Using human induced pluripotent stem cells to model cystinosis

Abstract

Cystinosis is a rare lysosomal storage disease caused by mutations in the CYSTINOSIN (CTNS) gene, encoding a cystine transporter located on the lysosomal membrane. Cystinosis leads to an accumulation of cystine within lysosomes in most cells of the body, however the kidney is the main organ affected. For poorly understood reasons, renal proximal tubule (PT) cells lose their functionality and degenerate in cystinosis, forming a characteristic “swanneck” lesion that leads to kidney failure before 10 years of age. Although, a drug-based therapy is available for cystinotic patients, the progression to kidney failure can only be delayed not prevented. A major challenge in the cystinosis field is the variation in cellular phenotypes between cell-based models of cystinosis and differences in the presentation of the disease in the mouse knockout. As such, there is no consensus on the pathways responsible for the cause of the disease. In order to develop a better human cell-based model of cystinosis, our lab has generated human pluripotent stem cells from a cystinosis patient (CTNS-iPS). The aim of this thesis is to characterise the cellular phenotype of CTNS-iPS cells and to develop protocols to differentiate these cells into cystinotic PT cells for further study. CTNS-iPS cells were found to exhibit classic cystinotic defects including increased cystine levels, an altered ratio of reduced to oxidised glutathione (indicative of oxidative stress), mitochondria with a fragmented morphology, and enlarged late endosomes/lysosomes that cluster in a perinuclear region. Approximately 25% of CTNS-iPS cells display massive multivesicular body-like vesicles that contain numerous electron-lucent vesicles when visualised by electron microscopy. Two protocols were established to convert iPS cells into PT cells. In the first, iPS cells were treated with Activin A, the Wnt agonist CHIR99021 (CHIR) and BMPs and then purified by FACS, based on binding to the PT cell specific lectin FITC-conjugated lotus tetragonolobus lectin. The resulting PT-like cells expressed numerous PT markers, displayed an epithelial morphology and could be maintained in culture for up to 28 days without losing their identity. In the second protocol, it was found that treating iPS cells with high levels of CHIR resulted in the formation of 3D tubules. When these structures were transferred into suspension bioreactors they grew into avascular kidney organoids comprised of 3-5 nephrons with renal tubules consisting of distinct proximal and distal segments and primitive glomeruli containing podocytes. Using this protocol, CTNS-iPS cells were differentiated into cystinotic kidney organoids and found to accumulate large multivesicular body-like vesicles in the renal tubules but did not display morphological signs of the swan-neck lesion. Taken together the data presented in this thesis shows that CTNS-iPS cells provide a new human cell-based model to study cystinosis. Development of PT-like monolayer and kidney organoid assay will significantly enhance our understanding of disease pathways in cystinosis. Kidney organoids provide novel means to study development of the swan-neck lesion and PT cell dysfunction in vitro and offer the potential for development of new and improved treatment of the disease.