Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney

Abstract

With the prevalence of end-stage renal disease rising 8% per annum globally1, there is an urgent need for renal regenerative strategies. The kidney is a mesodermal organ that differentiates from the intermediate mesoderm (IM) through the formation of a ureteric bud (UB) and the interaction between this bud and the adjacent IM-derived metanephric mesenchyme2 (MM). The nephrons arise from a nephron progenitor population derived from the MM (ref. 3). The IM itself is derived from the posterior primitive streak4. Although the developmental origin of the kidney is well understood2, nephron formation in the human kidney is completed before birth5. Hence, there is no postnatal stem cell able to replace lost nephrons. In this study, we have successfully directed the differentiation of human embryonic stem cells (hESCs) through posterior primitive streak and IM under fully chemically defined monolayer culture conditions using growth factors used during normal embryogenesis. This differentiation protocol results in the synchronous induction of UB and MM that forms a self-organizing structure, including nephron formation, in vitro. Such hESC-derived components show broad renal potential ex vivo, illustrating the potential for pluripotent-stem-cell-based renal regeneration.

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Figure 1: Sequential differentiation of primitive streak and intermediate mesoderm from human ESCs.
Figure 2: Stepwise temporal induction of ureteric and metanephric progenitors from hESCs in vitro.
Figure 3: Assessment of renal potential and evidence for nephron induction of hESC after CHIR99021–FGF9-directed differentiation.
Figure 4: The integration of hESC-derived kidney progenitors into re-aggregates of mouse kidney cells.
Figure 5: Evidence for self-organization after 3D culture of differentiated hESCs.

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Acknowledgements

We are grateful to E. Wolvetang for providing an iPSC line and D. Titmarsh, A. Hidalgo-Gonzalez and J. Cooper-White for supportive comments. This work was supported by the Queensland State Government through a National/International Research Alliance Project, the Australian Research Council as part of the Stem Cells Australia Strategic Research Initiative (SRI110001002) and the National Health and Medical Research Council of Australia (APP1041277). M.H.L. is a Senior Principal Research Fellow of the NHMRC.

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M.T. and M.H.L. conceived and planned the project and wrote the manuscript. M.B. and P.X.E. provided technical assistance with hESC culture, histology, microscopy and differentiation protocols. E.G.S. and A.G.E. provided targeted hESC lines and advised on design and execution. J.M.V. provided technical advice, support and analysis for ex vivo recombination assays.

Corresponding author

Correspondence to M. H. Little.

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M.H.L. consults for Organovo Inc.

Integrated supplementary information

Supplementary Figure 1 Posterior primitive streak induction.

a, Time course quantitative RT-PCR for pluripotent markers, OCT4 and NANOG after induction with BMP4/ActivinA (30/10 ng/ml), showing a reduction in pluripotent gene expression with time. Error bars are s.d. (n = 3 experiments). The source data for the graph are provided in Supplementary Table 1. b, IF for markers of ES cells, NANOG and ECAD, before (hESCs) and after (day 2) primitive streak induction using CHIR99021. (scale = 100 μm) c, IF for markers of posterior primitive streak, T and MIXL1 (GFP), after the primitive streak induction (day 2) using CHIR99021. MIXL1 was detected as GFP expression driven by the MIXL1 endogenous promoter. (scale = 100 μm) d, Levels of spontaneous OSR1 expression induced across time after culture if 3 different ratios of BMP4 and Activin A (ng/mL). hESCs were formed embryoid bodies with 3 different ratios of BMP4 and Activin A for 3 days then spontaneously differentiated under no growth factor condition until day 14. This demonstrates improved OSR1 expression in cells induced with high BMP4 and low Activin A (30/10). OSR1 marks IM and LPM.

Supplementary Figure 2 Influence of FGF signaling on induction of IM proteins.

a, IF for PAX2 protein on hESC cultures at day 6 treated with BMP4/Activin A to day 2 and FGF2 (200 ng/ml), FGF8 (200 ng/ml), FGF9 (200ng/ml) or no growth factors (no GFs) from day 2 to 6 in the presence or absence of the FGF signaling inhibitor, PD173074. (scale = 200 μm) b, Quantitative RT-PCR to examine the relative expression level of PAX2, LHX1 and OSR1 at day 6 of the same protocol as IF (a). Shaded bars show the effect of addition of the FGF inhibitor, PD173074. Error bars are s.d. (n = 3 experiments). The source data for graphs are provided in Supplementary Table 1. c, IF for the IM marker PAX2 and the marker of both LPM and IM, OSR1, on hESC cultures at day 6 treated with BMP4/Activin A (+FGF9 (B/A)) or 8 μM CHIR99021 (+FGF9 (CHIR)) to day 2 followed by 200 ng/mL FGF9 or no growth factors (no GFs) from day 2 to 6. Secondary antibody only control was used as a negative control (2° Ab only) (scale = 100 μm). The source data for graphs are provided in Supplementary Table 1. d, A table showing the percent of PAX2 and PAX2+ cells in total (total) or together with LHX1 and LHX1+ cells on hESC cultures at day 6 treated with 8 μM CHIR99021 to 2 days followed by 200 ng/mL FGF9 from day 2 to 6. Errors are s.d. (n = 5 fields in total from 3 experiments).

Supplementary Figure 3 The effect of BMP signaling on lateral-medial patterning of early mesoderm.

a, IFfor DAPI (blue) and PAX2 (red) at day 6 in the presence of 200ng/mL FGF9 with or without BMP4 (5 or 50 ng/mL) or the BMP antagonist NOG (25 or 250ng/mL) from day 2 to day 6. (scale = 200 μm) b, qRT-PCR to investigate the effect of this BMP/NOG gradient on the expression of PM (PARAXIS and TBX6) and LPM (FOXF1 and OSR1)markers at day 6. Error bars are s.d. (n = 3 experiments). The source data for graphs are provided in Supplementary Table 1.

Supplementary Figure 4 Schematic illustrating the anticipated gene expression of distinct progenitor and derivative cell populations during early kidney development.

PS, primitive streak; IM, intermediate mesoderm; MM, metanephric mesenchyme; NP, nephron progenitor / nephrogenic mesenchyme; RV, renal vesicle; DT, distal convoluted tubule; PT, proximal convoluted tubule; Pod, podocyte; ND, nephric duct; UB, ureteric bud/ureteric epithelium; CD, collecting duct; MET, mesenchymal to epithelial transition. All genes are indicated in italics. Shaded boxes indicate the timing and duration of expression for adjacent labeled genes. Specific genes marking DT, PT and Pod are indicated next to each cell type. The reciprocal induction of differentiation known to occur between the UB and NP is supported by the expression of FGF9 (nephrogenic mesenchyme survival) and Wnt9b (MET) and from the UB and GDNF (ureteric branching) by the NP.

Supplementary Figure 5 The positive effect of RA on ureteric epithelium formation.

a,EdU incorporation assay at day 12 of differentiation. 30 min exposure by EdU revealed that not only PAX2+ pre-epithelium structures but also PAX2 negative cells are proliferating. White arrowheads indicate EdU incorporation in PAX2+ cell. (scale = 100 μm) b, IM cells at day 6 after primitive streak induction using BMP4/Activin A were cultured for 11 days with FGF9 together with different RA concentrations. IF for UE markers, PAX2+ECAD+, showed UE structures were induced in a RA dose-dependent manner. (scale = 200 μm) c, RT-PCR at day 22 of differentiation using BMP4:Activin A/FGF9/FGF9:BMP7:RA protocol revealed the expression of genes indicative of differentiation into mature renal cell types, including SYNPO, NPHS1and WT1 for podocyte; AQP2 and SCNNB1 for distal tubule or collecting duct and AQP1 and SLC3A1 for proximal tubule. NC, negative control with no DNA template. g, IF of day 22 differentiation using BMP4/Activin A showing co-expression of two key podocyte markers; the slit-diaphragm protein SYNPO (green) and nuclear WT1 (red). Nuclei are also stained with DAPI (blue). (scale = 50 μm) The source data for graphs are provided in Supplementary Table 1.

Supplementary Figure 6 Differentiation of H9 hES cell line and iPS cell line towards renal lineages.

a, b, Immunofluorescence for DAPI (blue), PAX2 (red) or SIX2 (red) at Day 6 and Day 14 of differentiation on H9 hESC (a) and CRL2429 C11 iPS cells (b). (scale = 200 μm).

Supplementary Figure 7 The effect of 3D culture environment on self-organisation events.

a, Schematic of the replating assay. IM cells at day 6 were harvested and re-plated at high density or low density. Then cells were cultured for 12 days (6 days with 200 ng/mL FGF9 then another 6 days without growth factors). Cells plated at high density formed a uniform layer of cells while those plated at low density formed domed colonies. b, Induced IM cells at day 6 were re-plated to form monolayer or domed colonies at day 18. Cells were stained with ECAD for UE and WT1 for MM. More advanced structures are seen within domed colonies possibly due to the proximity of reciprocally inductive cell populations. (scale = 100 μm).

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Takasato, M., Er, P., Becroft, M. et al. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nat Cell Biol 16, 118–126 (2014). https://doi.org/10.1038/ncb2894

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