Paediatrics (RCH) - Theses

Permanent URI for this collection

Search Results

Now showing 1 - 1 of 1
  • Item
    Thumbnail Image
    Modelling Inherited Kidney Diseases with Kidney Organoids Derived by Directed Differentiation of Patient Induced Pluripotent Stem Cells
    Forbes, Thomas Alexander ( 2019)
    Genetic kidney diseases are a heterogeneous group of disorders with varying phenotypes dependent on the affected nephron segment. Next generation sequencing has increased our appreciation of the breadth of gene variants associated with these diseases. It has also identified large numbers of variants of unknown significance (VUS), which require functional genomic validation. There is an unmet need for novel therapies for genetic kidney diseases as most invariably progress to dialysis or transplantation without any form of targeted treatment. Laboratory based research of genetic kidney disease requires the recapitulation of a disease phenotype in animal and/or in vitro cellular disease models. Interspecies variation in anatomy, physiology and gene function limits the translation of animal models to human disease and clinical care. Classical two dimensional cell cultures lack the complexity and intercellular cross-talk of in vivo three dimensional tissue. Kidney organoids are three dimensional, miniature, multicellular, human, in vitro micro-tissues, offering distinct disease modelling advantages over other models. Furthermore, kidney organoids can be regenerated from induced pluripotent stem cells (iPSC) reprogrammed from patients with genetic kidney disease, potentially providing outcomes with personalised clinical relevance. As a novel platform, the capabilities and limitations of kidney organoids as disease models are not well understood. By differentiating and characterising kidney organoids from the iPSC of patients with inherited kidney diseases, this thesis aims to explore the application of kidney organoids to disease modelling. As proof of concept, kidney organoids were first generated from iPSC reprogrammed from a patient with compound heterozygous variants in IFT140, an already validated nephronophthisis (NPHP) genotype. An isogenic control was generated by precision CRISPR-Cas9 gene editing. In this project, differential primary ciliary morphology within organoid tubules and transcriptional profiling of organoid epithelium validated the ability of the organoids to model genetic disease. Attempts were then made to validate novel, candidate variants for other pedigrees with unresolved trio whole exome sequencing. In a proband with clinically suspected NPHP, DNAH5 was selected as a candidate gene, despite previously association with a motile ciliary phenotype. In this project, kidney organoids were unable to validate the patient variant as pathogenic. In addition, a number of lessons were learned regarding the necessary variant curation process prior to making a commitment to modelling with kidney organoids. In the final chapter, kidney organoids validated a novel genotype for the glomerular disease steroid resistant nephrotic syndrome, via international collaboration with the laboratory of Prof Friedhelm Hildebrandt. Glomeruli within kidney organoids differentiated from iPSC expressing a patient-derived, homozygous variant in NOS1AP, displayed aberrant development, increased podocyte apoptosis and reduced expression of PAR polarity proteins. Together these projects demonstrate the strengths and challenges of using kidney organoids as models of inherited renal disease. Kidney organoids stand to complement animal and 2D unicellular disease models rather than replace them. We proposed that patient-derived kidney organoids are best placed to model paediatric onset kidney diseases with the future potential of providing personalised therapeutic screening.