Advances in kidney organoid generation; insights gained through novel human iPS cell reporter line approaches and gene expression profiling
- Murdoch Children’s Research Institute, Parkville, Melbourne, 3052.
- Department of Pediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, 3052, Australia.
Development of directed differentiation techniques and stem cell-derived organoid culture methods are revolutionising the fields of regenerative medicine, disease modelling and drug screening. Based on our knowledge of human kidney organogenesis, we recently established a robust protocol to direct the differentiation of human pluripotent stem cells towards a kidney fate within complex, 3D kidney organoids (Takasato et al., 2014, 2016). These organoids display evidence of differentiation into many of the compartments present in early embryonic kidney, including nephrons, collecting duct, vasculature and interstitium. However, despite these impressive similarities, the structures within kidney organoids are still developmentally immature and they lack the continuous growth pattern seen in embryonic kidneys in vivo. Whilst we know that successful differentiation of pluripotent stem cells towards kidney progenitors and subsequent nephron patterning are critically reliant on the culture media and growth conditions, progress in this area is dependent on our ability to interrogate individual cell types and modify organoid conditions accordingly. Using CRISPR/Cas9-mediated gene-editing, we have generated a range of induced pluripotent stem cell lines carrying fluorescent reporters targeted to genes that mark specific kidney cell types and nephron segments, including nephron progenitors, proximal and distal nephron and collecting duct, enabling real-time monitoring of differentiation. Through the use of these reporter lines, in combination with detailed gene expression analyses, we have now been able to examine organoid development more deeply than ever before, enabling the perturbation of culture conditions to improve organoid growth and nephron patterning.