 200 kb region containing four candidate genes: PROCR, EDEM2, EIF6 and MMP24 (p = 2.14e‐06). The marker loci yielded odds ratios of 5.05 and 4.02 for ECA20 and ECA22, respectively. Associations between genetic MHC class II variants and papillomavirus‐induced tumors have been reported for human papillomavirus and cottontail rabbit papillomavirus infections. This suggests a common mechanism for susceptibility to tumor progression that may involve subversion of the host immune response. This study also identified a genomic region other than MHC that influenced papillomavirus‐induced tumor development in the studied population.

Mesenchymal Stem Cell Therapy: Clinical Progress and Opportunities for Advancement

AbstractMesenchymal 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.

Effect of needle diameter on the viability of equine bone marrow derived mesenchymal stem cells: LANG et al.

Objectives: Mesenchymal stem cells (MSCs) are frequently delivered via needle injection for treatment of musculoskeletal injuries. The purpose of this study was to evaluate the effect of needle diameter on the viability of MSCs. Methods: Equine bone marrow‐derived MSCs from 5 horses were suspended in PBS, and held at room temperature for 7 hours to mimic shipping conditions. Two replicate samples for each needle size (20, 22, 23, or 25‐gauge [ga]) were aspirated into a 3 mL syringe and re‐injected into the holding vial 3 times, to reproduce the resuspension of cells prior to injection in clinical cases. Cells were stained with fluorescein diacetate and propidium iodide to measure viability. Flow cytometry (FC) was performed to compare cell debris and intact cells between groups. Results: MSC viability was higher when cells were passed through a 20‐ga rather than a 25‐ga needle. Cell suspensions passed through a 20‐ga needle contained a larger percentage of intact cells, compared to 25‐ga samples. The percentage of debris present in cell suspensions tended to increase with decreasing needle diameter. Neither horse nor passage had a significant effect on viability. Conclusions: Cell damage is more likely when MSCs are passed through 25‐ga rather than 20‐ga needles. Clinical relevance: Use of needles larger than 25‐ga is recommended to maintain the viability of MSCs injected in horses.

Inflammatory licensed equine MSCs are chondroprotective and exhibit enhanced immunomodulation in an inflammatory environment

BackgroundInflammatory 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.MethodsInflammatory 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.ResultsIFN-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.ConclusionsIn-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.

Antigenicity of mesenchymal stem cells in an inflamed joint environment

OBJECTIVE To determine whether major histocompatability complex (MHC) class II expression in equine mesenchymal stem cells (MSCs) changes with exposure to a proinflammatory environment reflective of an inflamed joint. SAMPLE Cryopreserved bone marrow-derived MSCs from 12 horses and cartilage and synovium samples from 1 horse euthanized for reasons other than lameness. PROCEDURES In part 1 of a 3-part study, the suitability of a quantitative reverse transcriptase PCR (qRT-PCR) assay for measurement of MHC class II expression in MSCs following stimulation with interferon (IFN)-γ was assessed. In part 2, synoviocyte-cartilage cocultures were or were not stimulated with interleukin (IL)-1β (10 ng/mL) to generate conditioned media that did and did not (control) mimic an inflamed joint environment. In part 3, a qRT-PCR assay was used to measure MSC MHC class II expression after 96 hours of incubation with 1 of 6 treatments (control-conditioned medium, IL-1β-conditioned medium, and MSC medium alone [untreated control] or with IL-1β [10 ng/mL], tumor necrosis factor-α [10 ng/mL], or IFN-γ [100 ng/mL]). RESULTS The qRT-PCR assay accurately measured MHC class II expression. Compared with MHC class II expression for MSCs exposed to the untreated control medium, that for MSCs exposed to IL-1β was decreased, whereas that for MSCs exposed to IFN-γ was increased. Neither the control-conditioned nor tumor necrosis factor-α medium altered MHC class II expression. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that MSC exposure to proinflammatory cytokine IL-1β decreased MHC class II expression and antigenicity. Treatment of inflamed joints with allogeneic MSCs might not be contraindicated, but further investigation is warranted.

The immunomodulatory function of equine MSCs is enhanced by priming through an inflammatory microenvironment or TLR3 ligand

Mesenchymal stem cells (MSCs) have the therapeutic potential to treat a variety of inflammatory and degenerative disease processes, however the effects of the tissue environment on MSCs have been overlooked. Our hypothesis was that the immunomodulatory function of MSCs would be impaired by TLR4 stimulation or exposure to inflammatory macrophages, whereas their immunosuppressive properties would be enhanced by TLR3 stimulation. MSCs were exposed to polyinosinic:polycytidylic acid (poly I:C) to stimulate TLR3 receptors or lipopolysaccharide (LPS) to stimulate TLR4 receptors. MSC1 proinflammatory phenotype in human MSCs was associated with increased IL-6 and IL-8 and MSC2 regenerative phenotype was associated with increased CCL2 and CXCL10. MSC immunomodulatory function was assessed by measuring the ability of primed MSCs to suppress mitogen-stimulated T cell proliferation. Peripheral blood monocytes were isolated using CD14 MACs positive selection, differentiated into macrophages, and polarized using interferon-gamma (IFN-γ). Polarization was confirmed by increased gene expression of TNFα, CCL2, and CXCL10. Inflammatory macrophages were co-cultured with MSCs for 6h, and the resultant MSC phenotype was analyzed as described above. Both TLR3 and TLR4 priming and co-culture of MSCs with inflammatory macrophages resulted in increased expression of IL-6, CCL2, and CXCL10 in MSCs. Both TLR3 and TLR4 priming or exposure of MSCs to inflammatory macrophages significantly (p<0.05) enhanced their immunomodulatory function, demonstrated by a decrease in T cell proliferation in the presence of poly I:C primed MSCs (11%), LPS primed MSCs (7%), or MSCs exposed to inflammatory macrophages (12%), compared to unstimulated MSCs. Additionally, MHC class II positive MSCs tended to have a greater magnitude of response to priming compared to MHC class II negative MSCs. These results suggest that MSCs can be activated by a variety of inflammatory stimuli, but the recipient injured tissue bed in chronic injuries may not contain sufficient inflammatory signals to activate MSC immunomodulatory function. Enhancement of MSCs immunomodulatory function through inflammatory priming prior to clinical application might improve the therapeutic effect of MSC treatments.

Equine mesenchymal stromal cells from different tissue sources display comparable immune-related gene expression profiles in response to interferon gamma (IFN)-γ

Mesenchymal stromal cells (MSC) have the therapeutic potential to decrease inflammation due to their immunomodulatory properties. They can be isolated from various tissue sources such as bone marrow, adipose tissue, and blood, but it is unknown how the tissue source of origin affects the responses of MSC to inflammatory stimuli. Here, we conceptually addressed this question by evaluating the immune-related gene expression profiles of equine MSC from different tissue sources in response to interferon gamma (IFN-γ) stimulation, with the goal to determine if there is a preferable MSC source for clinical application in an inflammatory environment. The salient findings from this initial study were that the baseline expression of all immune related genes analyzed, with the exception of prostaglandin-endoperoxide synthase 2 (PTGS2), was variable in MSC depending on tissue source. Following IFN-γ stimulation, however, gene expression profiles became more similar across all tissue sources, suggesting that MSC from different sources will likely respond similarly in an inflammatory environment when used clinically.