0 10 3 10 2 10 4 10 5 A B iPSC - CTL1 iPSC - CTL2 iPSC - LND1 iPSC - LND2 SSEA3 10 5 10 4 10 3 10 2 0 iPSC - CTL1 0 10 3 10 2 10 4 10 5 TRA-1-81 iPSC - CTL2 10 5 10 4 10 3 10 2 0 0 10 3 10 2 10 4 10 5 iPSC - LND1 10 5 10 4 10 3 10 2 0 iPSC - LND2 10 5 10 4 10 3 10 2 0 0 10 3 10 2 10 4 10 5 Figure S1 - Ruillier et al- Revision SSEA3 TRA-1-81 SSEA3 TRA-1-81 SSEA3 TRA-1-81 iPSC - CTL1 iPSC - LND2 iPSC - CTL2 iPSC - LND1 Hoechst/SSEA-4/Oct-4 C Figure S1: CTL and LND donor-derived iPSC A - Bright field microscopy of colonies with typical iPSC morphology. Control HGPRT competent cells: CTL1 and CTL2. Lesch-Nyhan disease HGPRT- deficient cells: LND1 and LND2. Scale bar = 200 μm. B- Representative images of self-renewing HGPRT competent (CTL1 and CTL2) and deficient (LND1 and LND2) iPSC lines expressing the 2 pluripotency markers SSEA-4 (green) and Oct-4 (red) in colonies. Scale bar = 200 μm. C - Flow cytometry analysis of SSEA3 and TRA-1-81 pluripotency markers.
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A B iPSC - CTL1 iPSC - CTL2 iPSC - CTL2 · 2020. 2. 18. · of 4 technical replicates. CTG: Cell-TiterGlo. D- CTG estimation of cell viability after treatment of CTL and LND cell
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0 103102 104 105
A BiPSC - CTL1 iPSC - CTL2
iPSC - LND1 iPSC - LND2
SSEA
3
105
104
103
102
0
iPSC - CTL1
0 103102 104 105
TRA-1-81
iPSC - CTL2105
104
103
102
0
0 103102 104 105
iPSC - LND1
105
104
103
102
0
iPSC - LND2
105
104
103
102
0
0 103102 104 105
Figure S1 - Ruillier et al- Revision
SSEA
3
TRA-1-81
SSEA
3
TRA-1-81
SSEA
3
TRA-1-81
iPSC - CTL1
iPSC - LND2
iPSC - CTL2
iPSC - LND1
Hoe
chst
/SSE
A-4/
Oct
-4
C
Figure S1: CTL and LND donor-derived iPSC
A - Bright field microscopy of colonies with typical iPSC morphology. Control HGPRT
competent cells: CTL1 and CTL2. Lesch-Nyhan disease HGPRT- deficient cells: LND1 and
LND2. Scale bar = 200 μm. B- Representative images of self-renewing HGPRT competent
(CTL1 and CTL2) and deficient (LND1 and LND2) iPSC lines expressing the 2 pluripotency
markers SSEA-4 (green) and Oct-4 (red) in colonies. Scale bar = 200 μm. C - Flow cytometry
analysis of SSEA3 and TRA-1-81 pluripotency markers.
B
HG
PRT
activ
ity
(nm
ol N
ADH
/h/m
g of
pro
tein
)
0
2
4
6
CTL1
CTL2
LND1
LND2
iPSC
CTL1
CTL2
LND1
LND2
CTL1
CTL2
LND1
LND2
NSC Neurons
A#1
Con
trols
LNDHoe
chst
/Nes
tin/S
ox2
#2
Figure S2 - Ruillier et al- Revision
CTL1 CTL2 LND1 LND2C
Hoe
chst
/CU
X1/T
uj-1
Hoe
chst
/Tuj
-1/B
rn2
Figure S2: Phenotypical characterization of neurons differentiated from CTL and LND
stem cells
A - Representative images of neural stem cells (NSC) expressing Nestin (green) and Sox2 (red).
Scale bar = 200 μm. B - HGPRT enzymatic activity in CTL and LND-derived iPSC, NSC and
neurons. The results are expressed as nmol of NADH produced per hour and normalized to total
protein content. C – Immunostaining of the cortical superficial layer markers CUX1 and Brn2
in Tuj-1 positive neurons after 18 days of NSC differentiation. Scale bar: CUX1 = 25 μm, Brn2
= 50 μm.
Hypoxantine
Xanthine
Uric acid
IMP
InosineGuanosine Adenosine
AMPGMP
Guanine
GTP ATP
HGPRT HGPRT
Salv
age
path
way
Deg
rada
tion
De
novo
syn
thes
is p
athw
ay
PRA
PRPP
PPATGln
FGAM
FGAR
PFASGln
AZA
AZA
Figure S3 - Ruillier et al- Revision
B
Hyp
oxan
thin
e (μ
mol
/l)
0
20
40
60
80
100
CTLs
LNDs
A
0
20
40
60
80
100
Aza: 0.5 μM 2μM vGluts GABA vGluts GABA
% o
f neu
ron
sub-
type
s / D
MSO
LNDCTL
Hoe
chst
/vG
LUTs
/H
uC/D
Hoe
chst
/G
ABA
C DCTL1 CTL2 LND1 LND2
Figure S3: Inhibition of de novo synthesis using asazerine
A - Quantification of hypoxanthine synthesis in CTL and LND cells. B-. Inhibition of purine
de novo synthesis by Azaserine. C- Immunostaining of glutamatergic and GABAergic neuronal
sub-types in CTL and LND cells treated with DMSO 0.1% or azaserine 2 μM from day 5 of
differentiation. Scale bar = 25μm. D- Quantification of the different neuronal sub-types after
DMSO or azaserine treatment. Results are expressed as mean +/- SD of 2 cell lines in 3
biological replicate. C-D: The analysis of neuronal subtypes were performed at day 18 of
differentiation.
100
150
50
0-7 -6 -5 -4
EC50 = 0.52 μM
100
150
50
0% o
f rec
over
y (H
oech
st)
Log10 [M]
% of viability (H
oechst)
N6-MA
-7 -6 -5 -4
EC50 = 12.9 μM
% o
f rec
over
y (H
oech
st)
Log10 [M]
% of viability (H
oechst)
Thio-NADP
100
150
50
0
100
150
50
0
-7 -6 -5 -4
EC50 = 9.74 μM
% o
f rec
over
y (H
oech
st)
Log10 [M]
% of viability (H
oechst)
NADPH
100
150
50
0
100
150
50
0
-7 -6 -5 -4
EC50 = 6.52 μM
% o
f rec
over
y (H
oech
st)
Log10 [M]
% of viability (H
oechst)
SAM
100
150
50
0
100
150
50
0
-7 -6 -5 -4
EC50 = 18.5 μM
% o
f rec
over
y (H
oech
st)
Log10 [M]
% of viability (H
oechst)
NAD+
100
150
50
0
100
150
50
0
-7 -6 -5 -4
EC50 = 7.01 μM
% o
f rec
over
y (H
oech
st)
Log10 [M]
% of viability (H
oechst)
Ap4A
100
150
50
0
100
150
50
0
Figure S4 - Ruillier et al- Revision
Figure S4: Dose-response analysis of the 6 hit compounds efficiency using Hoechst
staining as a read-out.
The black curve corresponds to the percentage of recovery after treatment of LND1 NSC with
1.0 μM azaserine. The blue line shows the percentage of viability after treatment with the drug
alone without azaserine, representing the toxicity of the compound itself. Results are expressed
as mean ± SD of 4 technical replicates (2 biological replicates).
Non ed
ited 1
CRISPR-1
CRISPR-2
Non ed
ited 2
A
HGPRT
β-Actin
Non ed
ited 1
Non ed
ited 2
CRISPR-1
CRISPR-2
B
0
2
4
6
Non edited 1Non edited 2CRISPR-1CRISPR-2
C
HG
PRT
activ
ity
(nm
ol N
ADH
/h/m
g of
pro
tein
)
100
150
50
0-8 -7 -6 -5
% o
f via
bilit
y (C
TG)
Figure S5 - Ruillier et al- Revision
Azaserine
Log10 [M]
D
% o
f via
bilit
y (C
TG)
E
CTL1CTL2LND1LND2
100
150
50
0-3 -2 -1 0
CTL1CTL2LND1LND2
% o
f via
bilit
y (C
TG)
100
150
50
0-9 -8 -7 -6
HATLog10 [X]
RotenoneLog10 [M]
Figure S5: Primary hits compounds specificity.
A- Western-blot analysis of HGPRT protein expression in CRISPR/Cas-9 edited NSC-derived
from the human embryonic stem cells line SA001 (CTL3). Two non-edited clones and 2 clones
invalidated for HGPRT (CRISPR-1 and CRISPR-2) were compared. β-actin was used as a
loading control. B - Enzymatic HGPRT activity measurement in the same clones. C – Azaserine
selective toxic effect upon HGPRT-edited CRISPR NSC. Results are expressed as mean ± SD
of 4 technical replicates. CTG: Cell-TiterGlo. D- CTG estimation of cell viability after
treatment of CTL and LND cell lines with increasing concentrations of HAT medium
(Hypoxanthine, Aminopterin and Thymidine containing medium). The X axis represents HAT
medium concentration express as log10 (X), where the X is the dilution factor of the commercial
HAT complete medium. Results are expressed as mean ± SD of 4 technical replicates and 2
biological replicates. E- CTG estimation of cell viability after treatment of CTL and LND cell
lines with increasing concentrations of rotenone on CTL and LND NSC lines. X axis represents
compound concentration expressed as log10 (Rotenone concentration in molar M). Results are
expressed as mean ± SD of 4 technical replicates and 2 biological replicates.
LND1Azaserine + DMSO
Figure S6 - Ruillier et al- Revision
Hoe
chst
/GAB
AH
oech
st/G
ABA
Hoe
chst
/vG
luts
Hoe
chst
/vG
luts
Azaserine + SAM
Azaserine + DMSO Azaserine + SAMLND2
Figure S6: Analysis of neuronal subtypes rescued by SAM after azaserine treatment of
LND cells.
GABA: GABAergic neurons, vGluts: Glutamatergic neurons, SAM : S-adenosylmethionine.
Scale bar = 50 μm.
Table S1: List of CTL and LND hiPSC or hESC lines
Study code
Cell type
Donnor code
Cell of origin Provider Karyotype HGPRT mutation Clinical
description
CTL1 hiPSC GM01869 fibroblasts Coriell, USA 46, XY no unaffected
CTL2 hiPSC GM04603 fibroblasts Coriell, USA 46, XY no unaffected
CTL3 hESC SA001 Blastocyst Cellartis, Sweden 46, XY no unaffected
LND1 hiPSC GM02227 fibroblasts Coriell, USA 46, XY inv/del, ex6‐9