Cell Stem Cell, volume 14

Supplemental Information Redefining the In Vivo Origin of Metanephric Nephron Progenitors Enables Generation of Complex Kidney Structures from Pluripotent Stem Cells Atsuhiro Taguchi, Yusuke Kaku, Tomoko Ohmori, Sazia Sharmin, Minetaro Ogawa, Hiroshi Sasaki, and Ryuichi Nishinakamura

Figure S1 The Osr1+/Integrina8+/Pdgfra- population represents colony-forming nephron progenitor (related to Figure1)

Figure S1 (A) Targeting strategy for Osr1-GFP knock-in mice. EGFP was inserted into the Osr1 locus, such that the N-terminal

amino acids of Osr1 were fused to EGFP. B: BamHI.

(B) Southern blot analyses of wild-type (+/+) and heterozygous (+/–) Osr1-GFP mice. The genomic DNA was digested with BamHI

and hybridized with probe A.

(C) Genomic PCR analyses.

(D) Expressions of kidney-related genes in manually picked individual colonies. The relative expression against β-actin is shown

(four colonies per marker). The first lane of each cluster represents the Wnt4 feeder cells alone without colonies. Nephric lineage

markers: Pax2 and Sall1; proximal tubule marker: Slc5a1; Henle’s loop markers: Clcnka and Clcnkb; distal tubule marker: Pou3f3;

connecting tubule marker: s100g.

(E) FACS analyses of the Osr-GFP-positive cell ratios (lower panels) in the Itga8+/Pdgfra– populations of the embryonic tissues at

the indicated stages (upper panels).

Figure S2 Nephric colonies derived from anteriorly located Osr1-GFP+ cells at E9.5 (related to Figure2)

Figure S2 (A) Sheet-like colonies were formed on Wn4-expressing feeder cells from sorted E9.5 Osr1-GFP+ cells (left panel,

bright-field image). Immunostaining for Pax2 (middle panel) and LTL (right panel) at day 15 of culture. Scale bar, 200 μm.

(B) Expressions of kidney-related genes in manually picked individual colonies. The relative expression against β-actin is shown

(three colonies per marker). The first lane of each cluster represents the Wnt4 feeder cells alone without colonies. Nephric lineage

markers: Pax2 and Sall1; proximal tubule marker: Slc5a1; Henle’s loop markers: Clcnka and Clcnkb; distal tubule marker: Pou3f3;

connecting tubule marker: s100g.

(C) The anterior (7th to 22nd somite levels) and posterior (posterior from the 23rd somite level) parts of embryos were manually

dissected and 20,000 counts of Osr1+ cells were seeded on Wnt feeder cells. The colony-forming ratios of each part are listed on

the right as the mean ± s.e.m. (n=3).

(D) Six2-GFPCreER mice were crossed with mice carrying the tdTomato reporter allele, and injected with tamoxifen at E8.75 to

transiently activate Cre around E9.5. Embryos were collected at E11.5 and immunostained for E-cadherin (green) or Pax2 (green).

tdTomato-positive red cells were detected in the mesonephros, but not in the metanephros. Scale bars, 100 μm.

Figure S3 Microarray analysis for early stage kidney precursors (related to Figure 2, 3 and 4)

Figure S3 (A) Comparison of nephron progenitor marker gene expressions in sorted early stage renal progenitors. The relative

expression level against the normalized median value for each probe is shown as the log2 fold change. E8.5: sorted

Osr1-GFP-positive (GFP+) or -negative (GFP–) cells from the E8.5 body trunk of the embryo. E9.5: sorted Osr1-GFP-positive

(GFP+) or -negative (GFP–) cells from the E9.5 body trunk of the embryo. The GFP+ population was subdivided into the

Itga8+/Pdgfra– population and others. E11.5MM: sorted Osr1-GFP-positive (GFP+) or -negative (GFP–) cells from the E11.5

manually dissected metanephric mesenchyme. The GFP+ population was subdivided into the Itga8+/Pdgfra- population and others.

Note that the metanephric nephron progenitor marker genes, such as Osr1, Wt1, Pax2, Six2, Gdnf and Crym are expressed in both

the E9.5 and E11.5 Osr1+/Itga8+/Pdgfra– populations (shown in the blue and yellow rectangles, respectively), whereas the

posterior Hox genes like Hoxa10, a11, c10, d10, d11 and d12 are enriched in the E11.5 metanephric mesenchyme (shown in the

yellow rectangle).

(B) The caudal parts of E9.5 embryos (posterior from the 23rd somite level) were dissected manually, as shown in the middle panel.

The Osr1+ cells from the caudal parts of the embryos (right panel) were sorted by FACS and cultured.

(C) FACS analysis of Itga8/Pdgfra expression in the E9.5 Osr-GFP+ posterior intermediate mesoderm.

Figure S4 Nephron progenitor induction from E9.5 posterior intermediate mesoderm (related to Figure 2 and 4)

Figure S4 (A) Comparison of the expressions of Fgf, Bmp and Wnt-related genes among sorted early stage renal precursors by

microarray analyses. The relative expression level against the normalized median value for each probe is shown as the log2 fold

change. Osr1-GFP+/Itga8+/Pdgfra– colony-forming populations at E9.5 and E11.5 are shown in the blue and yellow rectangles,

respectively.

(B) The effects of growth factors or their inhibitors in addition to Y27632 were analyzed by colony-formation assays (upper left

panel) and quantitative RT-PCR. In the quantitative RT-PCR experiments, the relative expression against the condition with Y27632

alone is presented as the mean ± s.e.m. (n=3). Y: 10µM Y27632 (Rock inhibitor); F: 5ng/ml Fgf9; SU: 10µM SU5402 (Fgfr1

inhibitor); PD: 1µM PD0325901 (selective MEK1/2 inhibitor); C1: 1 µM CHIR99021 (canonical Wnt agonist); C10: 10 µM CHIR;

XAV: 10µM XAV939 (Wnt antagonist); A: 10ng/ml activin; SB: 10µM SB431542 (Smad2/3 inhibitor); B3: 3 ng/ml Bmp4; LDN:

100nM LDN93189 (Smad1/5/8 inhibitor); R: 0.1µM retinoic acid; BMS: 10µM BMS493 (pan-RAR antagonist).

(C) Cultured Wt1-GFP+ posterior intermediate mesoderm cells at E9.5 in the presence of Fgf9 and or 1 µM CHIR. (Scale bar;

100µm)

Figure S5 Nephron progenitor induction from E8.5 posterior nascent mesoderm (related to Figure 4)

Figure S5 (A and B) The finally adopted conditions are colored in yellow. CHIR: CHIR99021 (canonical Wnt agonist); A10: 10

ng/ml activin; SB: SB431542 (Smad2/3 inhibitor); LDN: LDN93189 (Smad1/5/8 inhibitor); XAV: XAV939 (Wnt antagonist); R:

0.1 μM retinoic acid; BMS: BMS493 (pan-RAR antagonist); F: 5 ng/ml Fgf9; SU: SU5402 (Fgfr1 inhibitor). The colony-forming

ratios in each condition are presented as the mean ± s.e.m. (n=3). The finally adopted conditions are colored in yellow.

(A) The effects of growth factors or their inhibitors on the posteriorization phase were analyzed by colony-formation assays. All

conditions include Y27632 (Rock inhibitor).

(B) The effects of growth factors or their inhibitors on the nephrogenic cocktail phase were analyzed by colony-formation assays.

All conditions include Y27632 (Rock inhibitor).

Figure S6 Three-dimensional kidney structures formed by ES cells (related to Figure 6)

Figure S6 (A) Hematoxylin and eosin staining of induced EBs cocultured with Wnt4 feeder cells (right panel: higher

magnification). The nephric tubules are outlined by dashed lines. G: glomerulus. Scale bars, 200 µm (left panel), 20 µm (right

panel).

(B) Hematoxylin and eosin staining of induced EBs without factors cocultured with spinal cord (middle and right panels: higher

magnification). The neural rosette-like structures are outlined by dashed lines. SP: spinal cord. Scale bars, 200 µm (left panel), 100

µm (middle panel), 50 µm (right panel).

(C–E) Immunostaining with an anti-DsRed antibody (C–E) and for the blood vessel marker CD34 (E). (C) Lower magnification

image. (D and E) Higher magnification images of nephric tubules (D) and a glomerulus (D, E) in the graft tissue. The nephric

tubules are outlined by dashed lines. G: glomerulus; t: transplanted tissue; k: host kidney. Scale bars, 20 µm.

Figure S7 Metanephric nephron progenitor induction from human iPS cells (related to Figure 7)

Figure S7 (A and B) Immunostaining of cytocentrifuged nephron progenitor cells. (A) Immunostaining for the intermediate

mesoderm marker Wt1 in differentiated hiPS cells harvested at day 11. (B) Immunostaining for metanephric mesenchyme markers

in differentiated hiPS cells harvested at day 14. Scale bars, 100 µm. The ratios of the respective marker-positive cells are shown in

the tables.

Table S1. Osr1-GFP+ cells after E9.5 contains colony forming renal progenitors (related to Figure 1)

E8.5 E9.5 E11.5 E15.5

tissue source caudal half of the

embryo caudal half of the

embryo metanephric

mesenchyme whole kidney

Osr1-GFP positive

cells(%) 31.9±2.9 31.5±6.3 97.9±0.78 26.7±1.9

Colony formation in

Osr1-GFP+ cells(%) 0 0.037±0.013 9.1±0.48 23.3±2.8

Table S1. For the E8.5 and E9.5 experiments, the caudal parts of E8.5 embryos (posterior to heart level) and E9.5 embryos

(posterior from the forelimb level) were harvested. For the E11.5 and E15.5 experiments, embryonic metanephroi were manually

dissected. After dissociation of the harvested cells, Osr1+ cells were sorted by FACS and seeded onto Wnt4 feeder cells. At day 8,

the numbers of colonies were counted.

Table S2. Osr1-GFP+/Integrinα8+/Pdgfrα- enriches colony forming progenitors (related to Figure 1)

E9.5

(caudal trunk) E10.5

(mesonephric region)

E10.5 (metanephric

region)

E11.5 (metanephric mesenchyme)

GFP+ population(%) 31.5±6.3 22.2±3.1 31.5±2.8 97.9±0.78 Itga8+/Pdgfra-

population in GFP+

population(%) 2.93±0.50 2.85±1.4 3.45±1.3 37.4±5.0

Colony forming ratio (%) 1.10±0.26 1.47±0.20 30.9±1.5 50.9±5.2

Table S2. E9.5 or E11.5 specimens were harvested as shown in Table S1. For the E10.5 experiments, the mesonephric region (from

anterior forelimb end to anterior hindlimb end) or metanephric region (anterior hindlimb end to posterior hind limb end) were

dissected manually. The harvested tissues were dissociated and immunostained with anti-Itga8 and anti-Pdgfra antibodies.

Osr1+/Itga8+/Pdgfra– cells were sorted by FACS and seeded onto Wnt4 feeder cells. At day 8, the numbers of colonies were

counted.

Supplemental Experimental Procedures

Generation of mutant mice

GFP was inserted into the AgeI site of exon 2, such that the N-terminal 12 amino acids of Osr1 were fused in-frame to GFP. The 5’

Hpa1–AgeI Osr1 genomic fragment (2.8 kb) fused to EGFP, as well as the 3’ BamHI–BamHI fragment (5.5 kb), were incorporated

into a vector containing Neo flanked by loxP sites and DTA in tandem, as reported previously (Takasato et al., 2004). The targeting

vector was electroporated into E14.1 ES cells, and three of 480 G418-resistant clones were correctly targeted as determined by

Southern blotting analyses using 5’ probes after BamHI digestion. The correctly targeted ES clones were used to generate germline

chimeras that were bred with C57BL/6J female mice. When Neo was deleted by crossing the Osr1-GFP mutant mice with mice

expressing Cre ubiquitously, the phenotypes and EGFP expression patterns were identical to those of the original mutant mice.

Genotyping of the offspring was performed by PCR using a forward primer, 5’-TATGTTGAGGGGGCAGTAGGTTC-3’, and two

reverse primers, 5’-GTTGGGCAGGTGGTCCGAGGGCA-3’ and 5’-TAGGTCAGGGTGGTCACGAGGGT-3’, producing

products of 320 bp for the wild-type allele and 420 bp for the mutant allele.

TnEGFP-CreERT2/+ (Acc. No. CDB0604K: http:// www.cdb.riken.jp/arg/mutant%20mice%20list.html), Wt1tm1(EGFP/cre)Wtp mice,

Six2tm3(EGFP/cre/ERT2)Amc mice and Gt(ROSA)26Sortm9(CAG-tdTomato)Hze mice were purchased from Jackson Laboratory. All animal

experiments were performed in accordance with institutional guidelines and ethics review committees.

In vitro colony-formation assay

In vitro colony-formation assays were performed as described previously (Osafune et al., 2006). Briefly, progenitor cells were

sorted by a FACS Aria II (Becton Dickinson) and plated onto NIH3T3 cells stably expressing Wnt4 at a low density (1,250–10,000

cells/well of 6-well plates).

Immunostaining

Specimens were fixed in 10% formalin, embedded in paraffin and cut into 6-µm sections. Immunostaining was carried out

manually for immunofluorescence staining or automatically using a BlueMap or DABmap kit and an automated Discovery System

(Roche). For fluorescence immunohistochemistry, paraffin-embedded sections were deparaffinized and autoclaved at 121°C for 5

min in citrate buffer (pH 6.0). After incubation in blocking solution for 1 h at room temperature, the sections were incubated

overnight with primary antibodies at 4°C, followed by incubation with secondary antibodies conjugated with Alexa Fluor 488, 568,

594, 633 or 647. Nuclei were counter-stained with DAPI (Roche). For frozen sections, samples were fixed with 4%

paraformaldehyde, embedded in optimal cutting temperature (OCT) compound (Tissue Tek) and cryosectioned at 10-µm thickness.

The immunostaining procedure was the same as that for paraffin section staining. For quantification of the induced nephron

progenitor ratio, EBs were dissociated and cytocentrifuged (Cytospin 2; Shandon), followed by immunostaining. Three randomly

selected fields in each slide were captured by confocal microscopy (TSC SP2 AOBS; Leica). The images were then processed and

quantified with ImageJ, a public domain Java image processing program.

Antibodies

The antibodies used were as follows: rabbit anti-Pax2 (Covance); fluorescein anti-LTL (FL-1321; Vector Laboratories); chicken

anti-GFP (Abcam); rabbit anti-GFP (Invitrogen); rabbit anti-Itga8 (Sigma); biotinylated goat anti-Itga8 (R&D Systems); rabbit

anti-Pdgfra (Cell Signaling Technology); mouse anti-Pdgfra (Takakura et al., 1997); rabbit anti-Wt1 (Santa Cruz Biotechnology);

mouse anti-Wt1 (Dako); rabbit anti-Six2 (Proteintech); mouse anti-Sall1 (Sato et al., 2004) (PPMX Perseus Proteomics); mouse

anti-E-cadherin (BD Biosciences); rabbit anti-Cdh6 (gift from Dr. Dressler (Cho et al., 1998); mouse anti-Aqp1 (Abcam); goat

anti-Jagged1 (Santa Cruz Biotechnology); goat anti-Megalin (Santa Cruz Biotechnology); rabbit anti-Brn1 (Santa Cruz

Biotechnology); rabbit anti-NCC (Millipore); rabbit anti-Podocin (gift from Dr. Asanuma (Lydia et al., 2012); guinea-pig

anti-Nephrin (Progen); rabbit anti Pecam1 (abcam); rat anti CD34 (abcam); rabbit anti DsRed (Clontech); rabbit anti VEGF

(abcam); goat anti Ephrin B2 (R&D Systems).

Quantitative RT-PCR

RNA was isolated using an RNeasy Plus Micro Kit (Qiagen), and then reverse-transcribed with random primers and Superscript III

(Invitrogen). Quantitative PCR was carried out using a Real-Time PCR System (Applied Biosystems) and Thunderbird SYBR

qPCR Mix (Toyobo). All samples were normalized by the β-actin expression using the relative standard curve method.

Flow cytometry analysis with immunostaining

Induced cell aggregates from embryonic tissues or ES cells were dissociated by incubation with 0.25% trypsin for 5 min. After

blocking in normal mouse serum (Thermo Scientific), cell surface marker staining was carried out in buffer comprising 1% bovine

serum albumin, 1×HBSS and 0.035% NaHCO3. Data were analyzed with FlowJo software (Treestar).

Microarray analysis

Microarray analyses were performed using an Agilent SurePrint G3 mouse gene expression (8×60K) microarray. The data were

normalized by GeneSpring GX software (Agilent). The microarray data have been deposited in the National Center for

Biotechnology Information Gene Expression Omnibus (GSE51054).

Mouse ES cell culture

Mouse ES cells (Osr1-GFP) were maintained on murine embryonic fibroblasts (MEF) in DMEM (Invitrogen) supplemented with

15% fetal calf serum, 0.1 mM 2-mercaptoethanol (Nacalai Tesque) and 1,000 U/ml leukemia inhibitory factor (ESGRO).

EB3-DsRed cells, gifts from Dr. Hitoshi Niwa (CDB RIKEN), were maintained as described previously (Usui et al., 2012). Before

the initiation of differentiation, the ES cells were passaged once onto feeder cell-free gelatin-coated dishes in DMEM (Invitrogen)

supplemented with 15% fetal bovine serum, 0.1 mM 2-mercaptoethanol, 1,000 U/ml leukemia inhibitory factor, 3 µM CHIR99021

(Wako) and 1 µM PD0325901 (Wako). ES cell differentiation was carried out in serum-free medium as follows. The ES cells were

dissociated with Accutase (ESGRO) and cultured in serum-free differentiation medium comprising 75% Iscove’s modified

Dulbecco’s medium (Invitrogen) and 25% Ham’s F12 medium (Invitrogen) supplemented with 0.5× N2 and 0.5× B27 (without

retinoic acid) supplements (Invitrogen), 0.5× penicillin/streptomycin, 0.05% bovine serum albumin, 2 mM glutamine (Invitrogen),

0.5 mM ascorbic acid (Sigma) and 4.5×10–4 M 1-thioglycerol (Gadue et al., 2006).

Human iPS cell culture

Human iPS cells (201B7) were maintained on MEF in Primate ES medium (ReproCELL) supplemented with 5 ng/ml recombinant

human basic Fgf (WAKO). The iPS cell differentiation was carried out in serum-free medium as follows. The iPS cells were

detached from feeder cells by adding type 4 collagenase (Invitrogen) to a final concentration of 1 mg/ml. The detached iPS cell

colonies were harvested into a conical tube and kept still for 10 min before removal of the supernatant, to deplete the feeder cells.

The sedimented iPS colonies were dissociated with Accutase (ESGRO) and cultured in serum-free differentiation medium

comprising DMEM/F12 (Invitrogen) supplemented with 2% (vol/vol) B27 (without retinoic acid), 2 mM L-glutamine, 1% (vol/vol)

ITS, 1% (vol/vol) nonessential amino acids (without retinoic acid), 90 μM β-mercaptoethanol and 0.5× penicillin/streptomycin

(Oldershaw et al., 2010).

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