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. Author manuscript; available in PMC: 2022 Jul 6.
Published in final edited form as: Cell Metab. 2020 Dec 9;33(2):379–394.e8. doi: 10.1016/j.cmet.2020.11.011

Figure 5. FAO and OXPHOS drives proximal tubule differentiation in human kidney organoid.

Figure 5.

(A) Experimental scheme for the generation of human kidney organoid. Briefly, hPSCs were first differentiated into posterior primitive streak (PPS) fate then to intermediate mesoderm (IM). Cells were aggregated (day 0, D0) and further differentiated in 3D culture into renal vesicle (RV) and nephron stage. At D20 of differentiated kidney organoids were stained for podocalyxin (PODXL: podocyte marker, yellow), Wilm’s tumor 1 (WT1, red), and Lotus tetragonolobus Lectin (LTL: PT marker, green). Scale Bar=200μM.

(B) Transcript expression levels (in bulk organoids) of PPARGC1A, SLC3A1, SLC5A12 and SLC27A2 on day 4, 8, 12, 16 and 20 of organoid differentiation. Data are represented as mean ± SEM. n = 2 independent experimental replicates analyzed from a pool of 12 organoids/group.

(C) Single-cell RNA-seq analysis of human kidney organoid. UMAP showing 9 distinct cell types identified by unsupervised clustering. Mesench: mesenchymal cells, CD: collecting duct, Endo: endothelial cells, cycling: cell cycling cells, Podo: podocytes, LOH: loop of Henle and PT: proximal tubule.

(D) Feature plots of key cell type markers (DES, COL21A1, GATA3; mesenchyme, NPHS1; podocytes, SLC3A1;PT cell, SLC12A1;LOH, PCNA, CCNA2;proliferating cells, PECAM11;endothelial cells).

(E) Expression SIX1 (nephron progenitor marker), MKI67 (proliferation maker) and SLC3A1 (PT cell marker) along the differentiation trajectory.

(F) Heatmap showing the expression changes of highly variable genes involved in FAO identified (Figure 4J, 4L) along the organoid cell differentiation trajectory.

(G) Expression level of genes associated with FAO (PPARGC1A, ACOX2, and CPT1A), and PT cell markers (ATP11A, ACOX12, SLC27A2, SLC34A1, SLC3A1, SLC5A2, and SLC6A19) in kidney organoids cultured in EGM and REGM media. The data are represented as mean ± SEM. n ≥ 2 independent experimental replicates from a pool of 12 organoids/group; *P < 0.05, **P < 0.01 ***P < 0.001 and ****P < 0.0001 paired Student’s t-test.

(H) Quantification of changes in the protein expression OXPHOS proteins in organoids cultured in EGM or REGM. Tubulin is used as loading control. The data are represented as mean ± SEM. n = 3 independent experimental replicates from a pool of 16 organoids/group; *P < 0.05, **P < 0.01 ***P < 0.001 and ****P < 0.0001 two-way ANOVA, followed by Bonferroni post-test.

(I) Representative immunofluorescence staining of LTL (green) and PODXL (red) in kidney organoids cultured in EGM and REGM. Scale Bar=400μM (EGM) and 500μM (REGM).

(J) Quantification of LTL positive cells in kidney organoids cultured in EGM or REGM. Y-axis represent relative fluorescence. The data are presented as mean ± SEM. n = 3 organoids/group.