The podocytes within the glomeruli of the kidney maintain the filtration barrier by forming interdigitating foot processes with intervening slit diaphragms, disruption in which results in proteinuria. Studies into human podocytopathies to date have employed primary or immortalised podocyte cell lines cultured in 2D. Here we compare 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids with conditionally immortalised human podocyte cell lines, revealing improved podocyte-specific gene expression, maintenance in vitro of polarised protein localisation and an improved glomerular basement membrane matrisome compared to 2D cultures. Organoid-derived glomeruli retain marker expression in culture for 96 h, proving amenable to toxicity screening. In addition, 3D organoid glomeruli from a congenital nephrotic syndrome patient with compound heterozygous NPHS1 mutations reveal reduced protein levels of both NEPHRIN and PODOCIN. Hence, human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modelling of human podocytopathies and screening for podocyte toxicity.

Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here we apply extrusion-based 3D cellular bioprinting to deliver rapid and high throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, 3D bioprinting enabled precise manipulation of biophysical properties including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitated the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production deliver improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.

to clarify that statements made comparing this and previous studies relate to the published analyses of data within those studies and not the detection of genes within the datasets themselves. The changes made are shown below and both the online full-text and PDF versions have been updated. In the Introduction, the following statement was changed: Original However, existing datasets have apparently not provided the transcriptional depth to identify the signalling pathways that operate within the human fetal kidney, and fail to detect several known ligand and receptor expression patterns in mouse. Corrected The analyses performed on existing datasets have not comprehensively identified the signalling pathway components known to be operating within the mouse fetal kidney. In the Discussion, the following statements were changed: Original For example, although >20,000 cells were profiled at P1 (Adam et al., 2017), several known signalling molecules with functionally validated roles in the nephrogenic niche such as Gdnf, Fgf20, Fgf9, Bmp7, Wnt4 and Fgf8 were not detected in that analysis, precluding further insight into signalling interactions. Corrected Although >20,000 cells were profiled at P1 (Adam et al., 2017), several known signalling molecules with functionally validated roles in the nephrogenic niche were not highlighted in that analysis. Original The improved resolution of gene expression in our study may be due to sequencing depth (∼3000 genes detected per cell), biological replication and differential expression analysis with the edgeR method, which has recently been shown to be a top performer in a comparison of 36 differential expression analysis methods for scRNA-seq data (Soneson and Robinson, 2018). Corrected The improved analysis of signalling interactions provided in this study may be due to sequencing depth (∼3000 genes detected per cell), biological replication and differential expression analysis with the edgeR method, which has recently been shown to be a top performer in a comparison of 36 differential expression analysis methods for scRNA-seq data (Soneson and Robinson, 2018).

The utility of human pluripotent stem cell–derived kidney organoids relies implicitly on the robustness and transferability of the protocol. Here we analyze the sources of transcriptional variation in a specific kidney organoid protocol. Although individual organoids within a differentiation batch showed strong transcriptional correlation, we noted significant variation between experimental batches, particularly in genes associated with temporal maturation. Single-cell profiling revealed shifts in nephron patterning and proportions of component cells. Distinct induced pluripotent stem cell clones showed congruent transcriptional programs, with interexperimental and interclonal variation also strongly associated with nephron patterning. Epithelial cells isolated from organoids aligned with total organoids at the same day of differentiation, again implicating relative maturation as a confounder. This understanding of experimental variation facilitated an optimized analysis of organoid-based disease modeling, thereby increasing the utility of kidney organoids for personalized medicine and functional genomics.

Kidney organoids have potential uses in disease modelling, drug screening and regenerative medicine. However, novel cost-effective techniques are needed to enable scaled-up production of kidney cell types in vitro. We describe here a modified suspension culture method for the generation of kidney micro-organoids from human pluripotent stem cells. Optimisation of differentiation conditions allowed the formation of micro-organoids, each containing six to ten nephrons that were surrounded by endothelial and stromal populations. Single cell transcriptional profiling confirmed the presence and transcriptional equivalence of all anticipated renal cell types consistent with a previous organoid culture method. This suspension culture micro-organoid methodology resulted in a three- to fourfold increase in final cell yield compared with static culture, thereby representing an economical approach to the production of kidney cells for various biological applications.