ESPN 54th Annual Meeting

ESPN 2022


 
Generation of ARPKD kidney organoids using patient-specific induced pluripotent stem cells
THERESA LEONIE FLUHR 1 MANSOUREH TABATABAEIFAR 1 FRANZ SCHAEFER 1

1- UNIVERSITY CLINIC HEIDELBERG
 
Introduction:

Autosomal-recessive polycystic kidney disease (ARPKD) is a severe paediatric kidney disorder characterised by the early development of multi-cystic kidneys and leading to a gradual decline in kidney function. Currently, the only treatment options for the resulting end stage renal disease remain dialysis or kidney transplantation.

Patient-derived organoids grown from our two new patient-specific iPSC lines can be used as a new approach for modelling ARPKD, understanding underlying pathomechanisms and identifying cyst-modifying compounds. 

Material and methods:

Peripheral blood mononuclear cells (PBMCs) form two patients with different PKHD1 mutations were used to generate patient-specific induced pluripotent stem cells according to the Yamanaka protocol. The iPSCs were subsequently grown into patient-derived kidney organoids using the StemDiff Kidney Organoid Kit (Stemcell Technologies). For cyst formation, PKA signalling inside the cells was amplified by treating the organoids with forskolin according to a protocol of Low et al. Cyst development was monitored by brightfield microscopy and compared to organoids derived from wildtype iPSCs functioning as a healthy control. The development of organ-specific cell types such as podocytes, tubular epithelial cells or endothelial cells was observed via immunofluorescence staining. 

Results:

 We were able to prove the development of glomeruli, proximal and distal tubules as well as vascular networks via immunofluorescence staining. While undergoing treatment with forskolin, cyst formation was visible in the patient-derived ARPKD organoids, whereas control organoids seemed to be nearly unaffected by the PKA induction.

Conclusions:

 Our patient-derived organoids are able to depict kidney organogenesis in vitro developing organ-specific structures and cell-types. Furthermore, they can be used as disease-in-a-dish models in which an ARPKD-like phenotype was inducible. This could be the basis of screening cyst-modifying compounds and developing new treatment strategies. The experimental setup is currently being adapted to enable a high throughput screening process.