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.

Effects of the new immunosuppressive agent AEB071 on human immune cells.

UNLABELLED: BACKGROUND. The novel immunosuppressive agent AEB071 is currently being evaluated for its capability to prevent rejection after kidney transplantation as a potential adjunct to calcineurin inhibitor-based regimen. AEB071 is a selective protein kinase C inhibitor and has been shown to be well tolerated in humans. We here present extensive in vitro studies that contribute to the understanding of AEB071 effects on human lymphocyte, natural killer (NK) cell and dendritic cell (DC) action. METHODS: The impact of AEB071 on several T-cell activation and costimulatory markers was assessed. Furthermore, assays were performed to study the effect on T-cell proliferation and intracellular cytokine production. Additionally, the effect of AEB071 on DC maturation and their capacity to stimulate allogeneic T-cells was examined. Also, an evaluation of AEB071 effects on the lytic activity of human NK cells was performed. RESULTS: We were able to show that T-cell proliferation and cytokine production rates are significantly reduced after AEB071 administration. Also, mitogen-induced T-cell activation characterized by expression levels of surface markers could be significantly inhibited. In contrast, the T-cell stimulatory capacity of AEB071-treated mature monocyte-derived DC (Mo-DC) is not reduced, and AEB071 administration does not prevent lipopolysaccharide (LPS)-induced Mo-DC maturation. It could be demonstrated that AEB071 significantly inhibited the cytotoxic activity of NK cells. CONCLUSIONS: The promising immunosuppressive agent AEB071 has a strong impact on T-cell activation, proliferation and cytokine production as well as NK cell activity, but not DC maturation in vitro, and therefore, seems to function T-cell and NK cell specific via protein kinase C (PKC) inhibition.

An anti-major histocompatibility complex class I intrabody protects endothelial cells from an attack by immune mediators.

OBJECTIVE: In vitro endothelialization has significantly improved the overall outcome of artificial prostheses in cardiovascular bypass surgery. A drawback of this tissue-engineering method remains the limited availability of suitable autologous endothelial cells (EC), especially in aged patients. Allogeneic EC with high proliferative capacity represent a potentially valuable alternative to a patient-specific vascular transplant. However, such cells carry the risk of being rejected due to Major Histocompatibility Complex (MHC) mismatches. METHODS: We investigated the effects of a very potent, intracellularly expressed antibody directed against MHC class I molecules, referred to as alpha-rat MHC I single chain variable fragment (sFv) intrabody. The intrabody was stably expressed in rat aortic EC (RAEC) following lentiviral vector-mediated gene transfer. The functional consequence of the MHC I down-regulation was tested in an allogeneic setting in two different in vitro assays. RESULTS: Stable expression of the alpha-rat MHC I sFv intrabody resulted in a highly efficient depletion of surface MHC I. Thereby those RAEC which displayed low MHC I levels over extended periods of time were protected against killing by allo-specific, cytotoxic T cells (CTL) and by allo-antibody/complement-mediated lysis. CONCLUSIONS: These results demonstrate that intrabody-mediated down-regulation of MHC I reduces the immunogenicity of RAEC which may provide a suitable alternative supply for the lining of vascular prostheses.

Evaluation of immunogenicity of rat ES-cell derived endothelial cells.

Evaluation of the immunogenicity of embryonic stem cell derived differentiated cells is important for their potential application in cell replacement therapies and transplantations. Low immunogenicity or even an immune privileged status would enable their general use in allogeneic settings and therefore supply an unrestricted source. Based on conflicting data in terms of immunogenicity published for mouse and human ES-derived cells, the rat model was used to complement the knowledge in this specific area by a set of in vitro test systems using endothelial ES cell derivatives.This chapter describes the strategies and methods used to analyze immunogenicity of rat ES cell derived endothelial cells (RESC) in comparison to adult mature rat endothelial cells (EC). In a first characterization step, the endothelial nature of rat ES cell derived endothelial cells was proved by labelling with von Willebrand factor (vWF) as well as testing tube formation capacity on an extracellular matrix. The RESC can be characterized by their constitutive or cytokine-induced expression level of the Major Histocompatibility Complex (MHC) antigens class I and class II by Fluorescence Activated Cell Sorter (FACS) technology. Moreover, regulation of transcription factors involved in the IFNγ signaling pathway could be evaluated by detecting either the phosphorylation status by specific intracellular antibody staining followed by flow cytometric measurement or by analyzing the mRNA expression level by quantitative RT-PCR. By stimulating the RESC with IFNγ and coculturing with Carboxy-fluorescein diacetate succinimidyl ester (CFDA-SE)-labelled CD4(+) rat T cells, the ability of RESC to induce proliferation was analyzed by FACS technology. Allo-reactive cytotoxic T cells were generated in a mixed lymphocyte culture (MLC) with lymph node cells from two MHC-disparate rat strains and used to determine the susceptibility of RESC to lytic processes. Therefore, RESC were labelled with calcein and the release of this fluorochrome after coculture was measured. To analyze the response to humoral attacks, RESC were incubated with allo-antibody containing sera and rabbit complement and then cell damage was assessed by 7-actinomycin D (7-AAD) incorporation into the DNA using FACS analysis.

Immune effects of mesenchymal stromal cells in experimental stroke.

Preclinical trials confirmed the potential of mesenchymal stromal cells (MSCs) to improve functional recovery after experimental stroke. Beneficial effects of MSCs are often attributed to their immunosuppressive/immunomodulatory functions. Surprisingly, the influence of MSCs on the immune system after stroke is poorly understood, but requires special consideration because cerebral ischemia is associated with stroke-induced immunodepression that predisposes to bacterial infections with increased mortality. In this study, we intravenously transplanted syngeneic murine bone marrow-derived MSCs (mMSCs) into C57BL/6 mice at 6 hours after transient middle cerebral artery occlusion (MCAo; 60 minutes) to investigate the impact of MSCs on stroke-induced immunodepression. Transplantation of syngeneic splenocytes or phosphate-buffered saline (PBS) served as controls. An immune status was determined by flow cytometry on days 3 and 14 after MCAo, which did not reveal any negative effects of cell transplantation on stroke-induced immunodepression. Although our mMSCs were found to exert immunosuppressive effects in vitro, stroke-mediated immune cell dysfunction was not altered by mMSCs in ex-vivo stimulation assays with lipopolysaccharide or concanavalin A. Moreover, systemic inflammatory cytokine levels (interleukin-6, tumor necrosis factorα, interferonγ, monocyte chemoattractant protein-1) remained unchanged in the sera of mice after cerebral ischemia and cell transplantation. These results reduce safety concerns about MSC administration in ongoing clinical stroke trials.

Immune privilege of endothelial cells differentiated from endothelial progenitor cells.

AIMS: The application of autologous endothelial progenitor cells (EPC) is a promising approach in cardiovascular regeneration, but the availability of cells in appropriate numbers is the limiting factor. Allogeneic EPC would be an alternative, and we therefore analysed the immunogenicity of EPC-derived endothelial cells (EC) to evaluate their potential usefulness. METHODS AND RESULTS: Circulating EPC from rat were differentiated into EC and characterized phenotypically and functionally. Major histocompatibility complex (MHC) expression in response to interferon-gamma was determined compared with rat aortic EC, and in vitro humoral and cellular allogeneic responses were analysed. To determine the in vivo effects, acellular aortic grafts were endothelialized in vitro with EPC-derived EC and transplanted in a complete allogeneic mismatch rat aortic interposition model. EPC-derived EC expressed endothelial-specific markers and low levels of MHC class I (MHC I), but no constitutive MHC class II (MHC II). When stimulated with interferon-gamma, they upregulated MHC I and moderately upregulated MHC II. They were protected against alloantibody/complement-mediated lysis and allospecific cytotoxic T lymphocyte activity. They were less potent in allogeneic stimulation of CD4 T cells than aortic EC. Seeding of EPC-derived EC into acellular grafts led to excellent endothelialization, and allogeneic aortic transplantation induced only mild inflammatory responses without signs of rejection. CONCLUSION: EPC-derived EC are protected against allospecific cellular immune responses and humoral-mediated attacks in vitro. When transplanted in vivo as a component of vascular grafts, these cells are not rejected, which makes them useful in therapeutic applications, especially vascular reconstruction.

Human immune responses to porcine xenogeneic matrices and their extracellular matrix constituents in vitro.

Several tissue engineering approaches for the treatment of cardiovascular diseases are based on a xenogeneic extracellular matrix. However, the application of engineered heart valves has failed in some patients, causing severe signs of inflammation by so far undetermined processes. Therefore we investigated the immune-mediated responses to porcine valve matrices (native, decellularized and glutaraldehyde-fixed) and to purified xenogeneic extracellular matrix proteins (ECMp). The induction of human immune responses in vitro was evaluated by analyzing the co-stimulatory effects of matrices and ECMp collagen and elastin on the proliferation of immune cell sub-populations via CFSE-based proliferation assays. The pattern of cytokine release was also determined. In porcine matrix punches we demonstrated strong immune responses with the native as well as the decellularized type, in contrast to attenuated effects with glutaraldehyde-fixed matrices. Furthermore, our results indicate that collagen type I (porcine and human) and human elastin were able to elicit proliferation in co-stimulation with anti-CD3 antibody, accompanied by a strong release of Th1 cytokines (IFN-gamma, TNF-alpha). In contrast, porcine elastin did not elicit any response at all. This low immunogenic potential of porcine elastin suggests its suitability for the creation of new tissue engineering heart valve scaffolds in the future.

Low immunogenicity of endothelial derivatives from rat embryonic stem cell-like cells.

Embryonic stem cells (ESC) are suggested to be immune-privileged, but they carry the risk of uncontrolled expansion and malignancy. Upon differentiation they lose their tumor-forming capacity, but they become immunogenic by the expression of a normal set of MHC molecules. This immunogenicity might trigger rejection after application in regenerative therapies. In this study MHC expression of and immune responses to endothelial derivatives of rat embryonic stem cell-like cells (RESC) under inflammatory conditions were determined in comparison to primary rat aortic endothelial cells (ECs). Cellular as well as humoral allo-recognition was analyzed in vitro. In addition, immune reactions in vivo were assessed by allo-antibody production and determination of interferon-gamma (IFNgamma)-secreting allo-reactive T cells. RESC derivatives expressed low but significant levels of MHC class I, and no MHC class II. In response to IFNgamma MHC class I expression was enhanced, while class II transactivator induction failed completely in these cells; MHC class II expression remained consistently absent. Functionally, the RESC derivatives showed a reduced allo-stimulatory capacity, protection against humoral allo-recognition in vitro and a slightly diminished susceptibility to cytotoxic T cell lysis. Furthermore, in vivo experiments demonstrated that these cells do not trigger host immune reactions, characterized by no allo-antibody production and no induction of allo-reactive memory T cells. Our results show that endothelial derivatives of RESC have a distinctive reduced immunogenic potency even under inflammatory conditions.

Stochastic variation in telomere shortening rate causes heterogeneity of human fibroblast replicative life span.

The replicative life span of human fibroblasts is heterogeneous, with a fraction of cells senescing at every population doubling. To find out whether this heterogeneity is due to premature senescence, i.e. driven by a nontelomeric mechanism, fibroblasts with a senescent phenotype were isolated from growing cultures and clones by flow cytometry. These senescent cells had shorter telomeres than their cycling counterparts at all population doubling levels and both in mass cultures and in individual subclones, indicating heterogeneity in the rate of telomere shortening. Ectopic expression of telomerase stabilized telomere length in the majority of cells and rescued them from early senescence, suggesting a causal role of telomere shortening. Under standard cell culture conditions, there was a minor fraction of cells that showed a senescent phenotype and short telomeres despite active telomerase. This fraction increased under chronic mild oxidative stress, which is known to accelerate telomere shortening. It is possible that even high telomerase activity cannot fully compensate for telomere shortening in all cells. The data show that heterogeneity of the human fibroblast replicative life span can be caused by significant stochastic cell-to-cell variation in telomere shortening.