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Cited by 211Pluripotent Stem Cells ResearchSingle-cell and spatial transcriptomicsRenal and related cancers

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Single cell census of human kidney organoids shows reproducibility and diminished off-target cells after transplantation

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Subramanian, Ayshwarya, Sidhom, Eriene-Heidi, Emani, Maheswarareddy, Vernon, Katherine, Sahakian, Nareh, Zhou, Yiming, Kost-Alimova, Maria, Slyper, Michal, Waldman, Julia, Dionne, Danielle, Nguyen, Lan T., Weins, Astrid, Marshall, Jamie L., Rosenblatt-Rosen, Orit, Regev, Aviv, Greka, Anna·2019·Nature Communications
DOI 10.1038/s41467-019-13382-0PMC PMC6884507

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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 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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Fig. 1 Mature kidney organoids from four different human iPSC lines contain major nephron cell classes. a Schematic of differentiation protocols for kidney organoids derived from human iPSCs by the JB and ML protocols (methods). Single-cell sequencing time-points as shown. b Table summarizing single cells profiled across four different human iPSC cell lines (AS, N1, N2, ThF) using two different protocols (ML, JB) across four time points (iPSC, Day 7 (D7), 15 (D15) and 29 (D29)). Replicates are indicated in parentheses. c t-SNE plot of composite single-cell transcriptomic profiles from all four iPSC D29 kidney organoids (left) and human adult kidney (right). Cluster color annotations as shown. d (left) Violin plots showing expression of canonical adult human kidney markers used to identify nephron clusters in D29 organoids: podocyte (NPHS2), proximal tubule (PT; LRP2), thick ascending limb/distal nephron (TAL; SLC12A1) and immature distal nephron (GATA3 + DT). Mesenchymal and off-target cells were also detected. (right) Violin plots of data-derived tubular markers revealed proximal to distal gradient of APOE expression, proximal marker SPP1, and distal markers WFDC2 and MAL. e Random forest classifier trained on D29 ML_ThF organoid gene expression profiles accurately predicted respective nephron cell classes in human first and second trimester fetal kidney (e, f) as compared to adult kidney (g). The Y-axis labels in each panel represent the annotated cell class in each human sample. The X-axis labels are predicted cell classes at D29 (ML_ThF organoids). The size and intensity of the red dot represents the % of cells in the Y-axis cell-types classified as the corresponding X-axis cell-class, e.g., all human adult podocytes are correctly classified as podocytes, while distal convoluted tubule (DCT) is predicted to be most similar to the GATA3 + DT organoid cell class.
Fig. 1 Mature kidney organoids from four different human iPSC lines contain major nephron cell classes. a Schematic of differentiation protocols for kidney organoids derived from human iPSCs by the JB and ML protocols (methods). Single-cell sequencing time-points as shown. b Table summarizing single cells profiled across four different human iPSC cell lines (AS, N1, N2, ThF) using two different protocols (ML, JB) across four time points (iPSC, Day 7 (D7), 15 (D15) and 29 (D29)). Replicates are indicated in parentheses. c t-SNE plot of composite single-cell transcriptomic profiles from all four iPSC D29 kidney organoids (left) and human adult kidney (right). Cluster color annotations as shown. d (left) Violin plots showing expression of canonical adult human kidney markers used to identify nephron clusters in D29 organoids: podocyte (NPHS2), proximal tubule (PT; LRP2), thick ascending limb/distal nephron (TAL; SLC12A1) and immature distal nephron (GATA3 + DT). Mesenchymal and off-target cells were also detected. (right) Violin plots of data-derived tubular markers revealed proximal to distal gradient of APOE expression, proximal marker SPP1, and distal markers WFDC2 and MAL. e Random forest classifier trained on D29 ML_ThF organoid gene expression profiles accurately predicted respective nephron cell classes in human first and second trimester fetal kidney (e, f) as compared to adult kidney (g). The Y-axis labels in each panel represent the annotated cell class in each human sample. The X-axis labels are predicted cell classes at D29 (ML_ThF organoids). The size and intensity of the red dot represents the % of cells in the Y-axis cell-types classified as the corresponding X-axis cell-class, e.g., all human adult podocytes are correctly classified as podocytes, while distal convoluted tubule (DCT) is predicted to be most similar to the GATA3 + DT organoid cell class.CC BY · PubMed Central
Fig. 2 IF validation of markers derived from the single-cell data in mature kidney organoids. a IF staining of an entire kidney organoid with segment specific markers as shown. b Schematic of kidney nephron with major cell types and canonical markers annotated. c Immunofluorescence staining of D29 kidney organoids for podocyte (WT1), proximal tubule (LTL), and distal tubule (CDH1 and GATA3) across two protocols (JB, ML) and four cell lines (AS, N1, N2, ThF). IF staining for validation of markers identified in the single-cell data: d NPHS1 colocalized with the podocyte-specific marker SYNPO and e LRP2 colocalized with the proximal tubular marker LTL (bottom). f IF staining validation for MEIS1-positive mesenchymal cells in D29 organoids. LAMA1 indicates basement membranes. MEIS1 staining of mesenchymal cells appropriately surrounds LAMA1-defined tubular nephron structures.
Fig. 2 IF validation of markers derived from the single-cell data in mature kidney organoids. a IF staining of an entire kidney organoid with segment specific markers as shown. b Schematic of kidney nephron with major cell types and canonical markers annotated. c Immunofluorescence staining of D29 kidney organoids for podocyte (WT1), proximal tubule (LTL), and distal tubule (CDH1 and GATA3) across two protocols (JB, ML) and four cell lines (AS, N1, N2, ThF). IF staining for validation of markers identified in the single-cell data: d NPHS1 colocalized with the podocyte-specific marker SYNPO and e LRP2 colocalized with the proximal tubular marker LTL (bottom). f IF staining validation for MEIS1-positive mesenchymal cells in D29 organoids. LAMA1 indicates basement membranes. MEIS1 staining of mesenchymal cells appropriately surrounds LAMA1-defined tubular nephron structures.CC BY · PubMed Central
Fig. 3 Variability in cell type proportions detected by scRNA-seq evident at D15. a, b Relative proportions of endothelial, nephron, mesenchymal and off-target cell clusters across all replicates of D29 organoids. Annotations as shown. c, d Comparison of cell-type composition between D29 organoids as determined by boxplots of the Jenson−Shannon Divergence (JSD) scores. Each point on the plot is a pair-wise (color) measure of JSD between two organoids. Legend indicates annotation for pairs of iPSC lines. The center line of the boxplots indicates the median, and the bottom and top lines of the box indicate the first and third quartiles respectively of the JSD scores. Outliers are indicated as dots beyond the whiskers; whiskers stretch up to +−1.5*IQR on both sides. c Organoid compositional differences are greater between lines than between different protocols for the same line or between replicates of the same line and protocol (within lines). d Organoid compositional heterogeneity is greatest in the off-target compartment followed by the mesenchyme and the nephron compartment in all three comparison groups (between lines, between protocols and within lines). t-SNE plot of single cells from e iPSC, f D7, and g D15 of the organoid differentiation protocol. h Comparison of relative cell type proportion across iPSC lines of cell clusters shown in (e−g). i Compositional differences are greatest between lines at D15 and the least at D7. j Compartment-specific cell-type proportions at D15 across all lines and replicates.
Fig. 3 Variability in cell type proportions detected by scRNA-seq evident at D15. a, b Relative proportions of endothelial, nephron, mesenchymal and off-target cell clusters across all replicates of D29 organoids. Annotations as shown. c, d Comparison of cell-type composition between D29 organoids as determined by boxplots of the Jenson−Shannon Divergence (JSD) scores. Each point on the plot is a pair-wise (color) measure of JSD between two organoids. Legend indicates annotation for pairs of iPSC lines. The center line of the boxplots indicates the median, and the bottom and top lines of the box indicate the first and third quartiles respectively of the JSD scores. Outliers are indicated as dots beyond the whiskers; whiskers stretch up to +−1.5*IQR on both sides. c Organoid compositional differences are greater between lines than between different protocols for the same line or between replicates of the same line and protocol (within lines). d Organoid compositional heterogeneity is greatest in the off-target compartment followed by the mesenchyme and the nephron compartment in all three comparison groups (between lines, between protocols and within lines). t-SNE plot of single cells from e iPSC, f D7, and g D15 of the organoid differentiation protocol. h Comparison of relative cell type proportion across iPSC lines of cell clusters shown in (e−g). i Compositional differences are greatest between lines at D15 and the least at D7. j Compartment-specific cell-type proportions at D15 across all lines and replicates.CC BY · PubMed Central
Fig. 4 Concordant expression of developmental programs across organoids from four human iPSC lines. a Heatmap of expression patterns for major nephrogenesis markers across organoid differentiation time points (iPSC D0, D7, D15, and D29, averaged across four cell lines, ML protocol) and human adult kidney. Expression values were row-normalized to obtain z-scores; red color indicates positive z-scores. b Canonical (NPHS2) and data-derived (CLDN5) podocyte marker genes superimposed in tSNE plots from D15 organoids (N2 line, ML protocol). c IF staining of D15 kidney organoid (N2 line, ML protocol) for CLDN5 as a marker of early podocyte differentiation derived from the single-cell data. Additional canonical podocyte markers (NPHS1, WT1) and DAPI staining as shown. d IF staining of D29 kidney organoid (AS line, ML protocol) for SOX17 and CD31, markers of endothelial cells.
Fig. 4 Concordant expression of developmental programs across organoids from four human iPSC lines. a Heatmap of expression patterns for major nephrogenesis markers across organoid differentiation time points (iPSC D0, D7, D15, and D29, averaged across four cell lines, ML protocol) and human adult kidney. Expression values were row-normalized to obtain z-scores; red color indicates positive z-scores. b Canonical (NPHS2) and data-derived (CLDN5) podocyte marker genes superimposed in tSNE plots from D15 organoids (N2 line, ML protocol). c IF staining of D15 kidney organoid (N2 line, ML protocol) for CLDN5 as a marker of early podocyte differentiation derived from the single-cell data. Additional canonical podocyte markers (NPHS1, WT1) and DAPI staining as shown. d IF staining of D29 kidney organoid (AS line, ML protocol) for SOX17 and CD31, markers of endothelial cells.CC BY · PubMed Central
Fig. 5 Genes associated with kidney diseases are expressed in expected compartments across organoids from four human iPSC lines. Heatmap of cell-type-specific gene expression (ThF line, ML protocol) in organoids and human adult kidney. Genes implicated in monogenic causes of a congenital abnormalities of the kidney and urinary tract (CAKUT), and b mendelian renal cystic diseases and tumor syndromes. c IF staining validates the expression of MUC1 and PAX2 in the distal tubule in D29 kidney organoids. CDH1 serves a distal tubular marker. Note appropriate apical expression of MUC1 in distal tubular epithelial cells.
Fig. 5 Genes associated with kidney diseases are expressed in expected compartments across organoids from four human iPSC lines. Heatmap of cell-type-specific gene expression (ThF line, ML protocol) in organoids and human adult kidney. Genes implicated in monogenic causes of a congenital abnormalities of the kidney and urinary tract (CAKUT), and b mendelian renal cystic diseases and tumor syndromes. c IF staining validates the expression of MUC1 and PAX2 in the distal tubule in D29 kidney organoids. CDH1 serves a distal tubular marker. Note appropriate apical expression of MUC1 in distal tubular epithelial cells.CC BY · PubMed Central
Fig. 6 Transplantation of human organoids into mouse diminishes off-target cells. t-SNE plots of organoids in prolonged in vitro culture reveal cell clusters in a D32 and b D51 organoids similar to clusters from D29 organoids. c Transplanted and vascularized organoid at D51. IF staining shows human podocytes (anti-human NPHS1 antibody) at the outer perimeter and mouse endothelial cells (anti-mouse Plvap antibody) lining the internal perimeter of a glomerular structure. Gray, human and mouse nuclei (DAPI). d Dotplots indicating ligand and receptor pairs involved in putative cross-talk between human podocytes and mouse endothelial cells of transplanted organoids. e Violin plots of expression levels for KLF6 and HES1 in the distal MAL+ cluster from D29, D32 and D51 organoids grown in vitro, compared to D32 transplanted organoids. f Violin plots demonstrate that PMEL+ melanoma cells and SOX2+ neuronal cells were diminished after transplantation. MYOG+ muscle cells and STMN2+ neuron-like cells persisted. g Random Forest Classifier shows the relation of cell clusters in transplant organoids compared to organoids grown in vitro. Melanoma and SOX2+ neuronal precursor cells are not detected in D32 transplant organoids. h IF staining validation for SOX2 (green) in D32 control organoids compared to diminished abundance in D32 transplanted organoids. Human and mouse nuclei (DAPI, gray).
Fig. 6 Transplantation of human organoids into mouse diminishes off-target cells. t-SNE plots of organoids in prolonged in vitro culture reveal cell clusters in a D32 and b D51 organoids similar to clusters from D29 organoids. c Transplanted and vascularized organoid at D51. IF staining shows human podocytes (anti-human NPHS1 antibody) at the outer perimeter and mouse endothelial cells (anti-mouse Plvap antibody) lining the internal perimeter of a glomerular structure. Gray, human and mouse nuclei (DAPI). d Dotplots indicating ligand and receptor pairs involved in putative cross-talk between human podocytes and mouse endothelial cells of transplanted organoids. e Violin plots of expression levels for KLF6 and HES1 in the distal MAL+ cluster from D29, D32 and D51 organoids grown in vitro, compared to D32 transplanted organoids. f Violin plots demonstrate that PMEL+ melanoma cells and SOX2+ neuronal cells were diminished after transplantation. MYOG+ muscle cells and STMN2+ neuron-like cells persisted. g Random Forest Classifier shows the relation of cell clusters in transplant organoids compared to organoids grown in vitro. Melanoma and SOX2+ neuronal precursor cells are not detected in D32 transplant organoids. h IF staining validation for SOX2 (green) in D32 control organoids compared to diminished abundance in D32 transplanted organoids. Human and mouse nuclei (DAPI, gray).CC BY · PubMed Central

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Authors
Subramanian, A., Sidhom, E.-H., Emani, M., Vernon, K., Sahakian, N., Zhou, Y., Kost-Alimova, M., Slyper, M., Waldman, J., Dionne, D., Nguyen, L. T., Weins, A., Marshall, J. L., Rosenblatt-Rosen, O., Regev, A., & Greka, A.
Affiliations
  • Broad Institute (US)
  • Brigham and Women's Hospital (US)
Corresponding author
Greka, Anna
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-11-29
Volume
10
Issue
1
Pages
5462-5462
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-019-13382-0
PMC
PMC6884507
OpenAlex
W2990026706
Cited by
211
References
96
Citations by year
2026: 12 · 2025: 27 · 2024: 36 · 2023: 28 · 2022: 39 · 2021: 44 · 2020: 18 · 2019: 6
FWCI
8.85
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
  • Nature
Funders
  • U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (DK099465, DK095045)
  • the Slim Initiative for Genomic Medicine in the Americas (SIGMA), and an NDSEG fellowship
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
  • Health Sciences › Medicine › Oncology › Renal and related cancers
Keywords
kidney organoids, induced pluripotent stem cells, single-cell RNA sequencing, reproducibility, organoid transplantation, kidney capsule transplantation
MeSH terms
Animals, Cell Differentiation, Cell Line, Humans, Kidney, Organoids, Transplantation, Heterologous, Reproducibility of Results, Kidney Transplantation, Sequence Analysis, RNA, Gene Expression Regulation, Developmental, Mice, Induced Pluripotent Stem Cells, Single-Cell Analysis

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