Meaghan Stolk

Ischemia-reperfusion injury: beneficial effects of mesenchymal stromal cells.

Purpose of reviewOrgan transplantation and other major surgeries are impacted by ischemia–reperfusion injury (IRI). Mesenchymal stromal cells (MSCs) recently became an attractive alternative therapeutic tool to combat IRI. The present review highlights the effects of MSCs in the preclinical animal models of IRI and clinical trials, and explains their potential modes of action based on the pathophysiological IRI cascade. Recent findingsThe application of MSCs in animal models of IRI show anti-inflammatory and anti-apoptotic effects, particularly for damage to the kidneys, heart and lungs. The mechanism of MSC action remains unclear, but may involve paracrine factors which could include the transfer of microvesicles, RNA or mitochondria. Although few clinical trials have reached completion, adverse effects appear minimal. SummaryMSCs show promise in protecting against IRI-induced damage. They appear to help recovery mainly by affecting the levels of inflammation and apoptosis during the organ repair process. In addition, they may mediate immunomodulatory effects on the innate and adaptive immune processes triggered during reperfusion and reduce fibrosis. Success in preclinical animal models has led to the initiation of ongoing clinical trials.

Immunomodulative Efficacy of Bone Marrow-Derived Mesenchymal Stem Cells Cultured in Human Platelet Lysate

Human mesenchymal stem cells (hMSCs) are considered to be a promising tool for novel cell-based therapies. Clinical applications in solid organ transplantation were hampered by the dependence on animal serum for hMSCs clinical scale expansion until substitution with human platelet lysate (HPL) became a promising alternative. Therefore we focused on a direct comparison of immunomodulatory properties of hMSCs cultured in HPL or fetal calf serum (FCS). Phenotypic characterization, detection of cytokine secretion and effects on alloantigen- and mitogen-induced lymphocyte proliferation as well as degranulation of cytomegalovirus-specific cytotoxic T cells were applied in potency assays. We demonstrated that HPL-cultured MSCs have comparable immunomodulatory capacities to their FCS-cultured counterparts. The observed immunomodulatory properties include a beneficial inhibitory effect on immune cell proliferation and an unaffected viral T cell immunity. Thus, culturing hMSCs in HPL generates an efficient and safe expansion combined with intriguing immunomodulatory properties making these cells an attractive cell therapeutic tool.

In vivo effect of bone marrow-derived mesenchymal stem cells in a rat kidney transplantation model with prolonged cold ischemia

Brain death and prolonged cold ischemia are major contributors to the poorer long‐term outcome of transplants from deceased donor kidney transplants, with an even higher impact if expanded criteria donors (‘marginal organs’) are used. Targeting ischemia‐reperfusion injury‐related intragraft inflammation is an attractive concept to improve the outcome of those grafts. As mesenchymal stem cells (MSCs) express both immunomodulatory and tissue repair properties, we evaluated their therapeutic efficacy in a rat kidney transplant model of prolonged cold ischemia. The in vitro immunomodulatory capacity of bone marrow‐derived rat MSCs was tested in co‐cultures with rat lymph node cells. For in vivo studies, Dark Agouti rat kidneys were cold preserved and transplanted into Lewis rats. Syngeneic Lewis MSCs were administered intravenously. Transplants were harvested on day 3, and inflammation was examined by quantitative RT‐PCR and histology. Similarly to MSCs from other species, rat MSCs in vitro also showed a dose‐dependent immunomodulatory capacity. Most importantly, in vivo administration of MSCs reduced the intragraft gene expression of different pro‐inflammatory cytokines, chemokines, and intercellular adhesion molecule‐1. In addition, fewer antigen‐presenting cells were recruited into the renal allograft. In conclusion, rat MSCs ameliorate inflammation induced by prolonged cold ischemia in kidney transplantation.

Detrimental effects of rat mesenchymal stromal cell pre-treatment in a model of acute kidney rejection

Mesenchymal stromal cells (MSC) have shown immunomodulatory and tissue repair potential including partial tolerance induction by pre-treatment of donor-specific cells in a rat heart transplantation model. Very recently, we could show that autologous MSC attenuated ischemia reperfusion injury in a highly mismatched donor–recipient rat kidney transplant model. Therefore, we investigated donor-specific MSC pre-treatment in this rat kidney transplantation model to study whether graft function could be improved, or if tolerance could be induced. Donor- and recipient-type MSC or phosphate buffered saline (PBS) as a control was injected i.v. 4 days before kidney transplantation. Mycophenolate mofetil immunosuppression (20mg/kg body weight) was applied for 7 days. Kidney grafts and spleens were harvested between days 8 and 10 and analyzed by quantitative RT-PCR and immunohistology. In addition, creatinine levels in the blood were measured and serum was screened for the presence of donor-specific antibodies. Surprisingly, application of both donor- and recipient-specific MSC resulted in enhanced humoral immune responses verified by intragraft B cell infiltration and complement factor C4d deposits. Moreover, signs of inflammation and rejection were generally enhanced in both MSC-treated groups relative to PBS control group. Additionally, pre-treatment with donor-specific MSC significantly enhanced the level of donor-specific antibody formation when compared with PBS- or recipient MSC-treated groups. Pre-treatment with both MSC types resulted in a higher degree of kidney cortex tissue damage and elevated creatinine levels at the time point of rejection. Thus, MSC pre-sensitization in this model impairs the allograft outcome. Our data from this pre-clinical kidney transplantation model indicate that pre-operative MSC administration may not be optimal in kidney transplantation and caution must be exerted before moving forward with clinical studies in order to avoid adverse effects.

Regenerative and immunogenic characteristics of cultured nucleus pulposus cells from human cervical intervertebral discs.

Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD) diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP) cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC) including changes in their immunogenic properties after 3-dimensional (3D)-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic) acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR) showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK) cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS), a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have disadvantageous implications on their potential therapeutic applications because they could be the targets of cytotoxic T-cell activity acting by Fas ligand-induced apoptosis.

Immune attributes of cardiac-derived adherent proliferating (CAP) cells in cardiac therapy

Cardiac‐directed cell therapies show potential to reduce mortality and morbidity in heart disease. However, high functional efficacy should be complimented with low immunogenicity, in particular if allogeneic cell sources are applied. Therefore, we aimed to examine cardiac‐derived adherent proliferating (CAP) cells with respect to their immunogenicity and immune modulatory features in vitro. Human CAP cells were isolated from cardiac biopsies and screened in a CFSE‐based proliferation assay in co‐cultures with phytohaemagglutinin (PHA)‐stimulated human peripheral blood lymphocytes (PBMCs) or mixed lymphocyte cultures (MLCs) to assess their potential to induce immune cell proliferation or activation by flow cytometry. Moreover, levels of pro‐ and anti‐inflammatory cytokines in supernatants of co‐cultures were analysed. The capacity of CAP cells to induce the generation of regulatory T cells (Tregs) was determined by flow cytometric measurement of FoxP3 expression. CAP cells of different donors (n = 5) showed low immunogenicity in co‐cultures with human allogeneic PBMCs. In addition, they induced no change in the normal alloantigen‐driven immune responsiveness in MLCs. However, CAP cells significantly reduced the induction of immune cell proliferation in PBMCs cultures stimulated with the polyclonal trigger PHA. Adding CAP cells into MLCs or PHA‐stimulated cultures resulted in significantly reduced levels of TNFα or IFNγ, respectively, compared to controls without CAP cells. At early time points (day 2), interaction of CAP cells with PBMCs resulted in elevated proportions of FoxP3+CD4+CD25high+ cells. The results indicate that CAP cells have low immunogenicity and could be advantageous in cardiac repair by reducing inflammatory cytokines and inducing regulatory T cells. Copyright

Stromal Cells Act as Guardians for Endothelial Progenitors by Reducing Their Immunogenicity After Co‐Transplantation

Regeneration of injured tissues requires effective therapeutic strategies supporting vasculogenesis. The lack of instantly available autologous cell sources and immunogenicity of allogeneic endothelial (progenitor) cells limits clinical progress. Based on the immunosuppressive potency of mesenchymal stem/progenitor cells (MSCs), we investigated whether crosstalk between endothelial colony‐forming progenitor cells (ECFCs) and MSCs during vasculogenesis could lower allogeneic T cell responses against ECFCs allowing long‐term engraftment in vivo. Immunodeficient mice received subcutaneous grafts containing human ECFCs alone, or pairs of human ECFCs/MSCs from the same umbilical cord (UC) to study vasculogenesis in the presence of human leukocyte antigen (HLA)‐mismatched human peripheral blood mononuclear cells (PBMCs). In vitro, cell surface marker changes due to interferon gamma (IFNγ) stimulation during ECFC/MSC coculture were determined and further effects on allostimulated T cell proliferation and cytotoxic lysis were measured. IFNγ‐induced HLA‐DR expression on ECFCs and MSCs, but both cell types had significantly less HLA‐DR in cocultures. ECFC‐induced T cell proliferation was abolished after MSC coculture as a result of HLA‐DR downregulation and indolamin‐2,3‐dioxygenase activation. Additionally, allospecific CD8+ T cell‐mediated lysis of ECFCs was reduced in cocultures. ECFC/MSC coapplication in immunodeficient mice not only promoted the generation of improved blood vessel architecture after 6 weeks, but also reduced intragraft immune cell infiltration and endothelial HLA‐DR expression following PBMC reconstitution. Crosstalk between UC‐derived ECFCs and MSCs after combined transplantation can lower the risk of ECFC rejection, thus enabling their coapplication for therapeutic vasculogenesis. Stem Cells 2017;35:1233–1245

Crosstalk between immune cells and mesenchymal stromal cells in a 3D bioreactor system.

INTRODUCTION Mesenchymal stromal cells (MSC), known for their high immune modulatory capacity are promising tools for several cell-based therapies. To better mimic the in vivo situation of MSC interactions with immune cells, we applied an artificial lymph node (ALN)-bioreactor culture system combining a miniaturized perfusion bioreactor with a 3D matrix-based cell culture of immune competent cells forming micro-organoids. METHODS Rat lymph node cells and allogeneic bone marrow-derived MSCs were seeded in a 20:1 ratio within the agarose matrix of the ALN-reactor. Lymphocytes were pre-incubated with Concanavalin A (ConA) and then co-cultured with MSC in the matrix with additional ConA in the perfusing medium. Live/dead staining showed survival of the co-cultures during the 8-day ALN-reactor run. Paraffin sections of bioreactor matrices were analyzed by proliferating cell nuclear antigen (PCNA)-specific stai-ning to determine MSC proliferation. Immune modulatory capacity was defined by daily analysis of cytokine secretion profiles (TNFa, IFNy, IL-1a, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-12p40/p70, GM-CSF). RESULTS Cytokine peak secretion at day 2 was significantly inhibited by MSCs for TNFa (96.8 ± 4.8%) and IFNy (88.7 ± 12.0%) in 3D co-cultures. In contrast, other cytokines (IL-1, IL-6, IL-12) were induced. Furthermore, we detected a significantly higher (58.8%) fraction of proliferating MSCs in the presence of immune cells compared to control bioreactors loaded with MSCs only. CONCLUSIONS In the future, this system might be an excellent tool to investigate the mechanisms of MSC-mediated immune modulation during simulated in vivo conditions.

Xeno-immunogenicity of ice-free cryopreserved porcine leaflets

BACKGROUND Undesirable processes of inflammation, calcification, or immune-mediated reactions are limiting factors in long-term survival of heart valves in patients. In this study, we target the modulatory effects of ice-free cryopreservation (IFC) of xenogeneic heart valve leaflet matrices, without decellularization, on the adaptive human immune responses in vitro. METHODS We tested porcine leaflet matrices from fresh untreated, conventionally cryopreserved (CFC), and IFC pulmonary valves by culturing them with human blood mononuclear cells for 5 d in vitro. No other tissue treatment protocols to modify possible immune responses were used. Matrices alone or in addition with a low-dose second stimulus were analyzed for induction of proliferation and cytokine release by flow cytometry-based techniques. Evaluation of the α-Gal epitope expression was performed by immunohistochemistry with fluorochrome-labeled B4 isolectin. RESULTS None of the tested leaflet treatment groups directly triggered the proliferation of immune cells. But when tested in combination with a second trigger by anti-CD3, IFC valves showed significantly reduced proliferation of T cells, especially effector memory T cells, in comparison with fresh or CFC tissue. Moreover, the cytokine levels for interferon-γ (IFNγ), tumor necrosis factor α, and interleukin-10 were reduced for the IFC-treated group being significantly different compared with the CFC group. However, no difference between treatment groups in the expression of the α-Gal antigen was observed. CONCLUSIONS IFC of xenogeneic tissue might be an appropriate treatment method or processing step to prevent responses of the adaptive immune system.

Effects on human heart valve immunogenicity in vitro by high concentration cryoprotectant treatment.

It has been shown previously that cryopreservation, using an ice‐free cryopreservation method with the cryoprotectant formulation VS83, beneficially modulated immune reactions in vivo and in vitro when compared with conventionally frozen tissues. In this study, we assessed the impact of a VS83 post‐treatment of previously conventionally frozen human tissue on responses of human immune cells in vitro. Tissue punches of treated and non‐treated (control) aortic heart valve tissue (leaflets and associated aortic root) were co‐cultured for 7 days with peripheral blood mononuclear cells or enriched CD14+ monocytes. Effects on cellular activation markers, cytokine secretion and immune cell proliferation were analysed by flow cytometry. Flow cytometry studies showed that VS83 treatment of aortic root tissue promoted activation and differentiation of CD14+ monocytes, inducing both up‐regulation of CD16 and down‐regulation of CD14. Significantly enhanced expression levels for the C‐C chemokine receptor (CCR)7 and the human leukocyte antigen (HLA)‐DR on monocytes co‐cultured with VS83‐treated aortic root tissue were measured, while the interleukin (IL)‐6 and monocyte chemoattractant protein (MCP)‐1 release was suppressed. However, the levels of interferon (IFN)γ and tumour necrosis factor (TNF)α remained undetectable, indicating that complete activation into pro‐inflammatory macrophages did not occur. Similar, but non‐significant, changes occurred with VS83‐treated leaflets. Additionally, in co‐cultures with T cells, proliferation and cytokine secretion responses were minimal. In conclusion, post‐treatment of conventionally cryopreserved human heart valve tissue with the VS83 formulation induces changes in the activation and differentiation characteristics of human monocytes, and thereby may influence long‐term performance following implantation. Copyright