Organoid single cell profiling identifies a transcriptional signature of glomerular disease

Harder, Jennifer L.; Menon, Rajasree; Otto, Edgar A.; Zhou, Jian; Eddy, Sean; Wys, Noel L.; O’Connor, Christopher; Luo, Jinghui; Nair, Viji; Cebrián, Cristina; Spence, Jason R.; Bitzer, Markus; Troyanskaya, Olga G.; Hodgin, Jeffrey B.; Wiggins, Roger C.; Freedman, Benjamin; Kretzler, Matthias

doi:10.1172/jci.insight.122697

Cited by 77 publications

(53 citation statements)

References 52 publications

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“…Primary output of unsupervised clustering by Seurat v3 yielded 13 clusters ( Figure 2C, S3A), seven of which were determined to consist of mesenchymal cells, while the others included glomerular epithelial cells at early and more mature stages, proximal and distal tubule, cycling cells, neurons, and endothelial cells ( Figure 2D-E, Figure S3A) as determined by canonical marker genes ( Figure 2D). The abundance of mesenchyme versus epithelial lineages in these whole-well differentiations is high but within the expected range for different iPSC lines and batches of organoids 7,14 . These clusters were present in both G0 and G1 organoids ( Figure S3B).…”

Section: Resultsmentioning

confidence: 70%

“…To model the G1 variants within a kidney context, we differentiated the G1 and G0 lines into kidney organoids using an adherent culture protocol we have previously established ( Figure 1B) [5][6][7]13,14 . Both G0 and G1 iPSC lines differentiated into kidney organoids without major structural differences, expressing markers of nephron structure including PODXL in glomerular epithelial cells, LTL in the proximal tubule, and E-cadherin in the distal tubule, in appropriately patterned and contiguous segments ( Figure 1C).…”

Section: Resultsmentioning

confidence: 99%

“…Using CRISPR-Cas9 mediated genome editing, we engineered iPSCs homozygous for the G1 risk allele and differentiated these cells into threedimensional kidney organoids. To evaluate cell-type specific effects of the APOL1 high-risk genotype, we also performed single-cell RNA-sequencing (scRNA-seq), which we and others have previously leveraged to uncover novel biology of how cell-specific phenotypes contribute to kidney development or disease in organoids and other models [10][11][12][13][14] . Here we present the application of genome-edited iPSC-derived kidney organoids and single-cell transcriptomics to profile APOL1-mediated effects on kidney organoids relevant to disease processes.…”

Section: Introductionmentioning

confidence: 99%

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ProfilingAPOL1Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

Liu

Radmanesh

Chung

et al. 2019

Preprint

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Running Title: APOL1 Variant Organoid scRNA-seq Word Count: 205 (Abstract), 1736 (Main Text, excluding Methods)ABSTRACT Background DNA variants in APOL1 associate with kidney disease, but the pathophysiological mechanisms remain incompletely understood. Model organisms lack the APOL1 gene, limiting the degree to which disease states can be recapitulated. Here we present single-cell RNA sequencing (scRNAseq) of genome-edited human kidney organoids as a platform for profiling effects of APOL1 risk variants in diverse nephron cell types. MethodsWe performed footprint-free CRISPR-Cas9 genome editing of human induced pluripotent stem cells (iPSCs) to knock in APOL1 high-risk G1 variants at the native genomic locus. iPSCs were differentiated into kidney organoids, treated with vehicle, IFN-g, or the combination of IFN-g and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared to isogenic G0 controls. ResultsBoth G0 and G1 iPSCs differentiated into kidney organoids containing nephron-like structures with glomerular epithelial cells, proximal tubules, distal tubules, and endothelial cells.Organoids expressed detectable APOL1 only after exposure to IFN-g. scRNA-seq revealed cell type-specific differences in G1 organoid response to APOL1 induction. Additional stress of tunicamycin exposure led to increased glomerular epithelial cell dedifferentiation in G1 organoids. ConclusionsSingle-cell transcriptomic profiling of human genome-edited kidney organoids expressing APOL1 risk variants provides a novel platform for studying the pathophysiology of APOL1mediated kidney disease.

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Section: Resultsmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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ProfilingAPOL1Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

Liu

Radmanesh

Chung

et al. 2019

Preprint

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“…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).…”

Section: The Balance Between Reproducibility and Cellular Complexitymentioning

confidence: 99%

“…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).…”

Section: Examining the Transcriptional Congruence Between Kidney Orgamentioning

confidence: 99%

Kidney organoids: accurate models or fortunate accidents

2019

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There are now many reports of human kidney organoids generated via the directed differentiation of human pluripotent stem cells (PSCs) based on an existing understanding of mammalian kidney organogenesis. Such kidney organoids potentially represent tractable tools for the study of normal human development and disease with improvements in scale, structure, and functional maturation potentially providing future options for renal regeneration. The utility of such organotypic models, however, will ultimately be determined by their developmental accuracy. While initially inferred from mouse models, recent transcriptional analyses of human fetal kidney have provided greater insight into nephrogenesis. In this review, we discuss how well human kidney organoids model the human fetal kidney and how the remaining differences challenge their utility.

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In Situ Detection of Kidney Organoid Generation From Stem Cells Using a Simple Electrochemical Method

et al. 2022

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Organoids that mimic the structural and cellular characteristics of kidneys in vitro have recently emerged as a promising source for biomedical research. However, uncontrollable cellular heterogeneity after differentiation often results in the generation of off-target cells, one of the most challenging issues in organoid research. This study proposes a new method that enables the real-time assessment of kidney organoids derived from stem cells. When placed on a conductive surface, these organoids generate unique electrochemical signals at ≈0.3 V with intensities proportional to the amount of kidney-specific cell types. Off-target cells (i.e., non-kidney cells) produce an electrical signature at 0 V that is distinguishable from other surrounding cell types, enabling non-destructive assessment of both the differentiation, and maturation levels of kidney organoids. The developed platform can be applied to other types of organoids and is thus highly promising as a tool for organoid-based drug screening, toxicity assessment, and therapeutics.

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Organoid single cell profiling identifies a transcriptional signature of glomerular disease

Cited by 77 publications

References 52 publications

ProfilingAPOL1Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

ProfilingAPOL1Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

Kidney organoids: accurate models or fortunate accidents

In Situ Detection of Kidney Organoid Generation From Stem Cells Using a Simple Electrochemical Method

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