Figure 1.
High-throughput Screening and Characterization of the Two Hit Compounds.
(A) Schematic diagram of the screening strategy to induce the differentiation of human iPSCs (hiPSCs) into intermediate mesoderm (IM). Stage 1 indicates the differentiation step from hiPSCs into mesendoderm. Stage 2 indicates differentiation from mesendoderm into IM. Chemical screening was performed on Stage 2 cells. (B) The chemical structures of the two hit compounds, AM580 and TTNPB. (C) Results of flow cytometric analyses of the induction of OSR1+ cells on culture day 8; treatments consisted of two days of Stage 1 with CHIR99021 and activin A, and five days of Stage 2 with AM580 or TTNPB, DMSO (negative control), or combined CHIR99021 and BMP7 (positive control). (D) Dose-response curve of OSR1+ cell induction on day 8 by AM580 and TTNPB. The data in (C) and (D) are means±SD of three independent experiments (n = 3).
Figure 2.
The Small Molecule Method for Differentiating hiPSCs into IM Cells.
(A) The results of the flow cytometric analyses comparing the induction efficiency of OSR1+ cells on culture days 6, 8, and 11 for various treatments with CHIR99021 and AM580 or TTNPB, and the growth factor method (CHIR99021 and activin A during Stage 1, CHIR99021 and BMP7 during Stage 2). (B) Results of flow cytometric analyses of OSR1+ cell induction on days 3 and 6 of the AM580, TTNPB, and growth factor methods. (C) Numbers of OSR1+ and total cells generated by the TTNPB and growth factor methods. (D) Results of the flow cytometric analyses showing the differentiation of OSR1+ cells on day 6 using the serum-free small molecules methods. (E) Results of RT-PCR analyses showing mRNA expression of IM and non-IM marker genes in undifferentiated hiPSCs before treatment on day 1, and in OSR1+ cells on day 6 after induction by the small molecule method. (F) Immunostaining for SALL1, WT1, LIM1, and PAX2 in the OSR1+ cells on day 6 of induction by the TTNPB method. Scale bars, 100 m. The data in (A–D) are means±SD of three independent experiments (n = 3). The data in (E) and (F) are representative of three independent experiments. See Figure S2 for additional data.
Figure 3.
Developmental Potential of hiPSC-derived IM Cells Generated by the Small Molecule Method.
(A) Schematic showing further in vitro and in vivo development of OSR1+ cells that were differentiated from OSR1-GFP knock-in hiPSCs (3D45), using the TTNPB method. (B) Results of RT-PCR analyses showing mRNA expression of marker genes for the developing kidney, gonad, and adrenal cortex in differentiated OSR1+ cells on culture day 14. (C) Differentiated cells stained on day 14 with antibodies or lectins against markers of IM derivatives: LTL and AQP1 for the proximal renal tubule, PDX and WT1 for glomerular podocytes, DBA and CK8 for the nephric duct and ureteric bud, &SMA for smooth muscle, ECAD as an epithelial marker, and GATA6 and HSD3β as markers of gonad or adrenal cortex. (D) Immunostaining of histological sections of four-week-old hiPSC-derived IM grafts generated by the TTNPB method transplanted into immunodeficient mice (NOD. CB17-Prkdcscid/J) for human mitochondria (HuMt) (green), LTL (red), AQP1 (purple) and all nuclei (blue). (E) Immunostaining for IM derivative markers (SALL4 for the nephric duct and ureteric bud, AQP2 for the collecting duct, and GATA4 for the gonads or adrenal cortex) of histological sections of cell aggregates generated from OSR1+ cells isolated on day 6 and grown in suspension culture for an additional 8 days. (F) Renal tubule-like structures formed inside the cell aggregates on day 14 stained for ECAD (green), LTL (red), laminin (purple), and nuclei (blue). The five panels on the right are magnified views of the solid box in the left panel. (G) Section immunostaining of organ culture samples collected on day 14, for human nuclei (HuNu, green), LTL (red), laminin (purple), and all nuclei (blue). The five panels on the right are magnified views of the solid box in the left panel. The data in (B, C), (D) and (E, F) are representative of the findings of three, five and four independent experiments, respectively. Scale bars, 100 m.
Figure 4.
The Small Molecule Method Functions via RARβ Receptors.
(A) Results of the flow cytometric analyses showing induction of OSR1+ cells by the small molecule method, when AM580 or TTNPB was replaced by all-trans retinoic acid (ATRA), adapalene, and CD1530. (B) Effects of adding RAR antagonists, BMS493, LE135, and MM11253, on the induction of OSR1+ cells by the TTNPB method. (C) The knockdown efficiency of siRNAs targeting RARB, and (D and E) effects on expression levels of OSR1 and the induction of OSR1+ cells by the TTNPB method. (F) Effects of adding a pan-RXR agonist (SR11237) or a pan-RXR antagonist (UVI3003) to the induction efficiency of OSR1+ cells by the TTNPB method. (G) The induction of OSR1+ cells by the small molecule method, when SR11237 replaced AM580 or TTNPB. (H) Results of qRT-PCR analyses showing OSR1 expression activated by the AM580 and TTNPB methods, and when ATRA, adapalene, or CD1530 were used instead of AM580 or TTNPB. OSR1-GFP knock-in hiPSCs prior to treatments were used to normalize the data. The data in (A–H) are presented as the mean±SD on culture day 6 of three independent experiments (n = 3).
Figure 5.
The BMP-Smad Signaling Pathway Regulates Development of IM.
(A) Results of the flow cytometric analyses examining the effects of adding noggin, dorsomorphin (DM), LDN193189 and DMH1 during Stage 2 on the induction of OSR1+ cells on culture day 6 in the AM580 and TTNPB methods. (B) Results of qRT-PCR analyses showing mRNA expression of OSR1 on culture day 6 of the AM580 and TTNPB methods, with or without noggin. (C) Copy numbers of BMP-2, -4, -5, -6, and -7 expressed in differentiation cultures. (D) Time course of BMP-4 and BMP-5 mRNA expression in the TTNPB method. OSR1-GFP knock-in hiPSCs prior to treatments were used to normalize the data. (E and F) Effects of adding noggin or DMH1 during Stages 1 and 2 on the induction of OSR1+ cells and the OSR1 expression levels analyzed on culture day 6. (G) Results of Western blot analyses examining phosphorylation levels of Smad1/5 in differentiation cultures of the small molecule method, with or without noggin or DMH1 added to Stages 1 and 2. (H) OSR1+ cell induction on culture day 6 in the small molecule method and following treatment with various combinations of recombinant BMP-4 and BMP-5 proteins. (I) Results of qRT-PCR analyses showing BMP-4 mRNA expression in differentiation cultures on day 6 of the small molecule method, with or without the addition of noggin or DMH1. Factor (-) indicates the rate of induction of OSR1+ cell (A and E), the phosphorylation level of Smad1/5 (G) and the mRNA expression level of BMP4 (I), on day 6 of the differentiation culture without growth factors or small molecules. OSR1-GFP knock-in hiPSCs obtained on day 6 following treatment of the TTNPB method without inhibitors were used to normalize the data shown in (B), (F) and (I). The data in (A–F, H, I) are means±SD of three independent experiments (n = 3). The data in (G) are representative of three independent experiments.
Figure 6.
The Small Molecule Method Can Produce IM Cells without the Mesendoderm Step.
(A) Induction of BRACHYURY+ mesendoderm cells generated from OSR1-GFP knock-in hiPSCs (3D45) after 24, 48, 72, and 96 hrs of treatment with CHIR99021 and TTNPB or activin A. (B) Results of the qRT-PCR analyses showing mRNA expression of the mesendoderm markers, BRACHYURY, GOOSECOID, and MIXL1, in differentiation cultures from the growth factor and TTNPB methods. (C) Effects of adding a RAR inhibitor, BMS493, to differentiation cultures of 3D45 cells treated with CHIR99021 and activin A, analyzed by anti-BRACHYURY immunostaining. (D) Induction of OSR1+ cells on culture day 11 of the growth factor method, with or without addition of BMS493 at various concentrations. (E) Results of qRT-PCR analyses showing expression of RARB mRNA. 3D45 cells on day 1, before treatment, were used to normalize the data shown in (B) and (E). The data in (A–E) are means±SD of three independent experiments (n = 3). Scale bars, 100 m.