A CRISP(e)R view on kidney organoids allows generation of an induced pluripotent stem cell–derived kidney model for drug discovery

Abstract

Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.

Reporter‐based fate mapping in human kidney organoids confirms nephron lineage relationships and reveals synchronous nephron formation

“…SIX2 + cells emerged soon after organoid formation and persisted until the termination of differentiation at day 25. Although this contrasts to other previously described differentiation protocols where a rapid loss of SIX2 + cells is observed soon after the formation of 3D kidney organoids [16], our findings are consistent with a recent study that employed a similar SIX2-EGFP reporter iPSC line and kidney organoid protocol, where SIX2 expression also persisted until the termination of differentiation [27]. Importantly, our findings do not support their hypothesis that the presence of these cells definitively represents a progenitor niche that may supply the organoid with more mature cells over time.…”

Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics

“…First, we must establish organoid reproducibility: the comparability and range of variability in cellular composition and state between different iPSC lines from normal individuals across replicates and protocols (building on previous efforts to draw comparisons between iPSC and embryonic stem cell (ESC) derived organoids (Wu et al, 2018) or bulk RNASeq data comparing iPSC derived organoids (Phipson et al, 2019)). This is critically important, because individual patient iPSCs offer significant advantages over ESCs for precision medicine and drug development projects (Boreström et al, 2018;Burrows et al, 2016;Takasato et al, 2016b). Second, we must define their faithfulness: how well organoids across many iPSC lines recapitulate kidney development and disease-associated genes at single cell resolution.…”

Kidney organoids: accurate models or fortunate accidents

“…The individual characterization of human renal cell types within organoids is being assisted via the development of fluorescent reporter tools generated using CRISPR/Cas9 gene editing (Sharmin et al 2016;Boreström et al 2018;Hale et al 2018;Howden et al 2018;van den Berg et al 2018;Vanslambrouck et al 2019). By targeting fluorescent reporter proteins to the loci of cell type-specific genes, it has been possible to visualize these cell types within the organoids in vitro (Sharmin et al 2016;Boreström et al 2018) and in vivo after transplantation (van den Berg et al 2018) as well as isolate and characterize individual cell types from within organoids using fluorescence activated cell sorting ( Fig. 3I-N; Vanslambrouck et al 2019).…”

“…We have recently used extrusion bioprinting to automate the process of generating a micromass culture at the stage of IM, facilitating the generation of large numbers of identical kidney organoids with a very low coefficient of variation as is required for screening (Higgins et al 2018). Such approaches have the potential of modification for high-content 3D screening when coupled with a rapid readout such as can be conferred via the use of fluorescent reporter hPSC lines (Boreström et al 2018;Howden et al 2018;Vanslambrouck et al 2019).…”