EMBO reports Fate mapping in human kidney organoids Sara E Howden et al

Expanded View Figures

A T2A EGFP pA
2 3 B
SIX2 LTL EPCAM GATA3
1 4

ex 1 ex 2 SIX2

ODNs ODNs ODNs 1+4

3+4 1+2
wt het hom
hom

hom
het

het
wt

wt

C EGFP EGFP + Brightfield

EGFP+ve
unsorted
EGFP+ve

EGFP-ve
EGFP-ve
unsorted
RT-
PCR
FACS

SIX2 GAPDH

D E
70 3 days
10 5 Het 60 4 days
% EGFP+ cells

Hom 50 5 days
10 4
40

10 3
30
20
0
10
0 10 3 10 4 10 5 0
EGFP 2 uM 4 uM 6 uM 8 uM
CHIR99021

Figure EV1. Characterization of SIX2EGFP iPSCs and SIX2EGFP kidney organoids.

A PCR analysis of SIX2 reporter iPSCs to confirm correct insertion, or heterozygous versus homozygous insertion of the EGFP reporter. ODNs used for each analysis are
marked.
B Immunofluorescence of SIX2EGFP/EGFP organoid demonstrating localization of endogenous SIX2 protein (green) within the interstitial but not epithelial compartment.
Low-magnification and high-magnification (inset) images are shown. Scale bar = 100 lm.
C RT–PCR analysis of sorted fractions from SIX2EGFP organoids shows highly enriched SIX2 expression in the EGFP+ but not EGFP cells. Scale bar = 50 lm.
D Flow cytometry analysis of SIX2EGFP/+ (heterozygous) versus SIX2EGFP/EGFP (homozygous) kidney organoids at day 13 of differentiation.
E SIX2 expression (measured as number of EGFP+ cells) positively correlates with CHIR99021 concentration and duration during the first stage of differentiation.

EV1 EMBO reports e47483 | 2019 ª 2019 The Authors

Sara E Howden et al Fate mapping in human kidney organoids EMBO reports

A
Day 10 4-OHT induction

10 5 10 5
EPCAM+
R1 =11.91%
11.91%

YG610/ 20- A
10 4 10 4
mCherry+ = 11.43%
P5 11.43%
10 3

Y
10 3

0 0

0 10 3 10 4 10 5 0 10 3 10 4 10 5
YG610/ 20- A B530/ 30- A

Day 18 4-OHT induction

P6 [TAM D7+ 11 d7+ 20_004]
P6 46.56% Events:23279
P4 [TAM D7+ 11 d7+ 20_004]
P4 6.55% Events:1526

10 5 10 5
EPCAM+
R2 0.59% = 0.59%
V450/channel

YG610/ 20- A
10 4 10 4
50- A

P7 =
mCherry+ 9.86%
9.86%
unrelated

10 3 10 3
1
mCherry

0 0

0 10 3 10 4 10 5 0 10 3 10 4 10 5
YG610/ 20- A B530/ 30- A
mCherry EPCAM

B C

25
12
% mCherry+ cells

%EPCAM+ cells

10 20
8 15
6
10
4
2 5
0 0
10 12 14 16 18 10 12 14 16 18
Day of 4-OHT induction Day of 4-OHT induction

Figure EV2. Flow cytometric analysis of SIX2CreERT2/CreERT2 kidney organoids.

A Representative plots for early (day 10) and late (day 18) 4-OHT-induced kidney organoids derived from SIX2CreERT2/CreERT2 iPSCs. The mCherry+ fraction was
plotted separately to determine the percentage of mCherry+ cells localized within EPCAM+ epithelial structures, following immunostaining with a directly
conjugated EPCAM antibody.
B, C No correlation between the time of 4-OHT treatment and the total number of mCherry+ cells (B) nor the overall fraction of EPCAM+ cells (C) was observed. Data
represent mean  SD, n ≥ 3.

ª 2019 The Authors EMBO reports e47483 | 2019 EV2