Additional file 1 of Generation of mitochondria-rich kidney organoids from expandable intermediate mesoderm progenitors reprogrammed from human urine cells under defined medium
Creators: name: Yuan, Yapei
name: Chen, Huan
name: Ou, Sihua
name: Cai, Baomei
name: Zhang, Ruifang
name: Qin, Yue
name: Pan, Mengjie
name: Cao, Shangtao
name: Pei, Duanqing
name: Hou, Fan Fan
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Marine Biology
subject: Cancer
subject: Hematology
subject: Computational Biology

description: Additional file 1: Fig. S1. Generation of 2D kidney organoids from hUCs of different donors. (A) Western blot for marker gene of the primitive streak, intermediate mesoderm, nephron progenitor, and segments of the nephron. PAX8 for tubule epithelial and nephron progenitor, CALBINDIN for distal tubule. (B) Gene expression analysis for markers of pluripotency (OCT4 and NANOG), endoderm (SOX17 and FOXA2), and ectoderm (SOX1 and FOX6) during differentiation is presented. Embryonic stem cells (ESCs), defined as endoderm (DE), and neural progenitor cells (NPCs) are shown as the positive control. The relative expression of each transcript to GAPDH expression is presented as the mean ± SEM (n = 6). (C) Volunteer information and 2D U-iKOs induction from urine cells of healthy volunteers and CKD patients. (D) Representative bright-field images for one healthy volunteer (UC-001) and one patient (CKD, PUC-011). Scale bars, 250 μm. (E) Heatmap of markers and signature genes of hUCs, U-iIMPLCs, U-iNPCs, and U-iKOs for the volunteers in B. (F) Immunostaining analysis of primitive streaks marker (T and TBX6) for U-iIMPLCs and nephron progenitor cells maker (PAX2 and SALL1) for U-iNPCs. Scale bars, 50 μm. (G) Comparison of culture conditions of kidney organoid systems in personalized ways. M-iKOs, iPSC-induced organoids with protocol by Morizane; T-iKOs, iPSC-induced organoids with protocol by Takasato. Fig. S2. Cell fate changes during U-iKOs differentiation by RNA-seq. (A) Scatterplot showing the differentially expressed genes between hUCs (blue) and U-iIMPLCs (red) in the left panel, U-iIMPLCs (blue) and U-iNPCs (red) in the middle panel, U-iIMPLCs (blue) and U-iKOs (red) in the right panel during differentiation. (B) Gene ontology (GO) analysis for upregulated expressed genes (blue) and downregulated expressed genes of U-iIMPLCs compared to hUCs with p value at the bottom. (C) GO analysis for upregulated expressed genes (blue) and downregulated expressed genes of U-iNPCs compared to U-iIMPLCs with p value at the bottom. (D) GO analysis for upregulated expressed genes (blue) and downregulated expressed genes of U-iKOs compared to U-iIMPLCs with p value at the bottom. Fig. S3. ScRNA-seq profiling subpopulations in 3D U-iKOs. (A) Violin plot shows the detected genes, UMI count, and percentage of mitochondrial genes between 3D and 2D U-iKOs. (B) Heatmap indicating Pearson’s correlations on the averaged profiles among cell types between 3D and 2D U-iKOs. (C) UMAP plot of podocyte subcluster in U-iKOs, each dot represents one cell, with color code for each cluster. (D) Heatmap of differentially expressed genes between clusters in C selected signature genes and GO term in the right. (E) UMAP plot of proximal tubule subcluster in U-iKOs, each dot represents one cell, with color code for each cluster. (F) Heatmap of differentially expressed genes between clusters in E selected signature genes and GO term in the right. (G) UMAP plot of the loop of Henle and distal tubule subcluster in U-iKOs, each dot represents one cell, with color code for each cluster. (H) Heatmap of differentially expressed genes between clusters in E selected signature genes and GO term in the right. Fig. S4. Characteristics of M-iKOs, T-iKOs, C-iKOs and fetal kidney. (A) Left: GO term of differentially expressed genes in M-iKOs with p value at the bottom. Right: Dotplot shows differentially expressed genes in M-iKOs. (B) Left: GO term of differentially expressed genes in T-iKOs with p value at the bottom. Right: Dotplot shows differentially expressed genes in T-iKOs. (C) Left: GO term of differentially expressed genes in C-iKOs with p value at the bottom. Right: Dotplot shows differentially expressed genes in C-iKOs. (D) Left: GO term of differentially expressed genes in fetal kidney with p value at the bottom. Right: Dotplot shows differentially expressed genes in fetal kidney. (E) Immunostaining analysis of U-iKOs markers for kidney function. LRP2 (megalin), a multi-ligand endocytic receptor; SLC12A1 (NKCC2), a kidney-specific sodium-potassium-chloride cotransporter. Scale bars, 50 μm. Fig. S5. Maturing organoids after CAM transplantation. (A) Methodology for the implantation of 3D U-iKOs into CAM. Embryonic day, ED. (B) Macroscopic views of implanted U-iKOs were maintained in CAM for 6 days. (C) H&amp;E staining of implanted U-iKOs implanted in the CAM for 6 days. CAM blood vessels were indicated with black asterisks. Scale bars, 100 μm. (n = 5). Fig. S6. 3D U-iKOs after FSK treatment. (A) Representative bright-field images of 3D U-iKOs treated with forskolin (FSK) for 4 days (day 24–28). Scale bars, 250 μm. (B) H&amp;E staining of U-iKOs in the absence (control) or presence of FSK. Scale bars, 100 μm. (C) Immunofluorescence analysis of U-iKOs treated with FSK for 4 days. Scale bars, 50 μm. (D) Gene expression analysis for REN and AGT by RT-qPCR. The relative expression of each transcript to GAPDH expression is presented as the mean ± SEM (n = 4). (E) Gene expression analysis for REN, AGT, and PRCP by RNA-seq. (F) Scatterplot showing the up-regulated genes (red) and down-regulated genes (blue) between control and FSK treatment groups. (G) GO term of the up-regulated genes (red) and down-regulated genes (blue) in the FSK treatment group compared with the control group with p value at the bottom. (H) Gene expression analysis for specifical genes after cisplatin, gentamicin, and FSK treatment in Fig.7I. The relative expression of each transcript to GAPDH expression is presented as the mean ± SEM (n = 4).