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Commercial in Confidence UNVEILING the BIOLOGY OF Commercial in Confidence UNVEILING THE BIOLOGY OF COLLECTING DUCT EPITHELIUM REPAIR AND REGENERATION Pamela Kairath Oliva Department of Paediatrics Faculty of Medicine, Dentistry, and Health Sciences The University of Melbourne Australia Submitted in total fulfilment of the requirement of the degree of Doctor of Philosophy December 2017 Commercial in Confidence ABSTRACT The healthy functioning of the kidney requires the orchestrated action of its two functional units, nephrons and the collecting duct system (CD). Therefore malfunction of either of these two essential compartments can lead to kidney failure. Diseases affecting the collecting duct system (CD), congenital abnormalities of the kidney and urinary tract (CAKUTs), are the most frequent cause of End-Stage Renal Disease (ESRD) in children. At this stage, the only available therapeutic options for treating kidney failure are dialysis or organ transplantation. Given the fact that only one in three patients will receive a transplant, and that dialysis comes with a high risk of mortality, the study of the mechanisms underlying the repair and regeneration of the collecting duct system is vital because it will facilitate new therapeutic strategies for treating kidney disease which are available to more people. The CD system originates from one of the two progenitor populations which give rise to the kidney, the ureteric bud (UB). The formation of the UB is a substantial part of the nephrogenesis process, and develops into a branched tree-like structure which will ultimately form the ducts of the urinary collecting system. This PhD thesis investigates recent concepts in normal kidney organogenesis, repair, and regeneration, and focuses on the CD system. In the first part of this research, we investigated the capacity of an endogenous kidney mesenchymal stem cell population (k-MSC), previously reported by our laboratory, which both arose from the collecting duct epithelium and also then integrated into the same compartment. To this end, we generated and examined the functional capacity of Pkd1 defective k-MSCs to trigger autosomal dominant polycystic kidney disease (ADPKD) into wild type mice. Given that micro-injection of double transgenic k-MSCsPkd1del2-4/TMTO+ did not produce significant evidence for cyst formation in the recipient mice, we therefore ruled out the possibility that k- MSCs represent a preferable population for effecting repair. Nevertheless, we showed the capacity to generate k-MSCs with mutant genes, which may prove useful as cellular models of human diseases. 2 Commercial in Confidence We then addressed the ambitious aim of recreating the ureteric bud population via the directed differentiation of human pluripotent stem cells (hiPSCs). Here we report a stepwise protocol for the differentiation of hiPSCs towards nephric duct (ND)/ureteric bud (UB) lineage. Optimisation cultures were established using fluorescently-tagged human iPSC lines as a readout of collecting duct differentiation cultures, which also provided a unique opportunity to trace cell lineage within organoids. Cells subjected to this differentiation protocol switched the expression of key genes thought to be of ND/UB lineage, and also showed the ability to epithelialise; this was seen using two- and three-dimensional approaches. The establishment of this methodology provides a valuable platform to now investigate the capacity of this epithelium to respond to a metanephric mesenchyme. We also anticipate that the use of this protocol will ultimately facilitate three-dimensional bio-printing and allow the recreation of larger kidney structures. This thesis has investigated areas currently applicable to regenerative medicine, including normal tissue development, turnover, repair, and regeneration, with a focus on the collecting duct epithelium. This research has concluded that k-MSCs do not represent a preferable population for effecting structural repair. It has also established the basis of a new methodology to recreate kidney tissue for therapeutic uses. 3 Commercial in Confidence DECLARATION I. The thesis comprises only original work towards the Doctor of Philosophy, II. Due acknowledgement has been made in the text of all other material used, III. The thesis is less than 100,000 words in length, exclusive of tables, figures, bibliographies and appendices. Pamela Kairath Oliva Signature: ____________________ December 2017 4 Commercial in Confidence PREFACE Pursuant to the regulations governing to the degree of Doctor of Philosophy at the University of Melbourne, I hereby submit that: Declaration by author: This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. Statement of parts of the thesis submitted to qualify for the award of another degree: None 5 Commercial in Confidence Publication during the candidature: 1. Little MH, Kairath P. Regenerative medicine in kidney disease. Kidney Int. 2016 Aug; 90(2):289-99. doi: 10.1016/j.kint.2016.03.030. Epub 2016 May 24. Review (Attached in appendix 1) 2. Little MH, Kairath P. Does renal repair recapitulate kidney development? J Am Soc Nephrol. 2017 Jan; 28(1):34-46. doi: 10.1681/ASN.2016070748. Epub 2016 Oct 26. Review (Attached in appendix 2) Patents: 1. Kairath P, Takasato M, Er X. P, Little MH. Directed differentiation of human pluripotent stem cells to Nephirc duct/Ureteric bud like-cells. Provisional submission in process. (Chapter 3 and 4). 6 Commercial in Confidence Contributions by others to the thesis: Prof. Melissa Little contributed to concept and design of the projects as well as interpretation of experimental data. She also performed GO analysis of single cell RNA-seq and provided critical proofreading for the thesis manuscript. Moreover, she provided constant and valued supervision. Dr. Joan Li contributed to design, analysis and interpretation of experimental data. She also performed microinjections and assistance in the analysis of k-MSCs (GFP+) (Chapter 2). Dr. Jessica Vanslambrouck provided advice in techniques and supervision meetings. Dr. Santhosh Kumar provided advice in techniques and supervision meetings. He also performed cell dissociation for RET/tdTomato+ organoid (Chapter 4). Dr. Sara Howden contributed to the generation of 1502.2 GATA3:m-Cherry and 2429 GATA3:m-Cherry iPSC lines (Chapter 3 and 4). Pei Er provided training and technical advice for the maintenance and banking iPSCs (Chapter 3 and 4). Irene Ghobrial provided technical assistance for iPSCs banking (Chapter 3 and 4). Luke Zapia performed analysis for single cell RNA-seq (Chapter 4). Dr. Alexander Combes performed GO analysis for single cell RNA-seq (Chapter 4). Virginia Nink conducted FACS sorting at University of Queensland (Chapter 2). Dr. Matthew Burton and Paul Lai conducted FACS sorting at Murdoch Children’s Research Institute (Chapter 3 and 4). 7 Commercial in Confidence ACKNOWLEDGEMENTS Firstly, I would like to thank my supervisor Prof. Melissa Little for her valuable mentoring, guidance, support, and time throughout my PhD journey. I am deeply grateful for her constant debate and criticism of the design of experiments and the analysis of the data, for the opportunities she gave me to assume new challenges, and for always encouraging me to do my best, which has not only sharpened my analytical skills but also helped me to discover my own research interests. Thank you to the members of the Little Lab, both past and present, for your help and companionship. Special thanks to Dr. Minoru Takasato for giving me the first directions in developmental and stem cell biology. Also, thanks to Pei Er for training me up in stem cell culture, and for her friendship over the years. I would also like to thank the co-supervisors who helped me during different stages of my PhD- Dr. Joan Li, Dr. Jessica Vanslambrouck, and Dr. Santhosh Kumar- for insightful discussions and feedback during PhD meetings. I would also like to thank the University of Queensland, where I did the first part of my PhD studies. Special mentions to Amanda Carozzi for her constant support, not only in the beginning but also during the transference of my candidature to University of Melbourne. I am also grateful to my committee members, Dr. Shireen Lamande and Dr. John Bateman, for their advice and guidance upon my arrival at the University of Melbourne. Last but not least, I would like to thank my family for their long-distance support and unconditional love. My most sincere and profound gratitude goes to my husband, Rodrigo, not only for believing in my capabilities and accompanying me to venture overseas to take up this challenge, but also for the endless encouragement, care, and love throughout
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