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.... Show moreNetrin-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-achip? 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 ?-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. Show less
Imberti, B.; Cerullo, D.; Corna, D.; Rota, C.; Locatelli, M.; Pezzotta, A.; ... ; Luyckx, V. 2020
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... Show moreMesenchymal 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 alpha-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. Show less
Leuning, D.G.; Witjas, F.M.R.; Maanaoui, M.; Graaf, A.M.A. de; Lievers, E.; Geuens, T.; ... ; Rabelink, T.J. 2019
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... Show moreThe 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. Show less
Rota, C.; Morigi, M.; Cerullo, D.; Introna, M.; Colpani, O.; Corna, D.; ... ; Remuzzi, G. 2018
Within this thesis several novel strategies to regenerate the human kidney are exploited. The first strategy is to improve kidney function is by mesenchymal stromal cell (MSC) therapy. In chapter 2... Show moreWithin this thesis several novel strategies to regenerate the human kidney are exploited. The first strategy is to improve kidney function is by mesenchymal stromal cell (MSC) therapy. In chapter 2 the current status of clinical trials with MSC therapy are discussed. In chapter 3 we show an extensive characterization of MSCs derived from human kidney (hkPSCs) compared to bone marrow derived MSCs (bmMSCs) and show that hkPSCs show organotypic expression signatures and functionality. For fluent clinical translation, we developed a clinical grade acceptable standard operation procedure (SOP) (chapter 4). In chapter 5 we show that the cytokine secretion profile of both hkPSCs and bmMSCs was closely related to cell morphology adaptation to culture surface topography and was stromal cell type specific. In chapter 6 we show that not only the kidney cortex but also the kidney capsule contains a stromal cell population. In chapter 7 we report the regeneration of kidney vasculature by repopulating the vascular compartment of human and rat kidney matrices with hiPSC-derived endothelial cells. We show efficient cell delivery, adherence and survival of these endothelial cells as a first, but critical, step towards a human bioengineered kidney. Show less
Leuning, D.G.; Beijer, N.R.M.; Fosse, N.A. du; Vermeulen, S.; Lievers, E.; Kooten, C. van; ... ; Boer, J. de 2018
