Periodic Reporting for period 4 - REGMAMKID (How to regenerate the mammalian kidney) Reporting period: 2020-05-01 to 2020-10-31 Summary of the context and overall objectives of the project Kidney-related disease remains one of the major health issues in modern society. Nowadays, Chronic kidney disease (CKD) is thought to affect around 10% of the EU population. In REGMAMKID our aim is to generate innovative strategies for kidney healing. Our project stands in the use of animal models with endogenous capacity to regenerate after injury (neonatal mice), together with human pluripotent stem cells. Taking advantage of these systems, REGMAMKID aims to identify endogenous molecular mechanisms responsible for kidney regeneration in mammals, and to define novel culture systems for kidney cell replacement and therapy. Conclusions of the action REGMAMKID has led to the development of fundamental knowledge on kidney development and regeneration in mammals. Taking advantage of animal models and human pluripotent stem cells (hPSCs) this project has led to the definition of novel approaches to derive renal-like cells and kidney organoids to understand kidney development and disease. REGMAMKID has resulted in a total of 10 research publications and 7 reviews, participation in 80 scientific meetings, and 900 dissemination activities (including press, TV, radio and online). The laboratory has grown from 2 to 12 people. The advances from REGMAMKID have allowed for understanding early steps of SARS-CoV-2 infection in hPSCs-kidney organoids identifying a clinical grade compound which nowadays is in a phase IIb clinical trial in COVID19 patients. REGMAMKID has attracted the attention of international collaborators and also allowed to attract young talent to the host laboratory (11 Master students, 6 PhD students, two senior scientists and 1 Marie Curie fellowship). In total, REGMAMKID has been promoted in more than 900 outreach activities. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far REGMAMKID has exploited the mouse model to understand early steps of kidney differentiation and regeneration. Then, exploiting hPSCs we have interrogated for the epigenetic/transcriptomic landscapes during renal differentiation in the human setting. REGMAMKID focus on the development of innovative strategies to enhance kidney differentiation from hPSCs (i.e. defining new biomimetic matrices and fabricating hydrogels with tailored stiffness mimicking kidney and embryonic milieu) has also allowed to generate kidney analogues by 3D bioprinting. In parallel, the knowledge generated in REGMAMKID has been key to establish a novel cellular platform for the introduction of an inducible module for the expression of Cas9 in hPSCs. All these developments have been of utmost importance to explore the definition of new procedures to explore genetic and systemic conditions leading to kidney disease (including polycystic kidney disease and congenital nephrotic syndrome). REGMAMKID advances have allowed interrogate early steps of SARS-CoV-2 infection in hPSCs-kidney organoids and to identify a clinical grade compound that nowadays is in a phase II clinical trial for COVID19 patients. Final period: overview of the results and their exploitation and dissemination REGMAMKID has allowed for the development of novel strategies to promote kidney regeneration. The project has focused on the derivation of renal-like cells and kidney organoids from hPSCs to further understand how the kidney develops and gets disease in mammals. REGMAMKID has also focused on the impact of genetic and systemic dysfunction(s) in these processes. We have defined experimental conditions to derive mesoderm progenitors from hPSCs (Biomaterials 2016) and, also, established new procedures to generate biological-derived hydrogels for applications in hPSCs differentiation into renal-like cells (Materials Today 2017). REGMAMKID also hypothesized that the emulation of the biophysical constrains encountered in the kidney native tissue may represent a straight-forward approach to externally control and guide hPSCs differentiation allowing to define a new methodology to derive kidney organoids transcriptomically matching the second trimester gestational kidney (Nature Materials, 2019). These results were recognized with the prize from Fundación Íñigo Álvarez de Toledo in basic nephrology (2019).Our work on Nature Materials also attracted the attention of the general public (more than 2.3 M€ audience reached, equivalent to 62 k€ in advertising). REGMAMKID also identified new approaches for kidney organoid differentiation through the presentation of systemic conditions boosting metabolic responses in the time course of organoid differentiation as a new approach to externally guide tubular-like cells generation (Cell Metabolism 2020). REGMAMKID also examined the sequence of transcriptomic changes that take place in the time course of hPSCs differentiation towards renal-like cells and kidney organoids using single cell RNA seq and demonstrated the cellular composition and differentiation extent of kidney organoids (Cell 2020). Importantly, and into the light of COVID19 outbreak REGMAMKID developments were applied to study SARS-CoV-2 infection in collaboration with Professors Josef Penninger and Ali Mirazimi. Our work, dentifedd a clinical-grade compound blocking virus infection about 1000-5000 times in hPSCs-kidney organoids (Cell 2020; EMBO Molecular Medicine 2020). Our Cell paper led to 760 dissemination activities in TV, radio and web. The identified compound is nowadays in a phase II b clinical trial in COVID19 patients (The Lancet Respiratory Medicine, 2020). These results have been presented in international webminars (Nature-Nikon 2020; 3D day Life Technologies, 2020, among others) attracting the attention of the general public (more than 410 M€ audience reached considering all dissemination activities performed) and have been recently awarded by ATRESMEDIA and AXA Foundation as the “best publication in biomedical research in Spain during 2020” (Constantes y Vitales Prize). The research conducted in REGMAMKID has also allowed for the preparation of reviews and collaborative works in the fields of hPSCs differentiation (Nephron, 2018, Curr Transpl Reports, 2018; Nature Cell Biology 2020), tissue engineering (FEBS, 2016), and mechanobiology (Nature, 2018; Nature Materials 2020). REGMAMKID has also allowed for the generation of genome edited hPSCs taking advantage of CRISPR/Cas9 technology to further generate stable and inducible knock out(s) for genes related to polycystic kidney disease and congenital nephrotic syndrome. These results have been presented in international conferences and served as a proof of concept four the recently awarded ERC Consolidator Grant proposal to the PI of REGMAMKID. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Our activities have led to the generation of fundamental knowledge about kidney differentiation by the definition of novel approaches for the derivation of 3D kidney organoids from hPSCs. Such platform represents a faithful in vitro system for kidney disease modeling using CRISPR/Cas9 technology, offering an attractive scenario for drug screening and toxicological studies in other organoid systems. The potential of such strategy is therefore expected to be of considerable interest for pharmaceutical companies. We have also developed fundamental knowledge on the derivation of new materials promoting renal-like cells and kidney organoid derivation from hPSCs. These approaches may have an impact in the definition of novel procedures for organoid derivation and thus represent a great value in the field of organoid and tissue engineering. A)Embryonic kidney of WT1-GFP mice B) Methods for embryonic kidney culture Confocal image of a kidney organoid generated in vitro by hPSC differentiation in 3D culture Confocal image from a hPSCs-kidney organoid exposed to glycolitic-promoting media
