Advanced Human Pluripotent Stem Cell Kidney Organoid Model for Investigating Development and Disease

Kidney function is essential for life and treatment for kidney failure costs the NHS £2 billion each year. Most scientists investigating kidney development and failure in disease use animal in vivo and ex-vivo models particularly mice. However, mouse kidneys are not the same as human kidneys, either in gross organisation or in the precise molecular composition during development. This may explain why mice with particular gene defects often do not show the same symptoms as a human with these changes. The aim of this proposal is to use very early stem cells (human pluripotent stem cells: hPSCs) to establish a human 3D model of the developing kidney at very small scale (micro-organoids). At the moment the standard kidney organoids we and others make, require a large number of cells/organoid (about 100,000+) and lack certain cell types including critically, cells of the immune system called macrophages. These macrophages play roles in development of the fetal kidney, roles that are different from their role in inflammation after birth. They promote blood vessel development and that of the outflow regions of the kidney, both of which develop poorly in the current hPSC-kidney models. We hypothesise that kidney organoids can be made dramatically better kidney models by introducing macrophages and improving nutrient/waste diffusion. We will generate macrophages from our hPSCs according to published methods, aided by our experienced collaborators, and test the effect of different proportions of these in improving our stem cell-kidney organoids, first in the conventional 'macro'-organoids, then in micro-organoids with 1/10th the number of cells or fewer. We will use this platform to ask whether immune macrophages can enhance kidney organoid developmental. In our study, the organoids will be grown in a way that compensates for lack of blood flowing through, by generating flow around the tiny kidney micro-organoids through culturing them in specially designed purpose built chambers. This model will allow the time in culture to be increased so that the kidney tissue remains healthy and develops further. The micro-organoids will be more complex and more similar in composition to developing kidneys than current stem cell derived macro-organoids. This human system will be better for understanding human kidney development and diseases affecting development of the human kidney, particularly those caused by detrimental changes in genes. It will replace the need to introduce similar gene changes into mice and look at their kidney development-which may anyway be affected differently. We can adapt the model for other tissues like liver gut or lung hPSC- or tissue-organoid models. Thus, this platform will be suitable for use in better understanding of human development and disease and by generating many micro organoids (scale up), for use in testing drug, which may alleviate kidney disease, or testing if they are harmful to the kidney. In the long term, it could provide a route for generating supplementary kidney tissue to aid ailing kidneys.

Animal in vivo and ex-vivo models do not faithfully model human kidney development or disease. Human pluripotent stem cell (hPSC)-organoids could substitute for use of transgenic animal but currently they are deficient in certain cells and immature as well as deteriorating on extended culture. The aim of this proposal is to establish a scaled down model from current hPSC-kidney organoids for future understanding of human development and disease and use in drug testing. The current model requires 100K+ cells/organoid and critically lacks macrophages. Tissue resident macrophages have been shown to enhance aspects of kidney development including epithelial branching and vascular development, two critical weaknesses in current models. So we will generate macrophages from our hPSCs according to published methods and first test in different proportions (0.5-4%) with our kidney cells in the conventional model (macro-organoids). We will ask whether macrophages can enhance kidney organoid developmental maturity. Currently organoid development is also limited by poor vascular development. The immaturity of vascular elements and large diffusion pathway contributes to deterioration in the organoid core. We will use microfluidic culture, with custom printed plates from the Manchester Royce Institute, and bioprinting of organoids in collaboration with Cellink, to generate a more reproducible system of perfused 1-10K cell micro-organoid arrays. Our scaled down organoids will be printed in novel supportive soft hydrogels with inclusion of macrophages. Reproducible microfluidic culture of bioprinted, size-limited, organoid arrays will allow quality control and extended kidney development. In future, this will greatly enhance human patient- or gene edited-iPSC disease models, replacing the need for transgenic animals for later authentic development/disease phenotypes. The platform will be applicable to other organoid models for testing drugs targeting aberrant molecular pathways.