Netrin-4 expression by human endothelial cells inhibits endothelial inflammation and senescence.

Netrin-4, recognized in neural and vascular development, is highly expressed by mature endothelial cells. The function of this netrin-4 in vascular biology after development has remained unclear. We found that the expression of netrin-4 is highly regulated in endothelial cells and is important for quiescent healthy endothelium. Netrin-4 expression is upregulated in endothelial cells cultured under laminar flow conditions, while endothelial cells stimulated with tumor necrosis factor alpha resulted in decreased netrin-4 expression. Targeted reduction of netrin-4 in endothelial cells resulted in increased expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. Besides, these endothelial cells were more prone to monocyte adhesion and showed impaired barrier function, measured with electric cell-substrate impedance sensing, as well as in an 'organ-on-a-chip' microfluidic system. Importantly, endothelial cells with reduced levels of netrin-4 showed increased expression of the senescence-associated markers cyclin-dependent kinase inhibitor-1 and -2A, an increased cell size and decreased ability to proliferate. Consistent with the gene expression profile, netrin-4 reduction was accompanied with more senescent associated ß-galactosidase activity, which could be rescued by adding netrin-4 protein. Finally, using human decellularized kidney extracellular matrix scaffolds, we found that pre-treatment of the scaffolds with netrin-4 increased numbers of endothelial cells adhering to the matrix, showing a pro-survival effect of netrin-4. Taken together, netrin-4 acts as an anti-senescence and anti-inflammation factor in endothelial cell function and our results provide insights as to maintain endothelial homeostasis and supporting vascular health.

Protective Effects of Human Nonrenal and Renal Stromal Cells and Their Conditioned Media in a Rat Model of Chronic Kidney Disease.

Mesenchymal stromal cells (MSCs) are emerging as a novel therapeutic option for limiting chronic kidney disease progression. Conditioned medium (CM) containing bioactive compounds could convey similar benefits, avoiding the potential risks of cell therapy. This study compared the efficacy of nonrenal and renal cell-based therapy with the corresponding CM in rats with renal mass reduction (RMR). Infusions of human kidney stromal cells (kPSCs) and CM-kPSCs, but not umbilical cord (uc) MSCs or CM-ucMSCs, reduced proteinuria and preserved podocyte number and nephrin expression in RMR rats. Glomerular fibrosis, microvascular rarefaction, and apoptosis were reduced by all treatments, while the peritubular microvascular loss was reduced by kPSCs and CM-kPSCs treatment only. Importantly, kPSCs and CM-kPSCs reduced NG2-positive pericytes, and all therapies reduced α-smooth muscle actin expression, indicating reduced myofibroblast expansion. Treatment with kPSCs also significantly inhibited the accumulation of ED1-positive macrophages in the renal interstitium of RMR rats. These findings demonstrate that the CM of ucMSCs and kPSCs confers similar renoprotection as the cells. kPSCs and CM-kPSCs may be superior in attenuating chronic renal injury as a cell source.

Vascular bioengineering of scaffolds derived from human discarded transplant kidneys using human pluripotent stem cell-derived endothelium.

The bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being investigated is the recellularization of kidney scaffolds. In this study, we present several key advances toward successful re-endothelialization of whole kidney matrix scaffolds from both rodents and humans. Based on the presence of preserved glycosoaminoglycans within the decelullarized kidney scaffold, we show improved localization of delivered endothelial cells after preloading of the vascular matrix with vascular endothelial growth factor and angiopoietin 1. Using a novel simultaneous arteriovenous delivery system, we report the complete re-endothelialization of the kidney vasculature, including the glomerular and peritubular capillaries, using human inducible pluripotent stem cell -derived endothelial cells. Using this source of endothelial cells, it was possible to generate sufficient endothelial cells to recellularize an entire human kidney scaffold, achieving efficient cell delivery, adherence, and endothelial cell proliferation and survival. Moreover, human re-endothelialized scaffold could, in contrast to the non-re-endothelialized human scaffold, be fully perfused with whole blood. These major advances move the field closer to a human bioengineered kidney.

Therapeutic potential of stromal cells of non-renal or renal origin in experimental chronic kidney disease.

BACKGROUND: Mesenchymal stromal cell (MSC)-based therapy is a promising strategy for preventing the progression of chronic kidney disease (CKD), with the potential to induce tissue regeneration. In search of the best cellular source we compared, in the rat model of adriamycin (ADR) nephropathy, the regenerative potential of human stromal cells of non-renal origin, such as bone marrow (bm) MSCs and umbilical cord (uc) MSCs, with that of newly discovered stromal cells of renal origin, the kidney perivascular cells (kPSCs) known to exhibit tissue-specific properties. METHODS: The therapeutic effect of repeated infusions of human bmMSCs, ucMSCs, kPSCs (1.5 × 106 cells/rats) or conditioned medium from ucMSCs was studied in athymic rats with ADR-induced nephropathy (7.9 mg/kg). The ability of the three stromal cell populations to engraft the damaged kidney was evaluated by detecting the presence of human nuclear antigenpos cells. Glomerular podocyte loss and endothelial damage, sclerotic lesions and inflammation were assessed at 14 and 28 days. In-vitro experiments with a transwell system were performed to investigate the effects of different stromal cell populations on parietal epithelial cells (PECs) activated or not with albumin or angiotensin II for 24 h. RESULTS: Infusions of non-renal and renal stromal cells resulted in a comparable engraftment into the kidney, in the peritubular areas and around the glomerular structures. All three cell populations limited podocyte loss and glomerular endothelial cell injury, and attenuated the formation of podocyte and PEC bridges. This translated into a reduction of glomerulosclerosis and fibrosis. Human ucMSCs had an anti-inflammatory effect superior to that of the other stromal cells, reducing macrophage infiltration and inducing polarisation towards the M2 macrophage phenotype. Conditioned medium from ucMSCs shared the same renoprotective effects of the cells. Consistent with in-vivo data, bmMSCs and kPSCs, but even more so ucMSCs, limited proliferation, migratory potential and extracellular matrix production of activated PECs, when cultured in a transwell system. CONCLUSIONS: Our data indicate that either non-renal or renal stromal cells induce renal tissue repair, highlighting ucMSCs and their conditioned medium as the most reliable clinical therapeutic tool for CKD patients.

The cytokine secretion profile of mesenchymal stromal cells is determined by surface structure of the microenvironment.

Mesenchymal stromal cells (MSC) secrete factors that contribute to organ homeostasis and repair in a tissue specific manner. For instance, kidney perivascular mesenchymal stromal cells (kPSCs) can facilitate renal epithelial repair through secretion of hepatocyte growth factor (HGF) while the secretome of bone marrow MSCs gives rise to immunosuppression. Stromal cells function in a complex 3-dimensional (3D) connective tissue architecture that induces conformational adaptation. Here we tested the hypothesis that surface topography and associated cell adaptations dictate stromal cell function through tuning of the cytokines released. To this end, we cultured human bone marrow and kidney perivascular stromal cells in the TopoWell plate, a custom-fabricated multi-well plate containing 76 unique bioactive surface topographies. Using fluorescent imaging, we observed profound changes in cell shape, accompanied by major quantitative changes in the secretory capacity of the MSCs. The cytokine secretion profile was closely related to cell morphology and was stromal cell type specific. Our data demonstrate that stromal cell function is determined by microenvironment structure and can be manipulated in an engineered setting. Our data also have implications for the clinical manufacturing of mesenchymal stromal cell therapy, where surface topography during bioreactor expansion should be taken into account to preserve therapeutic properties.

Renal Subcapsular Transplantation of PSC-Derived Kidney Organoids Induces Neo-vasculogenesis and Significant Glomerular and Tubular Maturation In Vivo.

Human pluripotent stem cell (hPSC)-derived kidney organoids may facilitate disease modeling and the generation of tissue for renal replacement. Long-term application, however, will require transferability between hPSC lines and significant improvements in organ maturation. A key question is whether time or a patent vasculature is required for ongoing morphogenesis. Here, we show that hPSC-derived kidney organoids, derived in fully defined medium conditions and in the absence of any exogenous vascular endothelial growth factor, develop host-derived vascularization. In vivo imaging of organoids under the kidney capsule confirms functional glomerular perfusion as well as connection to pre-existing vascular networks in the organoids. Wide-field electron microscopy demonstrates that transplantation results in formation of a glomerular basement membrane, fenestrated endothelial cells, and podocyte foot processes. Furthermore, compared with non-transplanted organoids, polarization and segmental specialization of tubular epithelium are observed. These data demonstrate that functional vascularization is required for progressive morphogenesis of human kidney organoids.

The human kidney capsule contains a functionally distinct mesenchymal stromal cell population.

We recently demonstrated that the adult human kidney cortex contains a perivascular stromal cell (kPSC) that shows organotypic properties and is important for repair and stabilisation of kidney function. Not only the kidney cortex but also the kidney capsule contains stromal cells that are important for the three dimensional organisation of the kidney during nephrogenesis. They provide the barrier function of the capsule which is critical for homeostatic processes such as pressure natriuresis. We postulated that stromal cells derived from the kidney capsule may therefore also have specific properties and functions. To this end, we isolated these capsule mesenchymal stromal cells (cMSC) from human cadaveric kidneys that were not suitable for transplantation. There were several similarities between cMSCs and kPSCs including support of vascular plexus formation, phenotypic marker expression and resistance against myofibroblast transformation. However, compared to kPSCs, cMSCs showed distinct mRNA and miRNA expression profiles, showed increased immunosuppressive capacity, and displayed strongly reduced HGF production, contributing to the inability to enhance kidney epithelial repair. Therefore cMSCs are a distinct, novel human kidney-derived MSC-population and these data underpin the large functional diversity of phenotypic similar stromal cells in relation to their anatomic site, even within one organ.

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells.

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Clinical-Grade Isolated Human Kidney Perivascular Stromal Cells as an Organotypic Cell Source for Kidney Regenerative Medicine.

Mesenchymal stromal cells (MSCs) are immunomodulatory and tissue homeostatic cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular stromal cells (PSCs) identified within several different organs share characteristics of bone marrow-derived MSCs (BM-MSCs). These PSCs may also possess tissue-specific properties and play a role in local tissue homeostasis. We hypothesized that human kidney-derived PSCs (hkPSCs) would elicit improved kidney repair in comparison with BM-MSCs. Here we introduce a novel, clinical-grade isolation method of hkPSCs from cadaveric kidneys by enriching for the perivascular marker, NG2. hkPSCs show strong transcriptional similarities to BM-MSCs but also show organotypic expression signatures, including the HoxD10 and HoxD11 nephrogenic transcription factors. Comparable to BM-MSCs, hkPSCs showed immunosuppressive potential and, when cocultured with endothelial cells, vascular plexus formation was supported, which was specifically in the hkPSCs accompanied by an increased NG2 expression. hkPSCs did not undergo myofibroblast transformation after exposure to transforming growth factor-ß, further corroborating their potential regulatory role in tissue homeostasis. This was further supported by the observation that hkPSCs induced accelerated repair in a tubular epithelial wound scratch assay, which was mediated through hepatocyte growth factor release. In vivo, in a neonatal kidney injection model, hkPSCs reintegrated and survived in the interstitial compartment, whereas BM-MSCs did not show this potential. Moreover, hkPSCs gave protection against the development of acute kidney injury in vivo in a model of rhabdomyolysis-mediated nephrotoxicity. Overall, this suggests a superior therapeutic potential for the use of hkPSCs and their secretome in the treatment of kidney diseases. Stem Cells Translational Medicine 2017;6:405-418.

Clinical translation of multipotent mesenchymal stromal cells in transplantation.

The prevalence of chronic kidney disease and end-stage renal disease is increasing each year and currently the best therapeutic option for end-stage renal disease patients is kidney transplantation. However, although short-term graft outcomes after transplantation have improved substantially as a result of new and more potent immunosuppressive drugs, the long-term survival has hardly changed. This most likely is caused by a combination of nonimmunologic side effects and sustained alloreactivity to the graft resulting in fibrosis. In addition, current immunosuppressive drugs have side effects, including nephrotoxicity, infections, and malignancies that compromise long-term outcomes. Consequently, there is a strong interest in immunosuppressive therapies that maintain efficacy, while reducing side effects. Because mesenchymal stromal cells have potent anti-inflammatory and antifibrotic properties, these cells are of particular interest as new candidates in transplant recipients. Mesenchymal stromal cells might play roles in the treatment of allograft rejection and fibrosis and in calcineurin minimization and induction protocols. In the present review we discuss both preclinical as well as clinical evidence of their therapeutic potential in kidney transplantation. In addition, challenges and obstacles for clinical translation are discussed.

Mesenchymal stromal cell therapy for cardio renal disorders.

Chronic kidney disease carries a very high mortality risk, in particular from cardiac diseases. Often heart failure and renal failure coincide, a phenomenon referred to as the cardio renal syndrome. In recent years, it has become clear that not only fibrotic repair but also restoration of damaged kidney and heart can occur and the use of cell therapy has been advanced as a means to activate endogenous repair mechanisms or even to re-introduce repairing tissue. In this perspective, mesenchymal stromal cells are of particular interest, since these cells have both immune modulating and reparative functions and are on the brink of entering the clinical arena. Indeed, MSCs can trigger numerous therapeutic biologic processes that contribute to both renal and cardiac repair; however exact mechanisms of actions are largely unknown. In the present review we have made a critical appraisal of the data available with respect to origin and function of MSCs, and we discuss both preclinical as well as clinical evidence on their therapeutic potential in kidney and heart disease.

Human Bone Marrow- and Adipose Tissue-derived Mesenchymal Stromal Cells are Immunosuppressive In vitro and in a Humanized Allograft Rejection Model.

BACKGROUND: Recent studies with bone marrow (BM)-derived Mesenchymal Stromal Cells (MSC) in transplant recipients demonstrate that treatment with MSC is safe and clinically feasible. While BM is currently the preferred source of MSC, adipose tissue is emerging as an alternative. To develop efficient therapies, there is a need for preclinical efficacy studies in transplantation. We used a unique humanized transplantation model to study the in vivo immunosuppressive effect of human BM-MSC and adipose tissue-derived MSC (ASC). METHODS: Gene expression of BM-MSC and ASC and their capacity to inhibit activated PBMC proliferation was evaluated. The in vivo immunosuppressive effect of BM-MSC and ASC was studied in a humanized mouse model. SCID mice were transplanted with human skin grafts and injected with human allogeneic PBMC with or without administration of BM-MSC or ASC. The effect of MSC on skin graft rejection was studied by immunohistochemistry and PCR. RESULTS: BM-MSC and ASC expressed TGFß, CXCL-10 and IDO. IDO expression and acitivity increased significantly in BM-MSC and ASC upon IFN-γ stimulation. IFN-γ stimulated BM-MSC and ASC inhibited the proliferation of activated PBMC in a significant and dose dependent manner. In our humanized mouse model, alloreactivity was marked by pronounced CD45+ T-cell infiltrates consisting of CD4+ and CD8+ T cells and increased IFN-γ expression in the skin grafts which were all significantly inhibited by both BM-MSC and ASC. CONCLUSION: BM-MSC and ASC are immunosuppressive in vitro and suppress alloreactivity in a preclinical humanized transplantation model.