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

Journal name:
Nature Cell Biology
Volume:
16,
Pages:
118–126
Year published:
DOI:
doi:10.1038/ncb2894
Received
Accepted
Published online

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.

At a glance

Figures

  1. Sequential differentiation of primitive streak and intermediate mesoderm from human ESCs.
    Figure 1: Sequential differentiation of primitive streak and intermediate mesoderm from human ESCs.

    (a) Schematic of developmental stages from inner cell mass to renal lineages. Genes shown in each stage represent specific markers of that stage. (b) FACS analysis (GFP and forward scatter (FSC)) showing the percentage of MIXL1–GFP-positive primitive streak cells induced with different ratios of BMP4/activin A (ng ml−1) or 8 μM of CHIR99021 after 3 days of culture. hESC, starting cells; No GFs (growth factors), 3 days culture with basal media. (c) Relative expression levels of SOX17, brachyury (T) and MIXL1 at day 3 for each ratio of BMP4 and activin A (ng ml−1) assessed by qRT–PCR analysis). (d) The same qRT–PCR analysis for different concentrations of CHIR99021. Error bars are s.d. (n = 3 experiments). (e) Schematic representation of the differentiation protocol used from hESC to IM. (f) RT–PCR at day 6 showing the expression of markers of IM (PAX2, LHX1, OSR1) in the presence or absence of 200 ng ml−1 FGF9 from day 2–6. (g) Quantification of the percentage of cells positive for PAX2 protein at day 6 in the presence or absence of 200 ng ml−1 FGF9 from day 2–6. Both differentiation protocols using BMP4/activin A (B/A) and CHIR99021 (CHIR) exceeded 80% induction efficiency. Error bars are s.d. (n = 5 fields in total from 3 experiments). (h) The presence and co-expression of PAX2 (red) and LHX1 (green) proteins at day 6 through primitive streak induction using either BMP4/activin A (B/A) or CHIR99021 (CHIR). Scale bars, 100 μm. (i) qRT–PCR showing the expression of markers of IM (PAX2, LHX1), PM (TBX6) and LPM (FOXF1) at day 6 across a concentration gradient of FGF9 from day 2 to 6. Error bars are s.d. (n = 3 experiments). (j) qRT–PCR showing the expression change of mesoderm markers at day 6 in the presence of FGF9 together with NOG or BMP4 from day 2 to 6. Error bars are s.d. (n = 3 experiments). (k) Immunofluorescence at day 6 showing a major IM population marked by PAX2 (red) and a non-overlapping PM marked by TBX6 (green). Scale bars, 100 μm. The source data for graphs are provided in Supplementary Table 1.

  2. Stepwise temporal induction of ureteric and metanephric progenitors from hESCs in vitro.
    Figure 2: Stepwise temporal induction of ureteric and metanephric progenitors from hESCs in vitro.

    (a) Schematic representation of the initial hESC-directed differentiation protocol used to induce kidney development (BMP4/activin AFGF9FGF9/BMP7/RA). Numbers below the line indicate the days of differentiation. (b) Time-course RT–PCR from day 0 to 17 for genes representing each stage of differentiation to kidney. These include genes for primitive streak (MIXL1, LHX1), IM (LHX1, PAX2, OSR1), MM (OSR1, SIX2, WT1, GDNF, HOXD11) and UE (PAX2, CRET, HOXB7). PAX6 was included to ensure that there was no evidence for ectodermal commitment. NC, negative control with no DNA template. (c) Time-course immunofluorescence from day 6 to 17 showing the formation of PAX2 (red) and ECAD (green) double-positive epithelial structures (upper panels) and WT1 (red)-positive populations surrounding these epithelial structures (lower panels). Scale bars, 200 μm. (d) Quantification of the proportion of WT1+ or SIX2+ cells present within hESC cultures across the directed differentiation time course. Co-expression of these proteins marks the MM/nephron progenitor population whereas WT1 protein is also expressed in subsequently differentiating nephrons. Error bars are s.d. (n = 3 experiments). The source data for the graph are provided in Supplementary Table 1. (e) Day 14 of the differentiation revealed the presence of MM (ECADSIX2+) around an ECAD+ UE (scale bar, 200 μm). (f) Schematic representation of the alternative hESC-directed differentiation protocol used to induce kidney development (CHIR99021FGF9). Numbers below the line indicate the days of differentiation. (g) Time-course RT–PCR from day 0 to 18 through differentiation using CHIR99021FGF9 representing each stage of differentiation to kidney as indicated in b. (h) Time-course immunofluorescence from day 0 to 18 through differentiation using CHIR99021FGF9 for proteins as indicated in c (scale bar, 200 μm). (i) Quantification as described in d after differentiation using CHIR99021FGF9. Error bars are s.d. (n = 5 fields in total from 3 experiments). (j) The presence of SIX2+ condensed mesenchymal cells surrounding ECAD+ UE structures at day 14 (scale bar, 100 μm). (k) Immunofluorescence microscopy at day 17 showing PAX2+GATA3+ UE at day 17 adjacent to a region of PAX2+GATA3 MM (scale bar, 50 μm).

  3. Assessment of renal potential and evidence for nephron induction of hESC after CHIR99021–FGF9-directed differentiation.
    Figure 3: Assessment of renal potential and evidence for nephron induction of hESC after CHIR99021FGF9-directed differentiation.

    (a) hESC-derived cells after day 12 of differentiation using CHIR99021FGF9 were cultured for a further 5 days with FGF9 together with different RA concentrations or without growth factors (No GFs). Immunofluorescence for PAX2 and ECAD proteins showed that UE structures were induced in a RA dose-dependent manner (scale bar, 200 μm). (b) qRT–PCR for major kidney markers (SIX2, HOXD11, HOXB7, FOXD1), a pluripotent marker (OCT4) and gonad/adrenal cortex markers (SOX9, SF1, GATA6). Gene expression levels at day 18 of differentiation using either the BMP4/activin A (B/A) or CHIR99021 (CHIR) protocol were normalized to GAPDH and then compared with levels in undifferentiated hESCs. Human fetal kidney RNA was used as a positive control. Error bars are s.d. (n = 3 experiments). The source data for the graph are provided in Supplementary Table 1. (c) Immunofluorescence showing that at day 12 of induction, some WT1+ MM cells (red) were also HOXD11+ (green). HOXD11 is a specific marker of the metanephric region, including both the MM and the renal stroma (HOXD11+WT1;  scale bar, 200 μm). (d) Low-magnification view of cultures after day 18 of differentiation (CHIR99021FGF9) using phase contrast and immunofluorescence for WT1 (red). Clusters of WT1+ mesenchyme surround the UE as would be seen in an embryonic kidney (scale bar, 200 μm). (eWT1+ and SIX2+ mesenchyme (red) tightly surrounding ECAD+ UE (green) at day 18 (scale bar, 50 μm). (f) Immunofluorescence confocal microscopy at day 18 showing PAX2+ECAD+ UE surrounded by early nephrons/renal vesicles as assessed by the presence of JAG1 and CDH6. The areas surrounded by a dashed line are PAX2+GATA3+ECAD+ UE structures. The areas indicated by a square bracket are magnified in the next right panels (scale bar, 25 μm; magnified scale bar, 10 μm).

  4. The integration of hESC-derived kidney progenitors into re-aggregates of mouse kidney cells.
    Figure 4: The integration of hESC-derived kidney progenitors into re-aggregates of mouse kidney cells.

    (a) Schematic of the re-aggregation assay of renal potential. Embryonic day 12.5–13.5 mouse kidneys were dissociated into single cells and combined with hESC-derived induced kidney cells of day 12–13, pelleted onto a filter membrane and cultured at an air–media interface for 4 days. The ratio of hESC-derived cells to mouse kidney cells was 4:96. (b) Re-aggregation assay using undifferentiated hESCs constitutively expressing GFP (ENVY cell line) as a negative control, showing undifferentiated hESC-derived large cyst formation (green). Scale bar, 200 μm. (c) Re-aggregation assay of mouse E12.5–13.5 kidney cells with hESC-derived cells after day 13 of differentiation. All integrated hESC-derived cells were detected by either human mitochondria antibody (HuMt) or a human nuclear antibody (HuNu; green). White arrowheads indicate integrated human cells into mouse renal structures. PAX2+ and CALB+ tubules represent UE. CDH6+ and JAG1+ structures represent renal vesicles. SIX2+ and WT1+ non-epithelial cells represent MM/nephron progenitors. All images show integration of hESC differentiated using the CHIR99021FGF9 protocol with the exception of the integration into CALB+ UE and SIX2+ MM where the hESC were differentiated using the BMP4/activin AFGF9FGF9/BMP7/RA protocol (scale bar, 50 μm).

  5. Evidence for self-organization after 3D culture of differentiated hESCs.
    Figure 5: Evidence for self-organization after 3D culture of differentiated hESCs.

    (a) Schematic of the process used for 3D culture. hESC-derived cells after day 18 of differentiation (CHIR99021FGF9) were collected and dissociated into single cells, pelleted and then cultured on a filter membrane at an air–media interface with 10% FCS/DMEM. After 4 days of culture, pellets were paraffin embedded and sectioned (scale bar, 200 μm). (b) Immunofluorescence of paraffin-embedded sections of the 3D cultured pellets showing the expression of a variety of key proteins (hESC-derived cells). ECAD (green) illustrates the presence of epithelium. PAX2+ epithelium represents UE whereas PAX2+ non-epithelium indicates MM and its derivatives. The co-expression of AQP2 with ECAD represents the formation of a derivative of UE, the collecting duct. WT1 staining shown here marks MM/nephron progenitors. Epithelial derivatives of MM/nephron progenitors include the renal vesicle, marked by JAG1 and proximal tubule, marked by AQP1 and SLC3A1. As a control, mouse embryonic day 13.5 kidney cells were dissociated, pelleted and then cultured in the same way as hESC-derived cells before being analysed (E13.5 mouse embryonic kidney (mEK) cells). Scale bars, 25 μm.

  6. Posterior primitive streak induction.
    Supplementary Fig. 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.

  7. Influence of FGF signaling on induction of IM proteins.
    Supplementary Fig. 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).

  8. The effect of BMP signaling on lateral-medial patterning of early mesoderm.
    Supplementary Fig. 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.

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

  10. The positive effect of RA on ureteric epithelium formation.
    Supplementary Fig. 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.

  11. Differentiation of H9 hES cell line and iPS cell line towards renal lineages.
    Supplementary Fig. 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).

  12. The effect of 3D culture environment on self-organisation events.
    Supplementary Fig. 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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Author information

Affiliations

  1. Institute for Molecular Bioscience, The University of Queensland, St Lucia 4072, Queensland, Australia

    • M. Takasato,
    • P. X. Er,
    • M. Becroft,
    • J. M. Vanslambrouck &
    • M. H. Little
  2. Murdoch Childrens Research Institute, The Royal Children’s Hospital, Flemington Road, Parkville 3052, Victoria, Australia

    • E. G. Stanley &
    • A. G. Elefanty
  3. Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton 3800, Victoria, Australia

    • E. G. Stanley &
    • A. G. Elefanty

Contributions

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.

Competing financial interests

M.H.L. consults for Organovo Inc.

Corresponding author

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Posterior primitive streak induction. (469 KB)

    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.

  2. Supplementary Figure 2: Influence of FGF signaling on induction of IM proteins. (844 KB)

    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).

  3. Supplementary Figure 3: The effect of BMP signaling on lateral-medial patterning of early mesoderm. (432 KB)

    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.

  4. Supplementary Figure 4: Schematic illustrating the anticipated gene expression of distinct progenitor and derivative cell populations during early kidney development. (168 KB)

    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.

  5. Supplementary Figure 5: The positive effect of RA on ureteric epithelium formation. (476 KB)

    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.

  6. Supplementary Figure 6: Differentiation of H9 hES cell line and iPS cell line towards renal lineages. (1,154 KB)

    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).

  7. Supplementary Figure 7: The effect of 3D culture environment on self-organisation events. (258 KB)

    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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  1. Supplementary Information (1,726 KB)

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Excel files

  1. Supplementary Table 1 (59 KB)

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  2. Supplementary Table 2 (47 KB)

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Additional data