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

Abstract

Human iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we used single cell RNA-Seq (scRNA-Seq) to profile 415,775 cells to show that organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes were largely reproducible across iPSC lines, time points, protocols, and replicates, cell proportions were variable between different iPSC lines. Off-target cell proportions were the most variable. Prolonged in vitro culture did not alter cell types, but organoid transplantation under the mouse kidney capsule diminished offtarget cells. Our work shows how scRNA-seq can help score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.

Kidney organoids: accurate models or fortunate accidents

“…Scale bars, 50 µM. Wu et al 2018;Combes et al 2019b;Phipson et al 2019;Subramanian et al 2019). We focus on our own protocol to illustrate how kidney organoid differentiation is performed and how we have validated the identity of component cell types within these structures.…”

“…They went on to map the expression of genes previously associated with kidney disease, suggesting significant potential for kidney organoids to recapitulate such transcriptional networks. A much larger single cell study (440,000 single cells) comparing the same two protocols is now available as preprint (Subramanian et al 2019) and similarly concluded that kidney organoids contain cell clusters recognizable as anticipated cell types within the developing kidney. They also concluded that the protocols are robust and reproducible by comparing different cell lines differentiated using the same methods (Subramanian et al 2019).…”

“…A much larger single cell study (440,000 single cells) comparing the same two protocols is now available as preprint (Subramanian et al 2019) and similarly concluded that kidney organoids contain cell clusters recognizable as anticipated cell types within the developing kidney. They also concluded that the protocols are robust and reproducible by comparing different cell lines differentiated using the same methods (Subramanian et al 2019). More recently, organoids generated using the Freedman protocol have been analyzed identifying the expression of podocyte genes known to be associated with glomerular disease (Harder et al 2019).…”

“…Cell types within kidney organoids were initially defined based on morphology and expression of established markers of mouse renal cell types by immunofluorescence. The application of single-cell profiling has enabled a deeper analysis of kidney organoid cell types, dramatically expanding the complement of markers used to identify organoid renal cell types and identifying varying proportions of off-target neural, glial, muscle progenitor, and melanocyte populations (Wu et al 2018;Combes et al 2019b;Harder et al 2019;Howden et al 2019;Subramanian et al 2019). Pseudotime analysis of organoid nephron cell types suggests that organoid nephron formation replicates the expected trajectory from nephron progenitor to podocyte and tubular endpoints (Combes et al 2019b).…”

“…In one study, based on the use of fluorescent reporter lines, multiphoton live imaging showed murine blood-flow through organoid glomeruli with evidence of human endothelial cell contribution to the neovasculature (van den Berg et al 2018). Recent data single-cell profiling such organoids after transplantation claim to show a reduction in off-target populations after transplantation (Subramanian et al 2019). Given the capacity for organoid to spontaneously vascularise, likely as a result of VEGF expression by the forming podocytes in the graft, this raises the prospect of generating hPSC-derived replacement tissue for the provision of renal replacement.…”