The centrosomal protein 83 (CEP83) regulates human pluripotent stem cell differentiation toward the kidney lineage
- Department of Nephrology and Medical Intensive Care, Charité-Universitätsmedizin Berlin, Germany
- Molecular and Translational Kidney Research, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Germany
- Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Egypt
- Berlin Institute of Health, Germany
- Department of Nephrology and Hypertension, Hannover Medical School, Germany
- Technology Platform Pluripotent Stem Cells, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Germany
- Department of Pediatrics, Section of Developmental Biology, University of Colorado School of Medicine, Anschutz Medical Campus, United States
Figures
Figure 1 with 2 supplements
Generation of centrosomal protein 83 (CEP83)-deficient human pluripotent stem cells.
(A) Schematic of the experimental approach to induce a deleting mutation in exon 7 of the CEP83 gene (as described in the methods section). (B) DNA extracted from pooled transfected cells was …
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Figure 1—source data 1
The file contains detailed original PCR gels and immunoblots.
- https://cdn.elifesciences.org/articles/80165/elife-80165-fig1-data1-v2.pdf
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Figure 1—source data 2
Excel sheet shows RT-qPCR data for mRNA expression of CEP83 in WT and knockout hiPSCs.
- https://cdn.elifesciences.org/articles/80165/elife-80165-fig1-data2-v2.xlsx
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Figure 1—source data 3
File contains uncropped PCR gels and immunoblots.
- https://cdn.elifesciences.org/articles/80165/elife-80165-fig1-data3-v2.zip
Figure 1—figure supplement 1
CEP83−/− human-induced pluripotent stem cells (hiPSCs) retain global iPCS cell gene expression signatures and express pluripotency markers.
(A) Alignment of the modified KO clones mRNA and expected amino acid sequences with WT revealed induction of stop codon on both strands of KO1 clone. While KO2 clone shows induction of stop codon on …
Figure 1—figure supplement 2
Phenotypical, molecular, and genetic characterization of CEP83−/− human-induced pluripotent stem cells (hiPSCs) versus the wildtype hiPSCs.
(A) CEP83−/− hiPSCs clones (KO1, KO2, and KO3) show similar morphology to the WT clones (WT1, WT2, and WT3) under the bright field microscope, scale bar = 200 µm. (B) Using bulk RNA sequencing data, …
Figure 2 with 1 supplement
Differentiation of CEP83−/− human-induced pluripotent stem cells (hiPSCs) to intermediate mesoderm cells (day 7) is associated with defective ciliogenesis.
(A) The schematic illustrates the applied differentiation protocol of hiPSCs, as previously described by Takasato et al., 2015. (B–C) WT and CEP83−/− cells on 7 days of culture (D7) of …
Figure 2—figure supplement 1
Loss of CEP83 in organoids results in defective ciliogenesis.
(A) Immunofluorescence staining of wildtype (WT) and CEP83−/− organoids for acetylated tubulin (green), CEP83 protein (red), and nuclear staining 4',6-diamidino-2-phenylindole dihydrochloride …
Figure 3 with 2 supplements
Defective kidney organoid differentiation from CEP83-deficient pluripotent stem cells.
(A, B) Brightfield images of organoids after a total of 25 days of culture (D25) indicate the formation of multiple kidney-like structures in WT organoids (A), whereas CEP83−/− organoids are …
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Figure 3—source data 1
The data shows the quantitative analysis of the percent of nephron formation per organoid in knockout organoids versus WT organoids.
- https://cdn.elifesciences.org/articles/80165/elife-80165-fig3-data1-v2.xlsx
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Figure 3—source data 2
The sheet shows the plotted TPM values of mRNA sequencing analysis in Figure 3L-H for the expression of renal epithelial marker genes in KO and WT at days 0, 7, and 25 of the differentiation.
- https://cdn.elifesciences.org/articles/80165/elife-80165-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
Whole mount immunostaining of the wildtype organoids shows positive staining for NPHS1 (podocyte marker), LTL (proximal tubule marker), and CDH1 (distal tubule marker).
Bar = 50 µm.
Figure 3—figure supplement 2
mRNA analysis of organoids differentiated for 7+ (18) days indicates marked differences in global gene expression in CEP83−/− (KO1–KO3) compared to wildtype (WT1–WT3) organoids.
(A) Heatmap displaying the expression of the top 1000 highly variable genes (see Methods, transcripts per million [TPM] ≥10) using bulk RNA within WT (WT1, WT2, WT3) and CEP83−/− (KO1, KO2, KO3) …
Figure 4 with 3 supplements
Gene expression differences of wildtype (WT) and CEP83−/− D7 monolayers based on bulk and single-cell transcriptomics.
(A) Principal component analysis (PCA) of WT (WT1, WT2) and CEP83−/− (KO1, KO2) cells at day 7 using the average gene expression of the top highly variable 1000 genes in pseudo-bulk scRNA sequencing …
Figure 4—figure supplement 1
Bulk RNA sequencing shows mild overall gene expression differences between WT and CEP83-deficient cells at day 7 of differentiation.
(A) Heat map of bulk RNA-seq data showing the most highly variable 1000 genes (see Methods, maximum transcripts per million ≥10) within wildtype (WT1, WT2, and WT3) and CEP83−/− (KO1, KO2, and KO3) …
Figure 4—figure supplement 2
Expression of intermediate mesoderm marker genes in WT and CEP83−/− human-induced pluripotent stem cells (hiPSCs) after 7 days of differentiation in a monolayer culture.
(A) mRNA expression of CEP83 was significantly downregulated in the CEP83−/− clones on day 7. The expression was investigated in bulk RNA seq data and confirmed by RT-PCR. (B–C) Using bulk RNA …
Figure 4—figure supplement 3
CEP83 loss induces apoptosis at day 7 of differentiation.
(A) Single-cell RNA sequencing data shows that the cell proportion of damaged cells (cluster 5) in the wildtype samples (WT1, WT2) is numerically lower than that in CEP83−/− cells (KO1, KO2). (B, C) …
Figure 5 with 3 supplements
Defective kidney progenitor differentiation from CEP83−/− cells after 7 days of monolayer induction.
(A, B, C) Proportions of cells from kidney progenitor clusters 1 (A), 3 (B), and 4 (C) among wildtype (WT1, WT2) and CEP83−/− (KO1, KO2) cells. (D, E, F) Violin plots of gene expression of kidney …
Figure 5—figure supplement 1
Expression of selected genes per cluster and per group (WT vs. CEP83−/−).
Please note downregulated expression of nephron progenitor genes PAX8, EYA1, and HOXB7 in clusters 1, 3, and 4 (marked in red) in CEP83−/− cells.
Figure 5—figure supplement 2
Violin plots of gene expression of kidney progenitor gene CITED1 within kidney progenitor clusters 1, 3, and 4 comparing wildtype (WT) and CEP83−/− (KO) cells.
N=2 per group. ****p<0.0001.
Figure 5—figure supplement 3
Violin plots of single-cell RNA sequencing show downregulated expression of genes encoding ciliary proteins in CEP83−/− cells, including (A) the basal body protein oral-facial-digital type I OFD1, (B) pericentriolar material-1 (PCM1), and (C) RAS oncogene family 11 A (RAB11A).
The three genes are essential for primary cilium formation, and their loss results in defective ciliogenesis (Mugford et al., 2008b; Mae et al., 2013; Mahlapuu et al., 2001). Data are derived from …
Figure 6 with 3 supplements
CEP83−/− cells upregulate expression of genes characteristic of early lateral plate mesoderm (LPM).
(A–I) Expression of early LPM markers OSR1 (A), FOXF1 (B), FOXF2 (C), FENDRR (D), HAND1 (E), HAND2 (F), CXCL12 (G), GATA5 (H), and GATA6 (I) in wildtype (WT) and CEP83−/− cells at day 0 (D0), day 7 …
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Figure 6—source data 1
The sheet shows the plotted TPM values of mRNA sequencing analysis in Figure 6 for the expression of lateral plate mesoderm marker genes between the KO and WT at days 0, 7, and 25 of the differentiation.
- https://cdn.elifesciences.org/articles/80165/elife-80165-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
CEP83−/− cells upregulate lateral plate mesoderm (LPM) genes in mesenchymal cells cluster and nascent nephron progenitor clusters.
The heatmap shows that the log fold change of the average expression (default setting in Seurat package) of LPM and intermediate mesoderm (IM) genes in the WT cells (8123 cells) and the knockout …
Figure 6—figure supplement 2
CEP83−/− organoids show significant enrichment compared to developmental zebrafish lateral plate mesoderm (LPM) single-cell RNA (scRNA) data.
The expression of the upregulated genes by CEP83−/− organoids on day 25 was compared with the top 20 genes per cluster of zebrafish LPM scRNA data (Prummel et al., 2020). The analysis showed …
Figure 6—figure supplement 3
Upregulation of hedgehog signaling components in CEP83−/− cells.
Bulk RNA sequencing data shows significant upregulation of (A) GLI1, and (B) PTCH1, at days 0, 7, and 25. n=3 clones per group. Data are mean ± SD. *p<0.05, **p<0.01, and ***p<0.001. ns = not …
Figure 7
CEP83−/− cells upregulate expression of genes characteristic of cardiomyocyte progenitors and vascular progenitors.
(A–G) Expression of cardiomyocyte markers ISL1 (A), TBX1 (B), and vascular progenitor markers SOX7 (C), SOX11 (D), NAP1L3 (E), LMO2 (F), and GATA2 (G) in wildtype (WT) and CEP83−/− cells at day 0 …
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Figure 7—source data 1
The sheet shows the plotted TPM values of mRNA sequencing analysis in Figure 7 for the expression of cardiomyocytes and vascular progenitors marker genes between the KO and WT at days 0, 7, and 25 of the differentiation.
- https://cdn.elifesciences.org/articles/80165/elife-80165-fig7-data1-v2.xlsx
Figure 8
Schematic model outlining the functional differences between wildtype and CEP83 knockout cells during the course of differentiation of human pluripotent stem cells toward kidney cells.
IM, intermediate mesoderm; LPM, lateral plate mesoderm.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/80165/elife-80165-mdarchecklist1-v2.docx
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Supplementary file 1
The table shows the primers list used in the qPCR.
- https://cdn.elifesciences.org/articles/80165/elife-80165-supp1-v2.docx
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Supplementary file 2
The table shows the list of primary antibodies used in IF staining.
- https://cdn.elifesciences.org/articles/80165/elife-80165-supp2-v2.docx
