Post on 17-Mar-2018
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)