Modeling human genetic kidney malformations caused by HNF1B mutation using pluripotent stem cells

UoM administered thesis: Phd

  • Authors:
  • Tengku Muhamad Faris Syafiq


Some human renal tract malformations (RTMs) are caused by mutations of genes active during formation of the metanephric kidney, so these are direct outcomes of perturbed kidney differentiation. Indeed, classic dissections by Edith Potter, 60 years ago, showed human dysplastic kidney cysts are dilated segments of poorly branched collecting ducts derived from the ureteric bud (UB). How these genes drive specific stages of human kidney development is unclear. Moreover, the precise biological aberrations in human RTMs associated with defined mutations are unknown. Critically, for translational medicine, such insights would facilitate designing novel therapies to enhance differentiation. One can not necessarily extrapolate from animal studies (i.e. mouse or zebrafish) due to differences in metabolic pathways and drug metabolites that can lead to variation in efficacy and toxicity so human models are urgently required. Recently, several new pluripotent stem cell (PSCs) protocols have been developed to generate human cells of the major kidney lineages, generating nephrons and collecting tubule structures. These protocols have been refined in Professor Kimber’s Laboratory (Takasato et al., 2016). Kidney disease is a major problem for health services, and so it may be possible to develop genetically defined RTM models using PSCs differentiated to kidney progenitors. Such model may be used to study pathobiology and design of new biological therapies. RTM patients with ‘renal cysts and diabetes syndrome’ (RCAD) caused by heterozygous hepatocyte nuclear factor 1-beta (HNF1B) mutations, identified by Professor Woolf, are ideal subjects for this research. HNF1B mutation can cause dysplastic kidney malformations containing poorly branched collecting ducts terminating in cysts, and lack of functional nephrons. I hypothesise that hPSCs carrying HNF1B mutation can be differentiated towards nephron progenitors in vitro expressing kidney transcriptional genes. These progenitors are expected to differentiate into a more complex 3D structure containing multiple cell types that can self-organise thereby can be used to recapitulate some morphological and functional aspect of the kidney as seen in the patient with cystic kidney disease. Towards this aim, I derived hiPSC lines from two siblings each born with bilateral cystic dysplastic kidneys and each harbouring heterozygous HNF1B mutation. I differentiated unaffected and mutant hiPSCs by inducing them to form kidney precursors over 7 days in 2D culture (CHIR99021 followed by FGF9/heparin), then pulsing them with CHIR99021 and maintaining them in 3D on transwells at an air-media interface for 18 days. Here, I reported that de novo generation of 3D kidney organoids in vitro via directed differentiation of HNF1B-derived iPSC lines resulted in lesser degree of structure complexity, increased diameter per organoid and lower expression of key kidney marker genes as compared to healthy organoids that contained epithelial nephron-like structures expressing markers of podocyte (WT1, NPHS1, NPHS2 and SYNPO) and surrounded by a Bowman space and capsule that resembles nephron in vivo. Deeper in the generated kidney organoid, I detected a variety of tubules, often HNF1B+/CDH1+ and PAX2+/CDH1+. Some were most likely to be in the ureteric bud/collecting duct lineage (e.g. GATA3+/CDH1+), while others were proximal tubules (HNF1A+/CUBN+) or loops of Henle (UMOD+) in the nephron lineage. Functional assay on CUBN+ proximal tubules revealed transporter capability to facilitate uptake of organic anion, 6CF but not cations, intracellularly in both HNF1B-affected and healthy organoids. Also, challenging organoids with cAMP has demonstrated tubule dilatation which is a normal feature in healthy organoids and the fact that it is severely decreased in HNF1B-affected organoids which indicated that those nephron tubules, although present, are lacking in functionality. To conclude, this research has partially proved the hypothesis that hPSCs can be utilised to study early kidney developmental program. The results has indicated phenotypic and functional aberrations in HNF1B-derived organoids that can be optimised in near future thereby making them appropriate models to study molecular and cellular pathobiology with a view to finding new therapies.


Original languageEnglish
Awarding Institution
Award date1 Aug 2020