Cell Stem Cell, Volume 21

Supplemental Information

Higher-Order Kidney Organogenesis

from Pluripotent Stem Cells

Atsuhiro Taguchi and Ryuichi Nishinakamura

Figure S1. In vivo analyses of the early WD development process (related to Figure 1).

B

CUB lineage markers

(similar to Sim1)Maturation markers(similar to E-cadherin)

Maturation markers(similar to Calb1)

Tip type markers(similar to Gfra1)

A

Hoxb7-GFP

Flk1

E8.75 E9.5 E10.5 E11.5E8.5

(A) GFP expression in Hoxb7-GFP transgenic mouse embryos at the indicated stages. Scale bars, E8.5,E8.75, E10.5, E11.5: 200 µm; E9.5: 500 µm.(B) Immunostaining of E9.5 Hoxb7-GFP embryos (left three panels). As we noticed that the Hoxb7-GFPtransgenic line leaked GFP fluorescence into part of the vascular endothelial population, we sorted WDprogenitors (Hoxb7-GFP+/Flk1- fraction) by flow cytometry. Right panel: analysis by flow cytometry.Arrows: WD; arrowheads: Flk1+ endothelial cells. Scale bars: 100 µm.(C) Examples of marker genes grouped by similarity to the indicated genes. WD: Hoxb7-GFP+ Wolffianduct; WD-A: anterior Wolffian duct; WD-P: posterior Wolffian duct; IM: Wt1-GFP+ intermediate mesodermat E9.5; IM-A: Wt1-GFP+ IM of anterior trunk region (domain of the future mesonephros); IM-P: Wt1-GFP+

IM of posterior region (posterior intermediate mesoderm; precursor of MM).

Hoxb7-GFP Flk1 Hoxb7-GFP/Flk1/Dapi

E8.

75W

D

E9.

5W

D-A

E9.

5W

D-P

E10

.5W

D-A

E10

.5W

D-P

E9.

5IM

-P

E9.

5IM

-A

E11

.5U

B

E8.

75W

D

E9.

5W

D-A

E9.

5W

D-P

E10

.5W

D-P

E11

.5U

B

E8.

75W

D

E9.

5W

D-A

E9.

5W

D-P

E10

.5W

D-A

E10

.5W

D-P

E11

.5U

B

E8.

75W

D

E9.

5W

D-A

E9.

5W

D-P

E10

.5W

D-A

E10

.5W

D-P

E11

.5U

B

Similarity Gene Symbol Gene Name

1.000 Sim1 single-minded homolog 1(Drosophila)

0.695 Tfap2a transcription factor AP-2,alpha

0.658 Pax2 paired box 20.654 Gata3 GATA binding protein 30.628 Lhx1 LIM homeobox protein 1

0.612 Emx2 empty spiracleshomeobox 2

Similarity Gene Symbol Gene Name

1.000 Cdh1 cadherin 1

0.932 Clcnka chloride channel Ka

0.914 Slc25a23 solute carrier family 25,member 23

0.856 Cux2 cut-like homeobox 2

0.791 Krt8 keratin 8

0.786 Hnf1b HNF1 homeobox B

0.785 Pdpn podoplanin

0.752 L1cam L1 cell adhesionmolecule

0.711 Tbx3 T-box 3

Similarity Gene Symbol Gene Name

1.000 Calb1 calbindin 1

0.860 F2rl1 coagulation factor II (thrombin)receptor-like 1

0.845 Adamts16

a disintegrin-like andmetallopeptidase (reprolysintype) with thrombospondin type1 motif, 16

0.826 Wnt9b wingless-type MMTVintegration site 9B

0.819 Cldn3 claudin 30.765 Cldn8 claudin 80.735 Tbx2 T-box 20.724 Tfcp2l1 transcription factor CP2-like 10.714 Prdm16 PR domain containing 16

0.707 Epcam epithelial cell adhesionmolecule

Similarity Gene Symbol Gene Name

1.000 Gfra1glial cell line derivedneurotrophic factor familyreceptor alpha 1

0.799 Etv5 ets variant gene 5

0.784 Etv4ets variant gene 4 (E1Aenhancer binding protein,E1AF)

0.775 Krt20 keratin 20

0.769 Pou4f2 POU domain, class 4,transcription factor 2

0.765 Tbx22 T-box 22

0.757 Wnt11 wingless-related MMTVintegration site 11

Cut-off range [0.6,1.0], Entry genes:105

Cut-off range [0.7,1.0], Entry genes:266

Cut-off range [0.7,1.0], Entry genes:42

Cut-off range [0.75,1.0], Entry genes:388

E10

.5W

D-A

Figure S2. Screening of WD specific signals and cell surface markers (related to Figure 2).

GeneSymbol

E8.75WD

E9.5WD-A

E9.5WD-P

E10.5WD-A

E10.5WD-P

E11.5UB

E9.5IM-A

E9.5IM-P

Lgr5 4.10 1.84 3.12 2.99 2.65 2.46 -1.30Lef1 -0.45 0.48 0.82 0.82 -0.42 0.18 1.34

Axin2 0.63 0.78 0.74 1.46 1.87 -0.83 -0.91 0.82Fgfr1 -0.35 0 -0.02 -0.06 -0.16 -0.08 -0.03 -0.11Fgfr2 0.85 1.09 1.56 2.18 2.01 1.83 0 0.20Fgfr3 0.01 -0.75 -0.46 -0.87 -0.75 -0.43 0.07 -0.05Fgfr4 0.59 2.07 1.84 3.04 2.78 2.83 0.02 -0.19Etv4 3.60 1.82 3.57 0 2.69 2.59 0.56 0.89Etv4 1.90 0 1.40 -1.40 0.78 1.93 -1.55 -0.27Etv5 0.93 -0.43 0.81 -0.86 0 1.32 -0.72 0.29

Spry1 -0.33 0.32 1.34 -0.32 0.44 2.04 -0.51 -0.48Aldh1a1 2.29 2.57 6.69 2.73 6.58 6.61 1.51Aldh1a3 5.81 1.32 8.04 4.39 7.67 7.84

A

B

C

GeneSymbol

E8.75WD

E9.5WD-A

E9.5WD-P

E10.5WD-A

E10.5WD-P

E11.5UB

E9.5IM-A

E9.5IM-P

Cxcr4 2.98 2.37 2.57 -0.61 1.31 1.71 -1.62 -1.03Icam2 3.86 0.46 2.24 0 -0.54 2.40Itgb3 2.91 0 4.38 0.14 2.18 0.91 -1.77 -0.81Kit 1.81 -0.31 1.16 -0.89 0.42 0.10 -2.07 -1.81

L1cam -0.37 3.67 2.39 3.52 4.08 3.67Epcam -0.15 3.27 5.12 5.27 5.49 5.49 -2.26 -0.45Bcam 0 1.96 2.24 2.89 2.72 2.61 0.82 0.75Met -0.76 1.38 1.75 2.04 1.52 0.90 -1.97 -1.42

(A and B) Comparison of UB and MM progenitor marker gene expressions in sorted early-stageprecursors. Relative expression levels against the normalized median value for each probe are shown aslog2 fold changes. WD: Hoxb7-GFP+ Wolffian duct; WD-A: anterior Wolffian duct; WD-P: posteriorWolffian duct; IM: Wt1-GFP+ intermediate mesoderm at E9.5; IM-A: Wt1-GFP+ IM of anterior trunk region(domain of the future mesonephros); IM-P: Wt1-GFP+ IM of posterior region (posterior intermediatemesoderm; precursor of MM).(A) Gene lists related to signaling pathways.(B) Gene lists related to cell surface molecules.(C) FACS analysis of Hoxb7-GFP/Flk1 and Cxcr4/Kit expression in the E8.75 embryo proper. Themagenta dots show the Hoxb7-GFP+/Flk1- committed WD progenitor fraction. The green dots show all theother populations from the viable single cell fraction.

KitHoxb7-GFP

Cxc

r4

Flk1

UB A0 A1 A3 A10 A30

B0C10 4.0 3.2 9.1 22.0 25.1

B03C10 11.7 10.4 23.2 35.6 33.4

B1C10 25.5 30.3 25.1 22.5 12.9

NP A0 A1 A3 A10 A30

B0C10 50.4 47.3 47.2 49.0 47.4

B03C10 50.0 35.9 28.1 11.6 4.7

B1C10 5.2 1.6 0.9 0.4 0.4

Figure S3. Induction efficiency of UB and NP lineages by Step 1/2 modulation (related to Figure 3).

The upper table and graph show the induction efficiency of Cxcr4+/Kit+ committed WD progenitors frommouse ESCs at day 6.25. The lower table and graph show the induction efficiency of Itga8+/Pdgfra-

metanephric nephron progenitors from mouse ESCs at day 8.5. The applied concentrations of Step 1 Activin(A) and Step 2 Bmp4 (B) and CHIR99021 (C) are shown as follows: Activin and Bmp4: ng/ml; CHIR: µM.

Epiblast patterning

Epiblast patterning

B0C10B03C10

B1C10

0,05,0

10,015,020,025,030,035,040,0

A0 A1 A3 A10 A30

Cxcr4+/Kit+ (%) at day 6.25

B0C10B03C10

B1C10

0,0

10,0

20,0

30,0

40,0

50,0

60,0

A0 A1 A3 A10 A30

Itga8+/Pdgfra- (%) at day 8.5

Figure S4. mESC-derived UBs possess branching capacity (related to Figures 4 and 5).

A

B

NephronProgenitor(mouse ESC)

D0-2Epiblastinduction

D2-3Epiblastpatterning

D3-4.5MesodermInduction

D4.5-5.5Mesodermmaintenance

D5.5-6.5Posterior IMinduction

D6.5-9.25Metanephric NPinduction

Osr-GFP No factor A1B0 B0C10 YB0C10 YRA10B3C3 YF5C1

Hoxb7-GFP No factor A1B0 B0C10 YB0C10 YRA10B3C3 YF5C1

UretericBud(mouse ESC)

D0-2Epiblastinduction

D2-3Epiblastpatterning

D3-4.5MesodermInduction

D4.5-5.5Anterior IMInduction

D5.5-6.25Committed WDInduction

D6.25-9.25WD maturation

24hr

24hr

24hr

Osr1-GFP No factor A10B0 B03C10 RF100SB10 RC5F100 YRC1F5

YRC3F5G1

YRC3G2

Hoxb7-GFP No factor A3B0.3 B0C10 RC2F200 RC3F100 YRC1F5

YRC3F5G1

YRC3G2

D1 D2 D3 D4 D5 D6

213 4 5 6

D

E

iUB+SP iUB+iNP

Brig

htfie

ldH

oxb7

-GFP

CK8/Six2 CK8 CK8/Six2 CK8/Six2

C

CK8/Six2 CK8/Six2

F

CD31/Dapi CD31/Dapi

tdTomato

Hoxb7-GFPBright fieldG

Merged

tdTomato/Hoxb7-GFP(cytoplasm)/Six2(nuclei)/E-Cadherin/DapiH

(A) UB and nephron progenitor induction protocols for the Osr1-GFP and Hoxb7-GFP mESC lines. A: Activin (ng/ml); B:Bmp4 (ng/ml); C: CHIR99021 (µM); F: Fgf9 (ng/ml); R: retinoic acid (µM); Y: Y27632 (µM); G: Gdnf (ng/ml); SB:SB431542 (µM).(B) Upper panels: Bright-field images of induced UB branching in 50% Matrigel culture. Lower panels: Fluorescenceimages of the upper panels. Orange arrows and numbers show the generation of the indicated bifurcations. Scale bars,200 µm.(C) Reconstructed organoid from the induced UB and embryonic MM . The peripheral nephrogenic zone is shown. Notethat the Six2+ nephron progenitors surround the CK8+ ureteric tips to form the capping mesenchyme. The right panelshows a magnified image of the enclosed area in the left panel. Scale bars, 100 µm.(D) Reconstructed organoids from the indicated progenitor populations. Scale bars, 100 µm.(E) Reconstructed organoid from the iUB plus iNPs. The right two panels show magnified images of the ureteric tipregion. Scale bars, 100 µm.(F) Immunostaining of sectioned organoids. Left panel: E14.5 embryonic kidney. Right panel: Reconstructed organoid.Note that the embryonic kidney, but not the organoid, contains CD31+ vascular endothelial cells. Scale bars, 200 µm.(G, H) Reconstructed organoids from Hoxb7-GFP ESC-derived iNPs, iUB, and E11.5 tdTomato mouse embryonickidney SPs. (G) Low-magnification images of the organoids are shown. Scale bars, 100 µm. (H) Immunostaining ofsectioned organoids. The right panel shows a magnified image of the enclosed area in the left panel. Note that the Six2+

capping mesenchyme and E-Cadherin+ epithelial components (including developing nephron and ureteric epithelium)are negative for tdTomato signals. Scale bars, 50 µm (left panel), 20 µm (right two panels).

Figure S5 Reconstructed organoid resembles the E15.5 kidney in global gene expression profile(related to Figure 5).

(A) Comparison of kidney developmental gene expression profiles among mouse embryonic kidneys at variousdevelopmental stages (from E11.5 to P0), 7-day cultured E11.5 kidney, and reconstructed kidney organoids (day 0and day 7). Genes downregulated with developmental progression were selected to undergo hierarchical clusteringanalysis. ORG1, 2, 3: triplicate samples of reconstructed organoids. Day 0 represents the iUB+iNP+SP reaggregatedtissue before starting the organ culture.(B) Gene expression kinetics of mature nephric tubule (proximal tubule) and collecting duct markers examined bymicroarray analysis data. Relative expression levels against the normalized median value for each probe are shownas log2 fold changes.

A B

-8,00

-6,00

-4,00

-2,00

0

2,00

4,00

6,00

8,00

Proximal Tubule Markers

Hnf4a

Cdh16

Aqp1

Slc3a1

Slc6a13

Lrp2

Slc5a2

Cubn

Slc22a5

-5,00 -4,00 -3,00 -2,00 -1,00

0 1,00 2,00 3,00 4,00 5,00

Ureteric Trunk/Collecting DuctMarkers

Foxi1

Gsdmc

Wnt7b

Tbx2

Ehf

Pou6f1

Aqp2

Aqp3

UB B0 B1

A0 A1 A3 A10 A30 A0 A1 A3 A10 A30

L30C10 0.20 0.50 1.30 5.87 12.33 0.90 8.43 11.10 16.60 15.37

B0C10 0.27 1.77 6.57 13.93 26.93 1.03 27.67 31.70 41.23 38.80

B1C10 0.63 8.23 16.10 32.20 43.43 2.37 40.47 48.67 51.20 48.03

NP B0 B1

A0 A1 A3 A10 A30 A0 A1 A3 A10 A30

L30C10 9.67 12.27 10.47 19.57 17.50 6.87 10.40 11.90 11.70 13.73

B0C10 27.63 51.40 46.03 41.60 23.63 40.67 39.73 31.20 35.03 26.53

B1C10 40.23 24.23 15.53 3.37 3.57 35.70 7.00 6.37 4.43 2.83

Figure S6. Induction efficiency of UB and NP lineage by Step 1/2 modulation (related to Figure 6).

The upper table and graph show the induction efficiency of CXCR4+/KIT+ committed WD progenitor fromhiPSCs at day 6.25. The lower table and graph show the induction efficiency of ITGA8+/PDGFRA-

metanephric nephron progenitor from hiPSCs at day 12. The applied concentrations of Step 1 Activin (A)and Step 2 Bmp4 (B) and CHIR99021 (C) are shown as follows: Activin and Bmp4: ng/ml; CHIR: µM.

Epiblast patterning

Epiblast patterning

0,00

10,00

20,00

30,00

40,00

50,00

60,00

A0 A1 A3 A10 A30 A0 A1 A3 A10 A30

B0 B1

CXCR4+/KIT+ (%) at day6.25

L30C10

B0C10

B1C10

0,00

10,00

20,00

30,00

40,00

50,00

60,00

A0 A1 A3 A10 A30 A0 A1 A3 A10 A30

B0 B1

ITGA8+/PDGFRA- (%) at day 12

L30C10

B0C10

B1C10

Figure S7. PAX2 is cell-autonomously required for human iUB development (related to Figure 7).

KOControl

(A) Temporal kinetics of marker gene expressions in two independent control and knockout clones. Relative expressionlevels of the transcripts to β-actin expression are presented (n=3).(B) Immunostaining of a day 12.5 UB stage spheroid. High-magnification images are shown. Note that E-CADHERINprotein shows membranous accumulation in the control UB, but not in the knockout clone-derived UB. Scale bars, 10µm.(C) hiPSC-derived iNP and iUB were reconstructed and cultured in the organ culture settings. Fluorescence images ofthe encapsulated iUB tissues are shown in the lower panels. Arrowheads indicate the budding sites of the iUB. Scalebars, 200 µm.

0,01

0,1

1D6.25 D8.5 D10.5 D12.5

PAX8

Cont1 Cont2

KO1 KO2

0,001

0,01

0,1

1D6.25 D8.5 D10.5 D12.5

LHX1

Cont1 Cont2

KO1 KO2

0,001

0,01

0,1D6.25 D8.5 D10.5 D12.5

N-CAD

Cont1 Cont2

KO1 KO2

Dapi/E-CADHERIN Dapi/E-CADHERIN

A

B

C Day 0 Day 3 Day 7

Table S1. Related to Figures 1 to 7. Primer sequences.

Movie S1. Related to Figure 3. Z-stack imaging of reconstructed kidney organoid at day 7 of culture

 

Oligonucleotides mouse qRT‐PCR Primers  sequence   Actnb  CATCCGTAAAGACCTCTATGCCAAC  ATGGAGCCACCGATCCACA Pax2  AGGCATCAGAGCACATCAAATCAG  GGGTTGGCCGATGCAGATAG Lhx1  AATGTAAATGCAACCTGACCGAGAA  GCGAACCAGATCGCTTGGA Emx2  GATATCTGGGTCATCGCTTCCAA  GCTCCCACCACGTAATGGTTC Gata3  AGAGATTTCAGATCTGGGCAATGG  CAGGGACTGATTCACAGAGCATGTA En2  CGCTATTCTGACCGGCCTTC  AACTCAGCCTTGAGCCTCTGGA Wnt11  TGTGCGGACAACCTCAGCTAC  ATGGCATTTACACTTCGTTTCCAG Ret  TGAGCCCTCGGCAACATTC  GCCTCTGATGACAGCAATACTGGA Hnf1b  CGCGGTGACTCAGCTACAGAAC  TCACCAGGCTTGCAGTGGAC Wnt9b  TGGCTTTCGTGAGCATGGAG  AAAGACAGCCACGGTGTGGTAA Ecad  CACCGATGGTGAGGGTACACAG  GGCTTCAGGAATACATGGACAAAGA Calb1  CCTTTGTGGATCAATATGGACAGA  TCAGTTGCTGGCATCGAAAG Fgf5  TTGGAAATATTTGCTGTGTCTCAGG  GATCGCGGACGCATAGGTATTATAG T  CCATGCTGCAGTCCCATGA  GCTCACAGACCAGAGACTGGGATAC Cdx2  AGCTGCTGTAGGCGGAATGTATG  TCAGTGACTCGAACAGCAGCAA Tbx6  GGTCAGCCTGAGCTTGGAGA  GGTCCAGGCCAGTGACTGATAC Osr1  ACTGATGAGCGACCTTACACCTG  ACTTGTGAGTGTAGCGTCTTGTGGA Hoxb7  GACTTGGCGGCCGAGAGTAA  CCGAGTCAGGTAGCGATTGTAGTG Hoxd11  AAAAGAGCGGCGGCACA  GAGCATCCGAGAGAGTTGG Wt1  TGAAGACCCACACCAGGACTC  TGTGATGGCGGACCAATTC Six2  GCAACTTCCGCGAGCTCTAC  GCCTTGAGCCACAACTGCTG      human  qRT‐PCR Primers  sequence   ACTNB  TGGCACCCAGCACAATGAA  CTAAGTCATAGTCCGCCTAGAAGCA OSR1  GACATCTGCCACAAAGCCTTC  CCCACAGGTTCTATTTAGCATTTGA PAX2  AGGCATCAGAGCACATCAAATCAG  TCAGGGTTGGTGGATGCAGATA EMX2  AATGCGGCGAAGACTCTGG  TTTAGACGAGGGTCGCTTGTTG LHX1  GCGTCCAGTGCTGTGAATGTAA  GAAGCAGTTCAGGTGAAACACTTTG RET  TGCAATGCTGGAAGCAGGAG  ACGCCGCAAGGTCCAAGTAG HOXB7  TTGGCGGCCGAGAGTAACTT  GCGTCAGGTAGCGATTGTAGTGAA WNT11  ATGTGCGGACAACCTCAGCTAC  GATGGAGCAGGAGCCAGACA HNF1b  GCTGTGACTCAGCTGCAGAACTC  TGTACTGATGCTGCTGGTATCTGTG ECAD  ACTCGTAACGACGTTGCACCA  GGTCAGTATCAGCCGCTTTCAG CALB1  GATACTGACCACAGTGGCTTCATAG  GCCATCTCAGTTAATTCCAGCTTC PAX8  CTGAGGGCGTCTGTGACAATG  TGAATGGTTGCTGCACTTTGG NCAD  CGAATGGATGAAAGACCCATCC  GCCACTGCCTTCATAGTCAAACACT 

Table S1. (Related to Figures 1 to 7)