SAT-118 COMPARATIVE TRANSCRIPTOMIC ANALYSIS OF CULTURED PODOCYTES AND HUMAN KIDNEY ORGANOIDS AT SINGLE-CELL RESOLUTION

AbstractHuman 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 profile four human iPSC lines for a total of 450,118 single cells to show how organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes are largely reproducible across time points, protocols, and replicates, we detect variability in cell proportions between different iPSC lines, largely due to off-target cells. To address this, we analyze organoids transplanted under the mouse kidney capsule and find diminished off-target cells. Our work shows how single cell RNA-seq (scRNA-seq) can 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.

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

10.1101/516807 ◽

2019 ◽

Cited By ~ 4

Author(s):

Ayshwarya Subramanian ◽

Eriene-Heidi Sidhom ◽

Maheswarareddy Emani ◽

Nareh Sahakian ◽

Katherine Vernon ◽

...

Keyword(s):

Single Cell ◽

Human Kidney ◽

Cell Types ◽

Mouse Kidney ◽

Target Cells ◽

Rna Seq ◽

Kidney Capsule ◽

Human Ipsc ◽

Kidney Organoids

AbstractHuman 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 off-target 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.

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High throughput single cell RNA-seq of developing mouse kidney and human kidney organoids reveals a roadmap for recreating the kidney

10.1101/235499 ◽

2017 ◽

Cited By ~ 10

Author(s):

Alexander N. Combes ◽

Belinda Phipson ◽

Luke Zappia ◽

Kynan T. Lawlor ◽

Pei Xuan Er ◽

...

Keyword(s):

Single Cell ◽

Kidney Tissue ◽

Human Kidney ◽

Population Based ◽

Mouse Kidney ◽

Rna Seq ◽

Kidney Morphogenesis ◽

Human Ipsc ◽

Kidney Organoids

AbstractRecent advances in our capacity to differentiate human pluripotent stem cells to human kidney tissue are moving the field closer to novel approaches for renal replacement. Such protocols have relied upon our current understanding of the molecular basis of mammalian kidney morphogenesis. To date this has depended upon population based-profiling of non-homogenous cellular compartments. In order to improve our resolution of individual cell transcriptional profiles during kidney morphogenesis, we have performed 10x Chromium single cell RNA-seq on over 6000 cells from the E18.5 developing mouse kidney, as well as more than 7000 cells from human iPSC-derived kidney organoids. We identified 16 clusters of cells representing all major cell lineages in the E18.5 mouse kidney. The differentially expressed genes from individual murine clusters were then used to guide the classification of 16 cell clusters within human kidney organoids, revealing the presence of distinguishable stromal, endothelial, nephron, podocyte and nephron progenitor populations. Despite the congruence between developing mouse and human organoid, our analysis suggested limited nephron maturation and the presence of ‘off target’ populations in human kidney organoids, including unidentified stromal populations and evidence of neural clusters. This may reflect unique human kidney populations, mixed cultures or aberrant differentiation in vitro. Analysis of clusters within the mouse data revealed novel insights into progenitor maintenance and cellular maturation in the major renal lineages and will serve as a roadmap to refine directed differentiation approaches in human iPSC-derived kidney organoids.

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Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

10.1101/780684 ◽

2019 ◽

Author(s):

Esther Liu ◽

Behram Radmanesh ◽

Byungha H. Chung ◽

Michael D. Donnan ◽

Dan Yi ◽

...

Keyword(s):

Gene Expression ◽

Kidney Disease ◽

Single Cell ◽

Human Kidney ◽

Cell Types ◽

Genomic Locus ◽

Glomerular Epithelial Cell ◽

Risk Variants ◽

Ifn Γ ◽

Kidney Organoids

ABSTRACTBackgroundDNA 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 (scRNA-seq) 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-γ, or the combination of IFN-γ 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-γ. 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 APOL1-mediated kidney disease.SIGNIFICANCE STATEMENTGaps persist in our mechanistic understanding of APOL1-mediated kidney disease. The authors apply genome-edited human kidney organoids, combined with single-cell transcriptomics, to profile APOL1 risk variants at the native genomic locus in different cell types. This approach captures interferon-mediated induction of APOL1 gene expression and reveals cellular dedifferentiation after a secondary insult of endoplasmic reticulum stress. This system provides a human cellular platform to interrogate complex mechanisms and human-specific regulators underlying APOL1-mediated kidney disease.

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Comparative analysis of kidney organoid and adult human kidney single cell and single nucleus transcriptomes

10.1101/232561 ◽

2017 ◽

Cited By ~ 9

Author(s):

Haojia Wu ◽

Kohei Uchimura ◽

Erinn Donnelly ◽

Yuhei Kirita ◽

Samantha A. Morris ◽

...

Keyword(s):

Single Cell ◽

Human Kidney ◽

Cell Types ◽

Great Promise ◽

Full Potential ◽

Diverse Range ◽

Adult Human ◽

Cell Diversity ◽

Single Nucleus ◽

Kidney Organoids

AbstractKidney organoids differentiated from human pluripotent stem cells hold great promise for understanding organogenesis, modeling disease and ultimately as a source of replacement tissue. Realizing the full potential of this technology will require better differentiation strategies based upon knowledge of the cellular diversity and differentiation state of all cells within these organoids. Here we analyze single cell gene expression in 45,227 cells isolated from 23 organoids differentiated using two different protocols. Both generate kidney organoids that contain a diverse range of kidney cells at differing ratios as well as non-renal cell types. We quantified the differentiation state of major organoid kidney cell types by comparing them against a 4,259 single nucleus RNA-seq dataset generated from adult human kidney, revealing immaturity of all kidney organoid cell types. We reconstructed lineage relationships during organoid differentiation through pseudotemporal ordering, and identified transcription factor networks associated with fate decisions. These results define impressive kidney organoid cell diversity, identify incomplete differentiation as a major roadblock for current directed differentiation protocols and provide a human adult kidney snRNA-seq dataset against which to benchmark future progress.

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Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics

Kidney360 ◽

10.34067/kid.0000422019 ◽

2020 ◽

Vol 1 (3) ◽

pp. 203-215

Author(s):

Esther Liu ◽

Behram Radmanesh ◽

Byungha H. Chung ◽

Michael D. Donnan ◽

Dan Yi ◽

...

Keyword(s):

Kidney Disease ◽

Single Cell ◽

Expression Patterns ◽

Human Kidney ◽

Model Organisms ◽

Additional Stress ◽

Genomic Locus ◽

Glomerular Epithelial Cell ◽

Risk Variants ◽

Kidney Organoids

BackgroundDNA variants in APOL1 associate with kidney disease, but the pathophysiologic 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 (scRNA-seq) 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-γ, or the combination of IFN-γ and tunicamycin, and analyzed with scRNA-seq to profile cell-specific changes in differential gene expression patterns, compared with 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-γ. 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 APOL1-mediated kidney disease.

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Single Cell Sequencing and Kidney Organoids Generated from Pluripotent Stem Cells

Clinical Journal of the American Society of Nephrology ◽

10.2215/cjn.07470619 ◽

2020 ◽

Vol 15 (4) ◽

pp. 550-556 ◽

Cited By ~ 3

Author(s):

Haojia Wu ◽

Benjamin D. Humphreys

Keyword(s):

Stem Cells ◽

Single Cell ◽

Pluripotent Stem Cells ◽

High Throughput Screening ◽

Kidney Development ◽

Toxicity Testing ◽

Human Kidney ◽

Cell Types ◽

Time Frame ◽

Kidney Organoids

Methods to differentiate human pluripotent stem cells into kidney organoids were first introduced about 5 years ago, and since that time, the field has grown substantially. Protocols are producing increasingly complex three-dimensional structures, have been used to model human kidney disease, and have been adapted for high-throughput screening. Over this same time frame, technologies for massively parallel, single-cell RNA sequencing (scRNA-seq) have matured. Now, both of these powerful approaches are being combined to better understand how kidney organoids can be applied to the understanding of kidney development and disease. There are several reasons why this is a synergistic combination. Kidney organoids are complicated and contain many different cell types of variable maturity. scRNA-seq is an unbiased technology that can comprehensively categorize cell types, making it ideally suited to catalog all cell types present in organoids. These same characteristics also make scRNA-seq a powerful approach for quantitative comparisons between protocols, batches, and pluripotent cell lines as it becomes clear that reproducibility and quality can vary across all three variables. Lineage trajectories can be reconstructed using scRNA-seq data, enabling the rational adjustment of differentiation strategies to promote maturation of desired kidney cell types or inhibit differentiation of undesired off-target cell types. Here, we review the ways that scRNA-seq has been successfully applied in the organoid field and predict future applications for this powerful technique. We also review other developing single-cell technologies and discuss how they may be combined, using “multiomic” approaches, to improve our understanding of kidney organoid differentiation and usefulness in modeling development, disease, and toxicity testing.

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