Rapid and Efficient Differentiation of Human Pluripotent Stem Cells into Intermediate Mesoderm That Forms Tubules Expressing Kidney Proximal Tubular Markers

Timed addition of exogenous factors modulates cell fate of CHIR-induced hPSCs. (A) Diagram depicting time course of differentiation. (B) Time course of gene expression in human PSCs treated with CHIR for 24 hours, CHIR for 48 hours, or DMSO (vehicle). Data shown represent mean±SEM (n=4). Representative immunostaining (C) and quantification (D) of markers of pluripotency, mesoderm, definitive endoderm, and ectoderm in human PSCs treated with DMSO (vehicle), CHIR for 24 hours, or CHIR for 48 hours, day 4 of differentiation. Data shown in graph represent mean±SEM (n>5). (E) Expression of BMP-4 by quantitative RT-PCR in hPSCs treated with CHIR for 24 hours, CHIR for 48 hours, or DMSO. Data shown represent mean±SEM (n=3). (F) Representative immunostaining for SOX17 and FOXA2 in hESCs and hiPSCs treated with CHIR for 24 hours followed by activin A 100 ng/ml for 3 days. Numbers represent the mean percentage±SEM of SOX17⁺ cells from at least two independent experiments. (G) Cells at the definitive endoderm stage were differentiated using a three-step protocol into hormone-expressing pancreatic endocrine cells producing insulin, proinsulin C-peptide, and somatostatin. Number represents the mean percentage±SEM of insulin⁺C-peptide⁺ cells from at least two independent experiments. (H) Diagram of directed differentiation of hPSCs into intermediate mesoderm. (I) Quantification of cells with positive immunostaining for PAX2. Data shown in the graph represent mean±SEM (n=2). (J) Immunostaining for PAX2 in hPSCs treated with or without CHIR for 24 hours followed by FGF2 100 ng/ml for 3 days, day 4. Scale bars, 100 μm. DAPI, 4′,6-diamidino-2-phenylindole.

FGF2 and RA induce PAX2⁺LHX1⁺ intermediate mesoderm cells. (A) Immunostaining for PAX2 and LHX1 in hPSCs cells treated with CHIR for 24 hours followed by FGF2 or FGF2+RA for 3 days, day 4 of differentiation. (B) Quantification of PAX2⁺ cells on days 2–7 of differentiation in hPSCs treated with CHIR for 24, 36, or 48 hours followed by FGF2+RA. Data shown represent mean±SEM (n>4). (C) Immunostaining for PAX2 and LHX1 and (D) quantification of PAX2⁺ cells in three hESC lines and two hiPSC lines treated with CHIR for 36 hours followed by FGF2+RA (ChFR), day 3. Data shown represent mean±SEM (n=3). (E) Quantification of PAX2⁺ or LHX1⁺ cells in hPSCs treated with CHIR for 36 hours followed by FGF2+RA or FGF2+RA+BMP7, day 3. Data shown represent mean±SEM (n=3). (F) Quantification of PAX2⁺ and LHX1⁺ cells by flow cytometry. (G) Quantitative RT-PCR of intermediate mesoderm genes in hPSCs treated with ChFR. Data shown represent mean±SEM (n=3). (H) Immunostaining for LHX1 and WT1 in hPSCs treated with ChFR. (I) Quantitative RT-PCR of non-IM genes in hPSCs treated with ChFR. Data shown represent mean±SEM (n=2). Scale bar, 100 μm. DAPI, 4′,6-diamidino-2-phenylindole.

PAX2⁺LHX1⁺ intermediate mesoderm cells form polar tubular structures expressing polycystin-2⁺ cilia and kidney proximal tubular markers. (A) Immunostaining time course from days 3 to 9 for PAX2, KSP, and LTL in hPSCs treated with FGF2+RA (ChFR) for 3 days, then cultured in media without additional growth factors for an additional 6 days. Scale bar, 50 μm. (B) Immunostaining for KSP and LTL in tubular structures formed by PAX2⁺LHX1⁺ IM cells, day 9. Inset shows tubular structure at higher magnification. Scale bar, 50 μm. (C) Brightfield stereomicroscope imaging of tubular structures, day 9. Scale bar, 200 μm. (D) Quantification of tubular structures formed from PAX2⁺LHX1⁺ IM cells, day 9. Data shown represent mean±SEM (n=4 for one hESC and n=4 for one hiPSC line). (E) Immunostaining for KSP, LTL, and laminin in tubular structures derived from two hESC lines and one iPSC line, day 9. Scale bar, 50 μm. (F) Immunostaining for LTL and N-cadherin in tubular structures derived from one hESC and one iPSC line, day 9. Scale bar, 50 μm. (G) Immunostaining for acetylated α-tubulin and polycystin-2 in tubular structures, day 9. Inset shows higher magnification of cilia localized to the apical surface. Scale bar, 50 μm. (H) Quantitative RT-PCR of genes associated with kidney development and mature kidney epithelial cells. Data shown represent mean±SEM (n=2). (I) Whole-mount immunohistochemistry for anti–human nuclear antigen (HNA) and laminin in chimeric kidney explant cultures. Dissociated hPSC-derived IM cells on day 3 (n=10) and day 9 (n=3) of differentiation were mixed with dissociated E12.5 mouse embryonic kidneys in single cell suspension, reaggregated by centrifugation, and cultured for 3 days in kidney explant culture. Arrowhead, laminin-bounded structures containing human and mouse cells. Scale bar, 50 μm. AQP1, aquaporin-1; AQP2, aquaporin-2; DAPI, 4′,6-diamidino-2-phenylindole; HNA, human nuclear antigen; UMOD, uromodulin.