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Development of human induced pluripotent stem cell derived kidney organoids for disease modelling of diabetic nephropathy with chemical mediators and CRISPR-Cas9 genome editing

Dalliston, Thomas William Mark; (2019) Development of human induced pluripotent stem cell derived kidney organoids for disease modelling of diabetic nephropathy with chemical mediators and CRISPR-Cas9 genome editing. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Diabetic nephropathy (DN) is a leading cause of morbidity and mortality worldwide, yet current disease models are failing to meet the need for new efficacious drugs. Kidney organoids differentiated from human induced pluripotent stem cells (hiPSCs) have recapitulated much of the kidney’s structural complexity, but currently reach only foetal-like maturity. Organ specific extracellular matrix (ECM) has been shown to promote differentiation and maturation of relevant cell types. Therefore, we aimed to create a three dimensional (3D), decellularised human kidney ECM-gel (K-gel) based kidney organoid differentiation system. We aimed to demonstrate the utility of the system for DN modelling by exposing the organoids to disease mediators (including hyperglycemia, angiotensin II and TGF-β), and through growth of organoids from hiPSCs modified with CRIPSR-Cas9 at genes linked to DN (UMOD and TIMP3). HiPSC derived kidney organoids differentiated in a Matrigel®-based 3D sandwich system showed segmented 3D expression of mature nephron markers and had the ability to uptake dextran at functional proximal tubular transporters. Initial differentiations using decellularised kidney ECM-hydrogel (K-gel) were unsuccessful in deriving typical organoids. Human kidneys decellularised by perfusion, with SDS, ddH2O and NaCl, proved more successful than immersion protocols for removal of DNA and preservation of ECM and renal microarchitecture. K-gel was able to form solidified layers and support hiPSC growth. TIMP3 and UMOD knock-out CRISPR-Cas9 plasmids were created, but genetically modified hiPSC lines were not achieved. Kidney organoids exposed to disease mediators displayed a DN specific gene expression profile of NOTCH1 upregulation concomitant with NOTCH3 downregulation; but did not show a robust fibrosis or EMT gene expression profile. With further refinement the protocols developed herein will enable the modelling of DN, or other kidney diseases, in an in vitro 3D renal organoid model generated through differentiation of hiPSCs in a decellularised human kidney derived ECM gel-based system.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Development of human induced pluripotent stem cell derived kidney organoids for disease modelling of diabetic nephropathy with chemical mediators and CRISPR-Cas9 genome editing
Event: UCL
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Surgery and Interventional Sci
URI: https://discovery.ucl.ac.uk/id/eprint/10080563
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