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Dreaming of no more renal dialysis: how self-derived tissue and cells can replace renal function

Final Report Summary - RESET (Dreaming of no more renal dialysis: how self-derived tissue and cells can replace renal function)

Chronic kidney disease (CKD) is a progressive clinical condition marked by deteriorating kidney function over time that requires renal replacement therapies to survive. The vast majority of the world’s population has no or limited access to these therapies, which consists of dialysis or renal transplantation. The increasing number of patients and the limited treatments provide an imperative need for developing new methods for generating transplantable kidneys.

The overall focus of RESET project was based on the concept to create new functional kidneys by tissue engineering technologies, starting from the whole kidney scaffold with intact three-dimensional geometry created by decellularization of a kidney from CKD patient. Reconstruction of the organ by reseeding with patient’s specific renal committed iPS cells, to obtain new transplantable organ with proper functional performance, was indicated as the final goal of the study.

During the project activities, we have achieved significant milestones in this process. Firstly, protocols for decellularization of rat kidneys have been developed and implemented resulting in well preserved three-dimensional rat and pig whole-kidney scaffolds. Ultrastructure of glomerular and tubular basement membranes was well preserved as assessed by TEM and SEM. Investigations based on microCT scans allowed us to establish the integrity, patency, and connections of the renal vascular network. These rat kidney scaffolds have been successfully implanted in rats, and perfused by blood for up to four weeks. However, they induced massive clotting and deposition of circulating cells. This expected finding further indicate that before implantation the renal scaffolds must be recellularized, especially with endothelial cells.

To this aim, we derived and characterized human iPS cells, starting from dermal fibroblasts transduced by lentiviral vectors encoding for Oct4, Klf4, Sox2, and c-Myc. We developed a two-stages inductive protocol to differentiate iPSCs into renal progenitor cell populations. Moreover, we also set up differentiation protocols to generate human endothelial cells or human podocytes from iPSCs. While renal progenitor cells were not able to reseed the kidney, endothelial cells, which showed developmental potential using self-forming mouse kidney organoids, successfully repopulated the vascular compartment of the rat renal scaffolds.

We developed and test different experimental strategies to recellularize kidney scaffolds, we also used mouse embryonic stem (mES) cells. Infusion of cell suspensions into the renal artery, vein and ureter allowed us to deliver of mES cells into the different parts of the nephron, both in vascular and tubular compartment. However, the volume of the scaffold occupied by cells was rather limited, as demonstrated by detailed morphometrical analysis. Experimental and theoretical approaches allowed us to identify the reason for the incomplete cell delivery. Actually, for the hydraulic permeability of basement membranes the infused fluid is filtered out of the vascular bed stopping cells into the glomerular capillaries.

These findings are fundamental for further development of efficient strategies for cell seeding in acellular organ scaffolds. In addition, they demonstrate kidney bioengineering is still far from the clinical reality. This information will better define the effective stage of scientific research in this field to avoid false expectations from the end-stage renal diseased patients.