Periodic Reporting for period 2 - ENGINORG (Engineering kidney organoids to study the interplay between Tissue Mechanics and Metabolism: from development to disease)
Reporting period: 2023-04-01 to 2024-09-30
Congenital anomalies of the kidney and the urinary tract (CAKUT) account for ~50% of the ethiology of chronic kidney disease in children worldwide. In ENGINORG we explore tissue-scale (nephron) morphogenetic process during the in vitro development and self-organization of human kidney organoids as a new approach to understand early morphogenetic changes related to CAKUT manifestation. To that, ENGINORG develops conceptual and technical approaches to mechanistically link physical and metabolic parameters with gene expression regulation and epigenetics during kidney organoid generation. The identification of the molecular mechanisms connecting these processes will define minimal design principles for the presentation of the proper control of cell-cell interactions, cell-matrix interplay, and cell organization (or cell sorting) for organoid generation. The knowledge generated here will ultimately serve to understand human kidney morphogenesis and CAKUT. Our specific interconnected Objectives are: 1) To generate bona fide hPSCs-organoids and renal hPSCs-derivatives by the presentation of controlled biophysical cues; 2) To understand and link how mechanics and metabolism guide hPSCs differentiation towards kidney organoids. Objective 3) To establish a microfluidic platform to assess how metabolic and mechanical cues regulate kidney development and disease.
The activities performed in the project’s first period have mostly covered the generation of hPSCs capturing CAKUT-genetic backgrounds. Furthermore, we have also taken advantage of genome editing for the generation of reporter cell lines for major kidney compartments, including metanephric mesenchyme (MM) derived cells, ureteric bud (UB), stroma and endothelium. We have also set up novel procedures sustaining for the generation of UB organoids and complex kidney organoids by the assembly of endothelial and UB organoids with nephron-like organoids. Taking advantage of these in vitro systems we have started to explore the impact of different CAKUT-related microenvironments (i.e. diabetic milieu and fibrotic-like, among others). Moreover, we are currently taking advantage of micropatterning substrates to present geometrical constrains and tailored stiffness to externally guide and control morphogenetic process during the in vitro development and self-organization of human kidney organoids.
Our activities are leading to conceptual and technical advances that will allow to mechanistically link how the presentation of controlled physical and metabolic constrains during organoid generation are further integrated and resolved through gene expression regulation and epigenetics. The identification of the molecular mechanisms connecting these processes will define minimal design principles for organoid generation. Such platform represents a faithful in vitro system for kidney disease modelling, offering an attractive scenario to understand and model CAKUT. At the present time we are also taking advantage of the conceptual advances generated in ENGINORG to include other differentiation pipelines to model CAKUT as heart and retina. The potential of such strategy will maximize the impact of ENGINORG and is expected to be of considerable interest for pharmaceutical companies interested in the fields of regenerative and personalized medicine.