Advanced in vitro Research Models to Study the Role of Endothelial Cells in Solid Organ Transplantation

The endothelium plays a key role in acute and chronic rejection of solid organ transplants. During both processes the endothelium is damaged often with major consequences for organ function. Also, endothelial cells (EC) have antigen-presenting properties and can in this manner initiate and enhance alloreactive immune responses. For decades, knowledge about these roles of EC have been obtained by studying both in vitro and in vivo models. These experimental models poorly imitate the immune response in patients and might explain why the discovery and development of agents that control EC responses is hampered. In recent years, various innovative human 3D in vitro models mimicking in vivo organ structure and function have been developed. These models will extend the knowledge about the diverse roles of EC in allograft rejection and will hopefully lead to discoveries of new targets that are involved in the interactions between the donor organ EC and the recipient's immune system. Moreover, these models can be used to gain a better insight in the mode of action of the currently prescribed immunosuppression and will enhance the development of novel therapeutics aiming to reduce allograft rejection and prolong graft survival.</p

Morphology and size of stem cells from mouse and whale: Observational study

Abstract Objective To compare the morphology and size of stem cells from two mammals of noticeably different body size. Design Observational study. Setting The Netherlands. Participants A humpback whale (Megaptera novaeangliae) and a laboratory mouse (Mus musculus). Main outcome measures Morphology and size of mesenchymal stem cells from adipose tissue. Results Morphologically, mesenchymal stem cells of the mouse and whale are indistinguishable. The average diameter of 50 mesenchymal stem cells from the mouse was 28 (SD 0.86) μm and 50 from the whale was 29 (SD 0.71) μm. The difference in cell size between the species was not statistically significant. Although the difference in bodyweight between the species is close to two million-fold, the mesenchymal stem cells of each were of similar size. Conclusions The mesenchymal stem cells of whales and mice are alike, in both morphology and size

On the interactions between mesenchymal stem cells and regulatory T cells for immunomodulation in transplantation

Experimental studies have established the use of mesenchymal stem cells (MSC) as a candidate immunosuppressive therapy. MSC exert their immunomodulatory function through the inhibition of CD4+ and CD8+ T cell proliferation. It is unknown whether MSC impair the immunosuppressive function of regulatory T cells (Treg). In vitro and in vivo studies suggest that MSC mediate their immunomodulatory effects through the induction of Treg. In this review we will focus on the interactions between MSC and Treg, and evaluate the consequences of these cellular interplays for prospective MSC immunotherapy in organ transplantation

Membrane particles from mesenchymal stromal cells reduce the expression of fibrotic markers on pulmonary cells

Background: Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with limited treatment options in which the telomere shortening is a strong predictive factor of poor prognosis. Mesenchymal stromal cells (MSC) administration is probed in several experimental induced lung pathologies; however, MSC might stimulate fibrotic processes. A therapy that avoids MSC side effects of transformation would be an alternative to the use of living cells. Membranes particles (MP) are nanovesicles artificially generated from the membranes of MSC containing active enzymes involved in ECM regeneration. We aimed to investigate the anti-fibrotic role of MP derived from MSC in an in vitro model of pulmonary fibrosis. Methods: Epithelial cells (A549) and lung fibroblasts, from IPF patients with different telomere length, were co-cultured with MP and TGF-β for 48h and gene expression of major pro-fibrotic markers were analyzed. Results: About 90% of both types of cells effectively took up MP without cytotoxic effects. MP decreased the expression of profibrotic proteins such as Col1A1, Fibronectin and PAI-1, in A549 cells. In fibroblasts culture, there was a different response in the inhibitory effect of MP on some pro-fibrotic markers when comparing fibroblast from normal telomere length patients (FN) versus short telomere length (FS), but both types showed an inhibition of Col1A1, Tenascin-c, PAI-1 and MMP-1 gene expression after MP treatment

Reparative effect of mesenchymal stromal cells on endothelial cells after hypoxic and inflammatory injury

Background: The renal endothelium is a prime target for ischemia-reperfusion injury (IRI) during donation and transplantation procedures. Mesenchymal stromal cells (MSC) have been shown to ameliorate kidney function after IRI. However, whether this involves repair of the endothelium is not clear. Therefore, our objective is to study potential regenerative effects of MSC on injured endothelial cells and to identify the molecular mechanisms involved. Methods: Human umbilical vein endothelial cells (HUVEC) were submitted to hypoxia and reoxygenation and TNF-α treatment. To determine whether physical interaction or soluble factors released by MSC were responsible for the potential regenerative effects of MSC on endothelial cells, dose-response experiments were performed in co-culture and transwell conditions and with secretome-deficient MSC. Results: MSC showed increased migration and adhesion to injured HUVEC, mediated by CD29 and CD44 on the MSC membrane. MSC decreased membrane injury marker expression, oxidative stress levels, and monolayer permeability of injured HUVEC, which was observed only when allowing both physical and paracrine interaction between MSC and HUVEC. Furthermore, viable MSC in direct contact with injured HUVEC improved wound healing capacity by 45% and completely restored their angiogenic capacity. In addition, MSC exhibited an increased ability to migrate through an injured HUVEC monolayer compared to non-injured HUVEC in vitro. Conclusions: These results show that MSC have regenerative effects on injured HUVEC via a mechanism which requires both physical and paracrine interaction. The identification of specific effector molecules involved in MSC-HUVEC interaction will allow targeted modification of MSC to apply and enhance the therapeutic effects of MSC in IRI. [Figure not available: see fulltext.

Treating Ischemically Damaged Porcine Kidneys with Human Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stromal Cells During Ex Vivo Normothermic Machine Perfusion

Pretransplant normothermic machine perfusion (NMP) of donor kidneys offers the unique opportunity to perform active interventions to an isolated renal graft before transplantation. There is increasing evidence that mesenchymal stromal cells (MSCs) could have a paracrine/endocrine regenerative effect on ischemia-reperfusion injury. The purpose of this study was to determine which cytokines are secreted by MSCs during NMP of a porcine kidney. Viable porcine kidneys and autologous whole blood were obtained from a slaughterhouse. Warm ischemia time was standardized at 20 min and subsequent hypothermic machine perfusion was performed during 2-3 h. Thereafter, kidneys were machine perfused at 37 degrees C during 7 h. After 1 h of NMP, 0, 10(7)cultured human adipose tissue-derived MSCs, or 10(7)cultured bone marrow-derived MSCs were added (n = 5 per group). In a fourth experimental group, 7-h NMP was performed with 10(7)adipose tissue-derived MSCs, without a kidney in the circuit. Kidneys perfused with MSCs showed lower lactate dehydrogenase and neutrophil gelatinase-associated lipocalin levels in comparison with the control group. Also, elevated levels of human hepatocyte growth factor, interleukin (IL)-6, and IL-8 were found in the perfusate of the groups perfused with MSCs compared to the control groups. This study suggests that MSCs, in contact with an injured kidney during NMP, could lead to lower levels of injury markers and induce the release of immunomodulatory cytokines.Nephrolog

Human Transplant Kidneys on Normothermic Machine Perfusion Display Endocrine Activity

Background. Normothermic machine perfusion (NMP) is an alternative to hypothermic machine perfusion (HMP) for donor kidney preservation before transplantation. Contrary to HMP, NMP allows for functional assessment of donor kidneys because normothermic conditions allow for metabolic activity. The kidneys are key producers of hormones. Yet, it remains unknown whether donor kidneys during NMP display endocrine functions. Methods. Fifteen donor kidneys were subjected to HMP followed by 2 h of NMP before transplantation. NMP perfusate was collected at 3 time points (0, 1, 2 h) for the measurements of prorenin/renin, erythropoietin (EPO), and vitamin D, and urine samples were collected at 1 h and 2 h for urodilatin measurement. Fifteen HMP perfusate samples were collected for the same measurements. Results. Kidneys on NMP secreted significantly more prorenin, renin, EPO, and active vitamin D than during HMP. EPO and vitamin D secretion remained stable during 2 h of NMP, whereas the prorenin release rate increased and renin release rate decreased after 1 h. Donation after brain death kidneys secreted more vitamin D and less EPO during NMP than donation after circulatory death kidneys. Twelve donor kidneys produced urine during NMP and released detectable levels of urodilatin. Kidneys exhibited a large variation in hormone release rates. No significant differences were found in hormone release capacity between delayed graft function (DGF) and non-DGF kidneys, and no significant correlations were found between hormone release rates and the duration of DGF or 1-mo posttransplant serum creatinine levels. Conclusions. Human transplant kidneys display endocrine activity during NMP. To explore whether correlations exist between hormone release rates and posttransplant kidney function, large numbers of kidneys are required.</p

Chondrogenically Primed Human Mesenchymal Stem Cells Persist and Undergo Early Stages of Endochondral Ossification in an Immunocompetent Xenogeneic Model

Tissue engineering approaches using progenitor cells such as mesenchymal stromal cells (MSCs) represent a promising strategy to regenerate bone. Previous work has demonstrated the potential of chondrogenically primed human MSCs to recapitulate the process of endochondral ossification and form mature bone in vivo, using immunodeficient xenogeneic models. To further the translation of such MSC-based approaches, additional investigation is required to understand the impact of interactions between human MSC constructs and host immune cells upon the success of MSC-mediated bone formation. Although human MSCs are considered hypoimmunogenic, the potential of chondrogenically primed human MSCs to induce immunogenic responses in vivo, as well as the efficacy of MSC-mediated ectopic bone formation in the presence of fully competent immune system, requires further elucidation. Therefore, the aim of this study was to investigate the capacity of chondrogenically primed human MSC constructs to persist and undergo the process of endochondral ossification in an immune competent xenogeneic model. Chondrogenically differentiated human MSC pellets were subcutaneously implanted to wild-type BALB/c mice and retrieved at 2 and 12 weeks post-implantation. The percentages of CD4(+) and CD8(+) T cells, B cells, and classical/non-classical monocyte subsets were not altered in the peripheral blood of mice that received chondrogenic MSC constructs compared to sham-operated controls at 2 weeks post-surgery. However, MSC-implanted mice had significantly higher levels of serum total IgG compared to sham-operated mice at this timepoint. Flow cytometric analysis of retrieved MSC constructs identified the presence of T cells and macrophages at 2 and 12 weeks post-implantation, with low levels of immune cell infiltration to implanted MSC constructs detected by CD45 and CD3 immunohistochemical staining. Despite the presence of immune cells in the tissue, MSC constructs persisted in vivo and were not degraded/resorbed. Furthermore, constructs became mineralised, with longitudinal micro-computed tomography imaging revealing an increase in mineralised tissue volume from 4 weeks post-implantation until the experimental endpoint at 12 weeks. These findings indicate that chondrogenically differentiated human MSC pellets can persist and undergo early stages of endochondral ossification following subcutaneous implantation in an immunocompetent xenogeneic model. This scaffold-free model may be further extrapolated to provide mechanistic insight to osteoimmunological processes regulating bone regeneration and homeostasis

Mesenchymal Stromal Cells Are Retained in the Renal Cortex Independently of Their Metabolic State After Renal Intra-Arterial Infusion

The regenerative capacities of mesenchymal stromal cells (MSCs) make them suitable for renal regenerative therapy. The most common delivery route of MSC is through intravenous infusion, which is associated with off-target distribution. Renal intra-arterial delivery offers a targeted therapy, but limited knowledge is available regarding the fate of MSCs delivered through this route. Therefore, we studied the efficiency and tissue distribution of MSCs after renal intra-arterial delivery to a porcine renal ischemia-reperfusion model. MSCs were isolated from adipose tissue of healthy male pigs, fluorescently labeled and infused into the renal artery of female pigs. Flow cytometry allowed MSC detection and quantification in tissue and blood. In addition, quantitative polymerase chain reaction was used to trace MSCs by their Y-chromosome. During infusion, a minor number of MSCs left the kidney through the renal vein, and no MSCs were identified in arterial blood. Ischemic and healthy renal tissues were analyzed 30 min and 8 h after infusion, and 1-4 x 10(4) MSCs per gram of tissue were detected, predominantly, in the renal cortex, with a viability >70%. Confocal microscopy demonstrated mainly glomerular localization of MSCs, but they were also observed in the capillary network around tubuli. The infusion of heat-inactivated (HI) MSCs, which are metabolically inactive, through the renal artery showed that HI-MSCs were distributed in the kidney in a similar manner to regular MSCs, suggesting a passive retention mechanism. Long-term MSC survival was analyzed by Y-chromosome tracing, and demonstrated that a low percentage of the infused MSCs were present in the kidney 14 days after administration, while HI-MSCs were completely undetectable. In conclusion, renal intra-arterial MSC infusion limited off-target engraftment, leading to efficient MSC delivery to the kidney, most of them being cleared within 14 days. MSC retention was independent of the metabolic state of MSC, indicating a passive mechanism