of 9
Chemicals
and
g
rowth factors used
in
the optimization of mNPSR
-
v2 medium
T
arget
ed
signaling pathways
Chemicals
and g
rowth factors
Wnt signaling pathway
CHIR99021
(
0.5μM, 1μM, 1.25μM, 1.5μM, 1.75μM,
2μM, 2.5μM, 3μM
)
, R
-
Spondin 1
(
50ng/m
L
,
100ng/m
L
)
,
IWR
-
1
(
0.5
μM,
2.
5μM, 5μM)
Hippo
/YAP
signaling pathway
XMU
-
MP
-
1
(
0.1μM, 0.3μM, 0.5μM, 1μM
)
, Verteporfin
(
0.1μM, 1μM
)
,
Lysophosphatidic acid (LPA)
(
1μM
)
,
Sphingosine
-
1
-
phosphate (S1P)
(
1μM
)
,
TRULI
(
1μM,
1
.5
μM
,
2μM,
2.5
μM
4μM, 10μM
)
Notch signaling
pathway
DAPT
(
2μM, 5μM, 10μM
)
TGF
-
β
and BMP
signaling pathway
Activin A
(
5ng/m
L
, 20ng/m
L
, 100ng/m
L
)
, SB431542
(
2μM, 10μM
)
, A83
-
01
(
0.05 to 0.5μM
)
,
LDN
-
193189
(
10 to 200 nM
)
, BMP4
(
5ng/m
L
,
20ng/m
L
, 100ng/m
L
)
, BMP7
(
10ng/m
L
, 50ng/m
L
,
100ng/m
L
)
FGF
signaling pathway
FGF1
(
100ng/m
L
)
, FGF2
(1
0ng/m
L
,
5
0ng/m
L
,
200ng/m
L
)
, FGF7
(
100ng/m
L
)
, FGF8
(
100ng/m
L
)
,
FGF9
(1
0ng/m
L
,
5
0ng/m
L
,
1
00ng/m
L
)
, FGF10
(
100ng/m
L
)
, FGF20
(
100ng/m
L
)
,
PD0325901
(
1μM
)
,
SB202190
(
5μM
)
,
SP600125
(
10μM
)
,
Retinoic acid
signaling pathway
All trans
-
Retinoic Acid
(
0.1μM, 1μM
)
, TTNPB
(
0.1μM,
1μM
)
LIF signaling pathway
LIF
(
1ng/m
L
, 5ng/m
L
, 10ng/m
L
)
,
JAKI
(
100nM
)
VEGF signaling pathway
VEGF
(
50ng/m
L
, 100ng/m
L
)
NF
-
κB signaling pathway
TNF
-
alpha
(
50ng/m
L
, 100ng/m
L
)
EGF
signaling pathway
EGF
(5
0ng/m
L
,
100ng/m
L
)
Insulin signaling pathway
IGF
-
1
(
100ng/m
L
)
, IGF
-
2
(
10ng/m
L
)
HGF signaling pathway
HGF
(
50ng/m
L
, 100ng/m
L
)
SCF/c
-
KIT signaling pathway
SCF
(
100ng/m
L
)
PDGF signaling pathway
PDGF
-
BB
(
100ng/m
L
)
GDNF
signaling pathway
GDNF
(
100ng/m
L
)
Others
Forskolin
(
10μM
)
, AICAR
(
0.1mM, 0.5mM
)
,
Y
-
27632
(
10μM
)
mNPSR
-
v2
m
edium
recipe
Basal medium
: DMEM/F12 (1:1) (1
X), Invitrogen, Cat. No. 11330
-
032.
Supplements
:
Reagent Name
Company
Cat. No.
Final
Concentration
GlutaMAX
-
I (100
X)
Invitrogen
35050
-
079
1
X
MEM NEAA (100
X)
Invitrogen
11140
-
050
1
X
2
-
Mercaptoethanol (55
mM)
Invitrogen
21985
-
023
0.1
1 m
M
Pen Strep (100
X)
Invitrogen
15140
-
122
1
X
B
-
27 Supplement
(50
X),
minus vitamin A
Thermo
Fisher Scientific
12587010
1
X
ITS Liquid Media
Supplement (100
X
)
Sigma
I3146
-
5ML
1
X
BMP
-
7
R&D
354
-
BP
-
010
50 ng/m
L
FGF
-
2
Peprotech
100
-
18B
200 ng/m
L
Heparin
Sigma
H3149
-
100KU
1 μg/m
L
Y
-
27632
Selleck Chemicals
S1049
10 μM
Mouse LIF
Millipore
ESG1107
1000 units/m
L
LDN193189
Selleck Chemicals
S2618
200 nM
A83
-
01
Cayman Chemical
9001799
50 nM
SB202190
Axon Medchem
1364
5 μM
DAPT
Cayman Chemical
13197
5 μM
CHIR99021
Selleck Chemicals
S1263
1.5 μM
Note:
1)
See also at the end of this file for
a working protocol
for making 100 ml of the medium.
2)
See also at the end of this file for an alternative to the commercially available 100X ITS
component from Sigma.
h
NPSR
-
v2 medium
recipe
Basal medium
:
DMEM/F12 (1:1) (1
X), Invitrogen, Cat. No. 11330
-
032.
Supplements
:
Reagent Name
Source
Cat. No.
Final Concentration
GlutaMAX
-
I (100
X)
Invitrogen
35050
-
079
1
X
MEM NEAA (100
X)
Invitrogen
11140
-
050
1
X
2
-
Mercaptoethanol (55
mM)
Invitrogen
21985
-
023
0.1
1 m
M
Pen Strep (100
X)
Invitrogen
15140
-
122
1
X
B
-
27 Supplement (50
X),
minus vitamin A
Thermo Fisher
Scientific
12587010
1
X
ITS Liquid Media
Supplement (100
X
)
Sigma
I3146
-
5ML
1
X
BMP
-
7
R&D
354
-
BP
-
010
50 ng/m
L
FGF
-
2
Peprotech
100
-
18B
200 ng/m
L
Heparin
Sigma
H3149
-
100KU
1 μg/m
L
Y
-
27632
Selleck Chemicals
S1049
10 μM
Human LIF
Millipore
LIF1050
10 ng/m
L
LDN193189
Selleck Chemicals
S2618
200 nM
A83
-
01
Cayman Chemical
9001799
50 nM
SB202190
Axon Medchem
1364
5 μM
DAPT
Cayman Chemical
13197
5 μM
CHIR99021
Selleck Chemicals
S1263
1.5 μM
TRULI
Cayman Chemical
36623
1.5
μM
Note:
1)
See also at the end of this file for
a working protocol
for making 100 ml of the medium.
2)
See also at the end of this file for an
alternative to the commercially available 100 X ITS
component from Sigma.
Preparation of 100 mL mNPSR
-
v2 medium:
93.065 mL
DMEM/F12
1 mL
GlutaMAX
-
I (100
X)
1 mL
MEM NEAA (100
X)
1 mL
Pen Strep (100
X)
200
μ
L
2
-
Mercaptoethanol (55
mM)
2 mL
B
-
27
Supplement (50
X), minus vitamin A
1 mL
ITS Liquid Media Supplement (100
X
)
200
μ
L
FGF
-
2
(FGF
-
2 Stock: 100
μ
g/mL)
100
μ
L
Heparin
(
Heparin
Stock:
1
m
g/m
L)
100
μ
L
Y
-
27632
(
Y
-
27632
Stock:
10
m
M
)
100
μ
L
LDN193189
(
LDN193189
Stock: 200
μM
)
100
μ
L A83
-
01 (A83
-
01 Stock: 50
μM
)
50
μ
L
BMP
-
7
(
BMP
-
7
Stock: 100
μ
g/mL)
50
μ
L DAPT (DAPT Stock: 10 mM)
10
μ
L
Mouse LIF
(
Mouse LIF
Stock: 10
7
units/m
L)
10
μ
L
SB202190
(
SB202190
Stock: 50 mM)
15
μ
L
CHIR99021
(
CHIR99021
Stock: 10 mM)
Preparation of 100 mL hNPSR
-
v2
medium:
93.05 mL
DMEM/F12
1 mL
GlutaMAX
-
I (100
X)
1 mL
MEM NEAA (100
X)
1 mL
Pen Strep (100
X)
200
μ
L
2
-
Mercaptoethanol (55
mM)
2 mL
B
-
27 Supplement (50
X), minus vitamin A
1 mL
ITS Liquid Media Supplement (100
X
)
200
μ
L
FGF
-
2
(FGF
-
2 Stock: 100
μ
g/mL)
100
μ
L
Heparin
(
Heparin
Stock:
1
m
g/m
L)
100
μ
L
Y
-
27632
(
Y
-
27632
Stock:
10
m
M
)
100
μ
L
LDN193189
(
LDN193189
Stock: 200
μM
)
100
μ
L A83
-
01 (A83
-
01 Stock: 50
μM
)
50
μ
L
BMP
-
7
(
BMP
-
7
Stock: 100
μ
g/mL)
50
μ
L
DAPT (DAPT Stock: 10 mM)
10
μ
L
Human LIF
(
Human LIF
Stock: 100
μ
g/mL)
10
μ
L
SB202190
(
SB202190
Stock: 50 mM)
15
μ
L
CHIR99021
(
CHIR99021
Stock: 10 mM)
15
μ
L
TRULI
(
TRULI
Stock: 10 mM)
Notes
on ITS component
:
The commercially available
ITS Liquid Media Supplement (100
X
)
(Sigma, Cat#
I3146
-
5ML
) w
as
used to make mNPSR
-
v2/hNPSR
-
v2 media based on the tables listed above. During the initial
submission, we used the commercial
ITS Liquid Media Supplement (100
X
)
to make mNPSR
-
v2/hNPSR
-
v2 media, and generated data shown in Figures 1
-
4 and 7, and Supplemental Figures
1
-
6 and 10
-
15. During the paper revision, we then found that preparing our own
ITS
solution
worked better than the commercially available ITS
Liquid Media Supplement (100
X
)
. Figures 5
and 6, and Supplemental Figures 7
-
9 were generated using NPC
s cultured with i
ndividual ITS
components prepared as below.
Reagent Name
Source
Cat. No.
Final Concentration
H
uman insulin
Sigma
91077C
-
100MG
10
μ
g/m
L
Transferrin,
Apo
-
, Human Plasma
Sigma
616395
-
100MG
5.5
μ
g/m
L
S
odium selenite
Sigma
S5261
-
10G
5
n
g/m
L
To
prepare
100
mL m/hNPSR
-
v2 medium
using individual ITS components, add
:
100
μ
L H
uman insulin
(H
uman insulin
Stock: 10 mg/mL)
100
μ
L
Transferrin, Apo
-
, Human Plasma
(
Transferrin, Apo
-
, Human Plasma
Stock: 5.5mg/mL)
10
μ
L S
odium
selenite
(S
odium selenite
Stock: 50
μ
g/m
L)
Summary of mNPC lines derived with mNPSR
-
v2 medium
mNPC cell lines
E13.5, E14.5,
E15.5, E16.5,
E18.5
Six2
-
GFP
mNPC
lines
E11.5 MM wild
type mNPC
lines
E11.5 MM
Cas9
-
GFP
mNPC lines
E11.5 MM
Six2
-
tdT
mNPC
lines
E12.5 wild
-
type whole
kidney mNPC
lines
E13.5, E15.5
Six2
-
tdT
whole
kidney mNPC
lines
E12.5
Wnt4
-
tdT
whole
kidney mNPC
lines
E12.5, E14.5, E16.5
Six2
-
GFP
clonal
mNPC lines
E11.5 MM
Cas9
-
GFP
clonal mNPC lines
E11.5 MM
Cas9
-
GFP
Pkd1
-
/
-
clonal mNPC
lines
E11.5 MM
Cas9
-
GFP
Pkd2
-
/
-
clonal mNPC
lines
Sources
Six2
-
GFP
mouse strain
Metanephric
Mesenchyme
(MM)
Whole kidney
Single mNPC Cell
Purification
method
FACS sorting
Manual
dissection
Enzymatic
dissociation
FACS sorting,
manual dissection
Derived cell
lines number
>12
>40
>15
>45
Passage #
P10
-
P15
P10
-
P31
P11
-
P13
P8
-
P10
In vitro
cultured
time (Days)
30
-
45
30
-
93
33
-
39
24
-
30
Fold of NPC
expansion
10
11
-
10
20
10
14
-
10
46
10
14
-
10
17
10
9
-
10
11
Average
passage cycle
(Days)
3
3
3
3
Doublings per
day
0.6542
-
0.6666
0.6291
-
0.6417
0.6711
-
0.7031
0.5838
-
0.5937
# of successful
differentiation
(nephron
organoid
induction)
>60
>200
>75
>250
Differentiation
competency
100%
100%
100%
100%
Oligonucleotides
qRT
-
PCR primers sequences
qRT
-
PCR Primers
(Mouse)
Gene Name
Forward Primer
Reverse Primer
Gapdh
CATGGCCTTCCGTGTTCCTA
CCTGCTTCACCACCTTCTTGAT
mNPC marker genes
Six2
AGGAAAGGGAGAACAGCGAGAA
GGACTGGACGACGAGTGGT
Wt1
CCACACCCCTACTGACAGTT
TCACTCTCATACCCTGTGCC
Osr1
CTGCCCAACCTGTATGGTTT
TGGCACTTTAGAAAAAGAGG
Eya1
GGACAGGCACCGTACAGCTACC
GTGTGCTGGATACGGCGAGCTG
Hoxd11
TGGAACGCGAGTTTTTCTTT
TTGCAGACGGTCCCTGTTCA
Pax2
AAGCCCGGAGTGATTGGTG
CAGGCGAACATAGTCGGGTT
Sall1
CTCAACATTTCCAATCCGACCC
GGCATCCTTGCTCTTAGTGGG
Gdnf
TCCAACTGGGGGTCTACGG
GCCACGACATCCCATAACTTCAT
Mouse pretubular aggregates (PTA) and renal vesicles (RV) marker genes
Pax8
GGCTCTACCTACTCTATCAA
CTGCTGCTGCTCTGTGAGTC
Lhx1
CTTCTTCCGATGTTTCGGTA
TCATGCAGGTGAAGCAGTTG
Mouse interstitial progenitor cell
(IPC)
marker gene
Foxd1
TGAGCACGGAGATGTCCGATG
CACCACGTCGATGTCTGTCTC
Differentiated
nephron marker genes
Slc12a1
TCATTGGCCTGAGGCGTAGTTG
TTTGTGCAAATAGCCGACATAGA
Slc12a3
ACACGGCAGCACCTTATACAT
GAGGAATGAATGCAGGTCAGC
Aqp1
AGGCTTCAATTACCCACTGGA
CTTTGGGCCAGAGTAGCGAT
Genes associated with cell cycle progression, mTOR signaling, and MYC activity
Cdc25a
ACAGCAGTCTACAGAGAATGGG
GATGAGGTGAAAGGTGTCTTGG
Pcna
TTTGAGGCACGCCTGATCC
GGAGACGTGAGACGAGTCCAT
Rrm2
TGGCTGACAAGGAGAACACG
AGGCGCTTTACTTTCCAGCTC
Mcm4
GAGGAAAGCAGGTCGTCACC
AGGGCTGGAAAACAAGGCATT
Tra2b
AATCCCGTTCTGCTTCCCG
TCGTGACCTTGTATAATGCCTTC
Mcm5
CAGAGGCGATTCAAGGAGTTC
CGATCCAGTATTCACCCAGGT
Mcm6
GCTGTTCCTAGACTTCCTGGA
CAACCAGCGTGTTTCTCTCAG
Bub1
AGAATGCTCTGTCAGCTCATCT
TGTCTTCACTAACCCACTGCT
Actr3
ACTGTGGCACGGGATATACAA
GTCACCCACTTTTGCAGACTC
Brca1
CTGCCGTCCAAATTCAAGAAGT
CTTGTGCTTCCCTGTAGGCT
Chek1
GTTAAGCCACGAGAATGTAGTGA
GATACTGGATATGGCCTTCCCT
Rrm2
TGGCTGACAAGGAGAACACG
AGGCGCTTTACTTTCCAGCTC
Brca2
ATGCCCGTTGAATACAAAAGGA
ACCGTGGGGCTTATACTCAGA
Hells
TGAGGATGAAAGCTCTTCCACT
ACATTTCCGAACTGGGTCAAAA
Hmgb3
AGGTGACCCCAAGAAACCAAA
GCAAAATTGACGGGAACCTCTG
Srsf3
GCGCAGATCCCCAAGAAGG
ATCGGCTACGAGACCTAGAGA
Dhcr24
CTCTGGGTGCGAGTGAAGG
TTCCCGGACCTGTTTCTGGAT
Cyb5b
GAGCCCTCCGTCACCTACTA
AGCTTTCAGTTGCATCAGCAC
Dhfr
CGCTCAGGAACGAGTTCAAGT
TGCCAATTCCGGTTGTTCAATA
Psma7
AGGAGGCGGTCAAAAAGGG
CATACAGACGTTATCGTCCAAGG
Wee1
GTCGCCCGTCAAATCACCTT
GAGCCGGAATCAATAACTCGC
Ezh2
TGCCTCCTGAATGTACTCCAA
AGGGATGTAGGAAGCAGTCATAC
Cdkn1a
CCTGGTGATGTCCGACCTG
CCATGAGCGCATCGCAATC
qRT
-
PCR Primers (Human)
hNPC marker genes
GAPDH
GTGGACCTGACCTGCCGTCT
GGAGGAGTGGGTGTCGCTGT
SIX2
AGGAAAGGGAGAACAACGAGAA
GGGCTGGA TGA TGAGTGGT
PAX2
CCCAAAGTGGTGGACAAGAT
GAAAGGCTGCTGAACTTTGG
EYA1
GGACAGGCACCATACAGCTACC
ATGTGCTGGATACGGTGAGCTG
HOXD11
TGGAACGCGAGTTTTTCTTT
CTGCAGACGGTCTCTGTTCA
OSR1
CTGCCCAACCTGTATGGTTT
CGGCACTTTGGAGAAAGAAG
SALL1
CCCCGGTTGCTAACAAAAGC
GAGGTTGTGATCGCTGAGGTA
WT1
GTGACTTCAAGGACTGTGAACG
CGGGAGAACTTTCGCTGACAA
GDNF
GGCAGTGCTTCCTAGAAGAGA
AAGACACAACCCCGGTTTTTG
Oligonucleotides
of
gRNA
and
Sanger_seq
primers
Oligonucleotides
SOURCE
IDENTIFIER
Oligonucleotides of gRNA
gRNA
-
EGFP_1: 5’
-
AAGGGCGAGGAGCTGTTCAC
-
3’
Zuo et al.,
2017
[1]
N/A
gRNA
-
EGFP_2: 5’
-
CTGAAGTTCATCTGCACCAC
-
3’
Zuo et al.,
2017
[1]
N/A
gRNA
-
EGFP_3:
5’
-
GGAGCGCACCATCTTCTTCA
-
3’
Zuo et al.,
2017
[1]
N/A
gRNA
-
Pkd1_1: 5’
-
GCTGCGCTGACGATGCCGCT
-
3’
This study
N/A
gRNA
-
Pkd1_2: 5’
-
CTGGCCGGAGACCCTGGGCG
-
3’
This study
N/A
gRNA
-
Pkd1_3: 5’
-
AGCGGCCGGAGCAATTGACG
-
3’
This study
N/A
gRNA
-
Pkd2_1:
5’
-
CGAGATGGAGCGCATCCGGC
-
3’
This study
N/A
gRNA
-
Pkd2_2: 5’
-
TCGCCCGCGCCGCGAGCGTC
-
3’
This study
N/A
gRNA
-
Pkd2_3: 5’
-
AGTGGCGCCCGGGCAGTCGG
-
3’
This study
N/A
gRNA
-
PKD2: 5’
-
CCGCGATAACCCCGGCTTCG
-
3’
This study
N/A
gRNA
-
SIX2:
5’
-
GGGCTCCTAGAACCCATTTG
-
3’
This study
N/A
gRNA
-
PAX2: 5’
-
ATGACCGCCACTAGTTACCG
-
3’
This study
N/A
Oligonucleotides of Sanger_seq primers
Primer_F_Pkd1_Sanger_seq_genotype
(CCCTCCTGAACTGCGGCT)
This study
N/A
Primer_R_Pkd1_Sanger_seq_genotype
(GGACCCAGTCATGATGCTCTA)
This study
N/A
Primer_F_Pkd2_Sanger_seq_genotype
(GCAAGCTACCCCGTAGGAATG)
This study
N/A
Primer_R_Pkd2_Sanger_seq_genotype
(GCGCAGGCAGTTGTCAAGC)
This study
N/A
Primer_F_PKD2_Sanger_seq_genotype
(GGAAGAAAGGAACATGGCTCC)
This study
N/A
Primer_R_PKD2_Sanger_seq_genotype
(TCTACGTCCATCTCCACCAC)
This study
N/A
Reference:
[1]
Zuo, E., Cai, Y.
-
J., Li, K., Wei, Y., Wang, B.
-
A., Sun, Y., Liu, Z., Liu, J., Hu, X., and Wei, W. (2017).
One
-
step generation of complete gene knockout mice and monkeys by CRISPR/Cas9
-
mediated
gene editing with multiple sgRNAs. Cell Research
27
, 933
-
945.