Identification of factors involved in the maintenance of embryonic stem cell self renewal and pluripotency 4

Chapter4


Materials and Methods










160

4 Materials and Methods


4.1 Cell culture


4.1.1 Routine cell line maintenance

All cell cultures were maintained at 37 °C with 5% CO2 and were fed daily.
Feeder-free E14 Mouse ES cells (ATCC, Manassas, VA: CRL-1821) were cultured on
0.1% gelatin-coated dishes in E14 proliferative medium containing DMEM/15% ES cell
tested FBS (Invitrogen), 0.1 mM MEM nonessential amino acids (Invitrogen), 2mM L-
glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), and penicillin–
streptomycin (Invitrogen) and Chinese hamster ovary-Leukaemia Inhibitory Factor
(CHO-LIF) (1000 U/ml).

Feeder-free TAG1 Mouse ES cells (Gift from Dr Kian Leong Lee) were
maintained on uncoated cell culture dishes in ES cell medium containing DMEM/20%
ES FBS (Invitrogen, Carlsbad, CA), 0.1 mM MEM nonessential amino acids (Invitrogen),
2mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), and penicillin–
streptomycin and Chinese hamster ovary-Leukaemia Inhibitory Factor (CHO-LIF) (1000
U/ml).

Feeder-free tet-off Nodal E14tg2a Mouse ES cells (Gift from Dr Yuichi Hori)
were maintained on uncoated cell culture dishes in E14 proliferative medium containing
DMEM/20% ES FBS (Invitrogen,Carlsbad, CA), 0.1 mM MEM nonessential amino acids
(Invitrogen), 2mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen),
161

and penicillin–streptomycin,Chinese hamster ovary-Leukaemia Inhibitory Factor (CHO-
LIF) (1000U/ml) and 1µg/ml Doxycyclin (Clontech,UK.). The

expression of the Nodal
transgene was suppressed in the presence

of 1µg/ml doxycyclin and activated when the
drug was removed from the medium.

Feeder-free undifferentiated HuES9 human ES cells were maintained on matrigel-
coated dishes in conditioned medium containing knockout DMEM/10% serum
replacement (Invitrogen), 0.1 mM MEM nonessential amino acids (Invitrogen), 1 mM l-
glutamine (Invitrogen), 0.1 mM -mercaptoethanol (Invitrogen), 8% plasmanate
(National University Hospital Pharmacy, Singapore) and 10 ng/ml human recombinant
basic fibroblast growth factor (bFGF; Invitrogen). Conditioned medium was obtained by
culturing mouse embryonic fibroblast (MEF) cells with HuES9 medium. The medium
was collected at 24 h intervals, filter sterilized, and further supplemented with 8 ng/ml
bFGF for HuES9 cell culture.

Mouse embryonic fibroblasts MEF and human embryonic kidney HEK 293T/17
(ATCC: CRL-11268) cells were maintained in DMEM supplemented with 10% heat-
inactivated FBS and penicillin–streptomycin.

4.2 RNAi design and construction of plasmids for shRNA synthesis

For RNAi design and construction of plasmids for shRNA synthesis, 19 base-pair
gene-specific regions were designed with the computer program “siRNA studio”, using
162

the algorithm of Reynolds et al (2004). Oligonucleotides were cloned into pSuper.puro
vectors. Between two to four shRNAs were designed to target each gene for screening
based on the Oct4 and Nanog promoter- luciferase assay. All sequences were analyzed by
BLAST to ensure specificity.

The first step of shRNA construction involved the linearization of the pSuper
vector. The vector was digested for 3 hours at 37
o
C. Component of the digestion mix was

as follow:

Component Volume (µl)
10x NEB buffer 2 (New England Biolab) 2
0.1% BSA (New England Biolab) 0.2
10,000 U/ml Bgl II (New England Biolab) 1
20,000 U/ml HindIII (New England Biolab) 0.5
Water 14.65
pSuper (1µg) (600 ng/µl) 1.65
Total 20
• Use 1µg DNA : 5-10 units enzymes

The oligos (Proligo, Singapore) were then annealed under the following conditions:

Temp (
o
C) used Time (min)
95 4
70 10
Reduce temp to 4 deg Celsius slowly @ 0.1 deg Celsius/sec
4 10

Annealing reaction composition Volume (µl)
Sense Oligos 1
Antisense Oligos 1
Annealing buffer (MgACO; KoAC; HEPES) 48
Total 50

Phosphorylation of annealed oligos was then carried out under the following conditions:

Temp (
o
C) Time (min)
37 30
70 10




163




Phosphorylation reaction composition Volume (µl)
Annealed Oligos 1
PNK (New England Biolab) 1
Ligase buffer (New England Biolab) 2
Water 6
Total 10

The following ligation mix was prepared and incubated overnight at 16
o
C.

Ligation reaction composition Volume( µl)
Oligos (100x dilution from previous step) 1
T4 Ligase (New England Biolab) 1
10x Ligase Buffer (New England Biolab) 1
Cut pSuper vector 1

Water 6
Total 10


For transformation, 3µl of ligation reaction mixture was added to 50µl of
DH5α bacteria (Invitrogen). The transformation mix was incubated on ice for 30min,
followed by heat shock for 45s. After 2 minutes of incubation on ice, 500µl of SOC
medium was added to the transformation mixture and incubated in a shaking incubator
for 1 hr. 200µl of bacteria were spread on Ampicilin agar plate (100µg/ml) and
incubated overnight. 3 clones were picked for each construct and inoculated in a 4 ml LB
broth bacteria culture. Plasmid isolation and purification using Miniprep Kit (Qiagen,
Valencia, CA) was carried out the following day and the concentration of DNA was
measured using a Nanodrop spectrophotometer. The constructs were then sequenced.

Sequencingreaction composition Volume (µl)
DNA (100 ng minimum)
Sequencing primers (either forward or reverse
primers) – 1µM
Water
Volume can be adjusted accordingly
Total 6

Sequencing primers
164


Forward 5’ gCTCCAAggAATCgCgggCCCA 3’
Reverse 5’ CgCCAAgCgCgCAATTAACCCT 3’
Control shRNA

Sense shRNA
GATCCCCGAACGGCATCAAGGTGAACttcaagagaGTTCACCTTGATGCCGTTCTTTTTA
Antisense shRNA
AGCTTAAAAAGAACGGCATCAAGGTGAACtctcttgaaGTTCACCTTGATGCCGTTCGGG

Oct4 shRNA

Sense shRNA
GATCCCCGAAGGATGTGGTTCGAGTAttcaagagaTACTCGAACCACATCCTTCTTTTTA
Antisense shRNA
AGCTTAAAAAGAAGGATGTGGTTCGAGTAtctcttgaaTACTCGAACCACATCCTTCGGG

Nanog shRNA

Sense shRNA
GATCCCCGAACTATTCTTGCTTACAAttcaagagaTTGTAAGCAAGAATAGTTCTTTTTA
Antisense shRNA
AGCTTAAAAAGAACTATTCTTGCTTACAAtctcttgaaTTGTAAGCAAGAATAGTTCGGG

Lefty2 shRNAs

shRNA1 sense
GATCCCCGTGAGCTTGTCCTAACTTAttcaagagaTAAGTTAGGACAAGCTCACTTTTTA
shRNA1 antisense
AGCTTAAAAAGTGAGCTTGTCCTAACTTAtctcttgaaTAAGTTAGGACAAGCTCACGGG

shRNA2 sense
GATCCCCGCATCAACGTACCATGTCAttcaagagaTGACATGGTACGTTGATGCTTTTTA
shRNA2 antisense
AGCTTAAAAAGCATCAACGTACCATGTCAtctcttgaaTGACATGGTACGTTGATGCGGG

shRNA3 sense
GATCCCCGGTGCATGCTGTAGATGTAttcaagagaTACATCTACAGCATGCACCTTTTTA
shRNA3 antisense
AGCTTAAAAAGGTGCATGCTGTAGATGTAtctcttgaaTACATCTACAGCATGCACCGGG

shRNA4 sense
GATCCCCGGAATAGGGGAAGCTTGAAttcaagagaTTCAAGCTTCCCCTATTCCTTTTTA
shRNA4 antisense
AGCTTAAAAAGGAATAGGGGAAGCTTGAAtctcttgaaTTCAAGCTTCCCCTATTCCGGG

Lefty1 shRNAs

shRNA1 sense
GATCCCCGCACGTGAGGACTCAGTATttcaagagaATACTGAGTCCTCACGTGCTTTTTA
shRNA1 antisense
AGCTTAAAAAGCACGTGAGGACTCAGTATtctcttgaaATACTGAGTCCTCACGTGCGGG

shRNA2 sense
GATCCCCGGATGTGCCTTTCATGCAAttcaagagaTTGCATGAAAGGCACATCCTTTTTA
165

shRNA2 antisense
AGCTTAAAAAGGATGTGCCTTTCATGCAAtctcttgaaTTGCATGAAAGGCACATCCGGG

shRNA3 sense
GATCCCCGTAGCCTCATCCCTAAATTttcaagagaAATTTAGGGATGAGGCTACTTTTTA
shRNA3 antisense
AGCTTAAAAAGTAGCCTCATCCCTAAATTtctcttgaaAATTTAGGGATGAGGCTACGGG

shRNA4 sense
GATCCCCGTATGCGAAGCACTTACATttcaagagaATGTAAGTGCTTCGCATACTTTTTA
shRNA4 antisense
AGCTTAAAAAGTATGCGAAGCACTTACATtctcttgaaATGTAAGTGCTTCGCATACGGG

Rex2 shRNAs

shRNA1 sense
GATCCCCGGGCATAACCGAAAGAAGTttcaagagaACTTCTTTCGGTTATGCCCTTTTTA
shRNA1 antisense
AGCTTAAAAAGGGCATAACCGAAAGAAGTtctcttgaaACTTCTTTCGGTTATGCCCGGG

shRNA2 sense
GATCCCCGTAATGAGCTTAGAAATGTttcaagagaACATTTCTAAGCTCATTACTTTTTA

shRNA2 antisense
AGCTTAAAAAGTAATGAGCTTAGAAATGTtctcttgaaACATTTCTAAGCTCATTACGGG

shRNA3 sense
GATCCCCGACAAATGCTTACTGACAAttcaagagaTTGTCAGTAAGCATTTGTCTTTTTA
shRNA3 antisense
AGCTTAAAAAGACAAATGCTTACTGACAAtctcttgaaTTGTCAGTAAGCATTTGTCGGG

shRNA4 sense
GATCCCCGTCAAGTTGGGGTAATCATttcaagagaATGATTACCCCAACTTGACTTTTTA
shRNA4 antisense
AGCTTAAAAAGTCAAGTTGGGGTAATCATtctcttgaaATGATTACCCCAACTTGACGGG

Zfx shRNAs

shRNA1 sense
GATCCCCGTTGTGGATTCCGACATAAttcaagagaTTATGTCGGAATCCACAACTTTTTA
shRNA1 antisense
AGCTTAAAAAGTTGTGGATTCCGACATAAtctcttgaaTTATGTCGGAATCCACAACGGG

shRNA2 sense
GATCCCCGGAGGACAACGAAATGAAAttcaagagaTTTCATTTCGTTGTCCTCCTTTTTA
shRNA2 antisense
AGCTTAAAAAGGAGGACAACGAAATGAAAtctcttgaaTTTCATTTCGTTGTCCTCCGGG

shRNA3 sense
GATCCCCGCAGCTGCTTACGGTAATAttcaagagaTATTACCGTAAGCAGCTGCTTTTTA
shRNA3 antisense
AGCTTAAAAAGCAGCTGCTTACGGTAATAtctcttgaaTATTACCGTAAGCAGCTGCGGG

shRNA4 sense
GATCCCCGTGTGACATGTGCGATAAAttcaagagaTTTATCGCACATGTCACACTTTTTA
shRNA4 antisense
166

AGCTTAAAAAGTGTGACATGTGCGATAAAtctcttgaaTTTATCGCACATGTCACACGGG


4.3 PCR based cloning of V5-tagged Lefty2 open reading frame into pCAG
IRES EGFP vector for generation of LEFTY2 conditioned medium and rescue
experiment

The pCAG_Lefty2V5 construct was generated by PCR based cloning from
RIKEN full length mouse Lefty2 complementary DNA (AK131960). The forward primer
contained a Nsi1 restriction site, a Kozak sequence acc and the transcription start codon

ATG; the reverse primer includes a Bcl1 restriction site and the termination codon TGA.
The reverse primers contained a V5 tag fused at the C-terminal of Lefty2 for detection in
western blots. A V5 tag at the C-terminus of Lefty2 was chosen instead of the N-terminus
because tagging at N-terminus may result in the V5 tag being cleaved off during LEFTY2
processing and secretion. The insert was amplified by PCR, purified, digested withNsi1
and Bcl1; the pCAG vector was cut with the same retriction enzymes, treated with
alkaline phosphatase, and purified. The vector and the insert were ligated in the ratio 1:3
overnight at 16
o
C, and transformed into E. coli (DH5) cells. 5 clones were picked and
inoculated into overnight cultures. Plasmids isolation and purification using Miniprep Kit
(Qiagen, Valencia, CA) was carried out the following day and the concentration of DNA
was measured on a Nanodrop. The constructs were then verified through sequencing.

Cloning primer sequences used:
NsiILefty2V5orf_F
5’ CAATGCATaccatgaagtccctgtggctttgc 3’
167

BclILefty2V5orf_R
5’ ggagaTGATCAttaACGCGTAGAATCGAGACCGAGGAGAGGGTTAGGGAT
AGGCTTACCcagatctatccccctgggtat 3’

4.4 Generation of LEFTY2 conditioned medium

HEK 293T/17 (ATCC: CRL-11268) cells were seeded in growth medium 24
hours before transfection at a density of 6.28 × 10
6
cells per 15-cm culture dish. The cells
were transfected with 60µg of V5 tagged Lefty2 overexpression plasmids using 60µl of

Lipofectamine 2000 (Invitrogen). Conditioned medium was obtained by harvesting media
from the Lefty2 overexpressing 293T cells 16 hours after transfection. The medium was
collected at 24 h intervals for up to 4 days and filter sterilized using 0.22µM filters. To
control for factors other than Lefty2 that could have been released into the medium by
293T cells, a control conditioned medium was produced and collected from 293T cells
transfected with 60µg empty CAG vector in the exact manner described above.

4.5 ES cell transfection

4.5.1 Mouse ES cells and HEK293

Transfection of plasmids into mouse ES cells and HEK293 cells were performed
using Lipofectamine2000 (Invitrogen).

168


4.5.2 Human ES cell transfection

For transfection of siRNAs into the human ES cell line hues9, LEFTYA or non
targeting control siRNA (Dharmacon) was introduced into cells using Dharmafect 2
(Dharmacon), according to the manufacturer’s recommendation. Briefly, 100nM of
siRNA was used for each transfection of 2.5 × 10
5
cells in suspension, and subsequently
plated onto a 12-well tissue culture plate in the presence of MEFs. Retransfection was
performed on adherent cells every 24 hours, and RNA extraction and analysis was carried
out on the fifth day.

4.6 Oct4/Nanog promoter–firefly luciferase assays

Oct4 promoter- luciferase and Nanog promoter-luciferase constructs were
generated as described previously by Chew et al. (2005). ES cells were seeded 24 hr
before transfection at a density of 2.0 × 10
4
cells per well in gelatinized 96-well culture
plates. For RNAi screening experiments, gene-specific shRNA (pSuper.puro, 1g) was
cotransfected with Oct4 promoter–luciferase (75ng) and pRL-SV40 (1ng; Promega,
Madison, WI). Similarly, gene-specific shRNA (pSuper.puro, 1g) was cotransfected
with Nanog promoter–luciferase (75 ng) and pRL-SV40 (1ng; Promega, Madison, WI). A
ratio of 1µg DNA : 1µl Lipofectamine2000 was used Firefly and Renilla luciferase
activities were measured with the dual-luciferase reporter system (Promega) on the
second day of selection on the Centro LB960 Luminometer (Berthold Technologies,
169

Oakridge, TN). The pRL-SV40 plasmid served as an internal control

for normalizing the
transfection efficiency. The data generated from gene-specific shRNA cells were
expressed relative to non-targeting shRNA control transfection, after normalization to
Renilla luciferase readings.

4.7 shRNA transfection and RNAi assays

RNAi assays were carried out in either 6 or 12 well formats. For 12 well format,
2X10
5
mouse ES

cells were seeded whereas 4X10
5
mouse ES

cells were seeded for 6 well
format in growth medium. shRNA transfections were carried out the next day. 2µg and 5
µg shRNAs were used to transfect cultures on 12 and 6 well plate respectively. A ratio of
1µg DNA: 1µl Lipofectamine2000 was used. Drug selection using 1µg/ml of puromycin
(Sigma) 24 hours post-transfection was carried out to eliminate untransfected cells for the
entire duration of the experiment. The cells were fed daily with fresh growth medium
supplemented with fresh puromycin. AP staining, immunoflourescence staining and RNA
extraction and analysis were carried out on the fourth day of selection







170

4.8 Rescue experiment for Lefty2 RNAi

4.8.1 Lefty2 RNAi rescue experiment using shRNA resistant Lefty2 overexpression
construct

2X10
5
mouse ES

cells were seeded on 12 well plates in growth medium. The next
day, the cells were transfected with shRNAs- Lefty2 shRNA3 or scrambled shRNA, and
CAG vectors-empty or with V5 tagged Lefty2 open reading frame at two different ratios
of 2µg shRNA:3µg CAG vectors or 2µg shRNA:4µg CAG vector. A ratio of 1µg DNA:
0.5µl Lipofectamine2000 was used for transfection. Dual drug selection of 1µg/ml of
puromycin and 300µg/ml of Gentamycin G418 (Gibco) selection was introduced 24 hour
post transfection to select for cells transfected simultaneously with both the shRNA and
the shRNA resistant overexpression construct for three days. The samples were fed daily
with fresh growth medium supplemented with fresh puromycin and G418. RNA
extraction and analysis was carried out on the fourth day of selection.

4.8.2 Lefty2 RNAi rescue experiment with LEFTY2 conditioned medium

4X10
5
mouse ES

cells were seeded on 6 well plates in growth medium. The next
day, the cells were transfected with 5µg of shRNAs. 24 hour post transfection, the
transfection mix was replaced with growth medium conditioned with LEFTY2. At the
same time, drug selection with 1µg/ml puromycin was started and performed for three
171

days. The samples were fed daily with fresh growth medium supplemented with fresh
puromycin. AP staining was carried out on the fourth day of selection.

4.8.3 Lefty2 RNAi rescue experiment with the chemical inhibitor, SB431542 for
ALK4/5/7

4X10

5
mouse ES

cells were seeded on 6 well plates in growth medium. The next
day, the cells were transfected with 5µg of shRNAs. 24 hour post transfection, the
transfection mix was replaced with growth medium supplemented with 1µM SB431542
(Tocris Bioscience, Bristol, U.K.). At the same time, drug selection with 1µg/ml
puromycin was started and performed for three days. The samples were fed daily with
fresh growth medium supplemented with fresh puromycin and fresh SB431542. RNA
extraction and analysis was carried out on the fourth day of selection

4.9 Alkaline phosphatase staining

Detection of alkaline phosphatase, which is indicative of the nondifferentiated
state of ES cells, was carried out using a commercial ES Cell Characterization Kit from
Chemicon or a home-made AP kit (See appendix for components). Briefly, the cells were
washed three times with PBS prior to fixing in 4% formaldehyde for 1min. Before the
stain solution was added, the cells were again rinsed thrice with PBS to remove the
fixative. The samples were then incubated for 15min in the dark at room temperature,
with the stain rinsed thrice with PBS and observed under a Leica

microscope.

172

4.10 Secondary ES cell-colony replating assay/ Colony formation assay

ES cells were transfected with Lefty2 or scrambled shRNA control or empty
pSUPER shRNA constructs and selected 24 h later with puromycin at 1.0µg/ml over 5
days in growth medium without LIF. At the end of 5 days growth in selective medium,

few cells remained in the untransfected control wells indicating that selection was
effective. The surviving cells were trypsinized and resuspended in ES cell medium. 300
or 500 cells were plated onto mouse feeder layers in six-well plates for secondary ES
cell-colony formation. After 7days, emerging colonies were stained with the Wright-
Giemsa (Sigma, St. Louis, MO) stain. The undifferentiated colonies were defined based
on morphology and staining and the numbers of secondary colonies were counted for all
samples and compared.

4.11 RNA isolation, reverse transcription (cDNA synthesis) and real-time PCR
analysis

Cells were rinsed twice in ice-cold PBS. To minimize genomic DNA
contamination, total RNA was extracted with Trizol reagent (Invitrogen) and further
column purified with the RNeasy minikit (Qiagen, Valencia, CA). cDNA was
synthesized with 1.0µg total RNA using the High Capacity cDNA Archive kit (Applied
Biosystems, Foster City, CA). For each qPCR reaction, cDNA samples were first diluted
10 times in water. Endogenous mRNA levels of pluripotency and differentiation markers
were measured with inventoried Taqman probes or qPCR primers using the ABI Prism
173

7900HT Sequence Detection System 2.2 (Applied Biosystems, Foster City, CA). For
measurement made with Taqman probes, the cDNA was mixed with 5.0µl TaqMan
Universal PCR Master Mix reagent (Applied Biosystems) and 0.5µl of a single TaqMan
probe. For measurement made with qPCR primers, the cDNA was mixed with 5.0µl
TaqMan Universal PCR Master Mix reagent (Applied Biosystems, Foster City, CA) and
0.5µl of a single TaqMan probe.

For measurement made with qPCR primers, the real-time PCR mixture contained
1µl of the reverse transcription reaction in a total volume of 10µl, consisting of 5µl
SYBR Green mix reagent (Applied Biosystems), 50 nM forward primer, and 50 nM

reverse primer. Each sample was analyzed in duplicate. Results were normalized to -
actin and analyzed using the SDS 2.2 software. The experimental samples were further
normalized to the appropriate experimental samples.


4.12 Protein extraction and western blotting

To obtain protein extracts, cells were trypsinized from culture dishes harvested in
chilled PBS, centrifuged at 10,000g for 4 min at 4 °C, washed again in PBS and
incubated for 30 min on ice in ice cold lysis buffer supplemented with protease (Roche
Diagnostics, Singapore) inhibitors. For western blotting of phosphoSMAD2, the lysis
buffer was additionally supplemented with 1/100 phosphatase (Sigma, Singapore) and
50µM MG132 proteosome inhibitor (Calbiochem, UK). Lysates were cleared by
centrifugation at 12,100g, 4°C for 25 min and the supernatant was snap frozen in liquid
174

nitrogen and stored at -80
o
C. Protein concentrations were determined using Bradford
reagent (Bio-Rad, Hercules, CA). Total protein was separated by SDS–PAGE on
NuPAge gels (Invitrogen) and transferred to Hybond-P PVDF membrane (GE Healthcare,
Piscataway, NJ). The membrane was probed with specific antibodies and antibody–
protein complex as detected by HRP-conjugated antibodies and ECL luminol reagents
(Santa Cruz Biotechnology)

Antibodies

PRIMARY
ANTIBODY
COMPANY CATALOGUE

NUMBER
DILUTION RAISED IN
P-SMAD2 Calbiochem 566415 1/500 Rabbit
P-SMAD2 Abcam ab5487-50 1/500 Rabbit
SMAD2 Zymed
(Invitrogen)
51-1300 1/1000 Rabbit
P-SMAD3 Cell Signaling
Technology
9514S 1/1000 Rabbit
SMAD2/3 (N-
19)
Santa Cruz
Biotechnology
SC-6200

1/200 Goat
SMAD4 (B-8) Santa Cruz
Biotechnology
SC-7966

1/1000 Mouse
Nanog Chemicon ab5731 1/1000 Rabbit
Actin
(I-19)
Santa Cruz
Biotechnology
SC-1616 1/2500 Goat
OCT3/4 (N-19) Santa Cruz
Biotechnology

SC-8628 1/2000 Goat
CDX2 (clone
88)
BioGenex AM392-5M

1/200 Mouse
P-Cadherin AB-
1 (Clone56C1)
Thermo
Scientific
MS-1741-S0 1/100 Mouse
LEFTY Abcam ab30955-100 1/500 Rabbit
V5 Invitrogen P/N46-0705 1/5000 Mouse
Nodal R&D Systems AF1315 1/250 Mouse
Brachyury (N-
19)
Santa Cruz
Biotechnology
SC-17743 1/200 Goat
PCNA Santa Cruz
Biotechnology
SC-56342 1/5000 Mouse


175

SECONDARY
ANTIBODY
COMPANY CATALOGUE
NUMBER

DILUTION
AND
APPLICATION

RAISED IN

Anti-mouse
HRP
Santa Cruz
Biotechnology
SC-2005 1/1000 for Nodal

1/3000 for
SMAD4

1/5000 for V5

1/5000 for
PCNA

Goat
Anti-goat HRP Santa Cruz
Biotechnology
SC-2033

1/1000 for
SMAD2/3

1/3000 for Actin

1/3000 for
OCT3/4

1:1000 for
Brachyury

Donkey
Anti-rabbit HRP Santa Cruz
Biotechnology
SC-2004 1/1000 for P-
SMAD2

1?2000 for P-
SMAD3

1:2000 for
SMAD2

1:2000 for
LEFTY
Goat


4.13 Immunofluorescence staining and microscopy



Cell cultures were fixed in cold 4% paraformaldehyde at room temperature for 30
minutes, washed three times with cold PBS and permeabilized with 0.3% Triton X-100
for 5 minutes. The samples were then blocked with 1% BSA and 5% FBS in PBS for half

176

an hour. ES cells were stained with the primary antibody of interest, washed three times
with cold PBS followed by incubation with the appropriate secondary antibodies, Alexa
Fluor 594 or 488 (Molecular Probes, Carlsbad, CA) for an hour. DAPI (Molecular
Probes, Carlsbad, CA) was used to counter stain for nuclei and was added together with
the secondary antibody at a dilution of 1:1000. The antibodies were diluted in the
blocking solution. Images were captured with a Zeiss microscope and analyzed.

PRIMARY
ANTIBODY
COMPANY CATALOGUE
NUMBER
DILUTION RAISED IN
Nanog Chemicon ab5731 1/1000 Rabbit
Ki-67 Abcam ab15580 1/1000 Rabbit
Actin
(I-19)
Santa Cruz
Biotechnology
SC-1616 1/2500 Goat
CDX2 (clone
88)
BioGenex AM392-5M

1/200 Mouse
P-Cadherin AB-
1 (Clone56C1)
Thermo
Scientific

MS-1741-S0 1/100 Mouse


SECONDARY
ANTIBODY
COMPANY CATALOGUE
NUMBER
DILUTION RAISED
IN
Alexa Flour 488
anti-mouse IgG
(H+L)
Molecular probes,
Invitrogen
A11017 1/400 Goat
Alexa Flour 594
anti-rabbit IgG
(H+L)
Molecular probes,
Invitrogen
A11072 1/400 Goat
Alexa Flour488
anti-goat IgG
(H+L)
Molecular probes,
Invitrogen
A11055 1/400 Donkey
DAPI Molecular probes,
Invitrogen
D-21490 1/1000 -

177

4.14 Manipulation of Nodal signaling

Manipulation of Nodal signaling was carried out in chemically defined condition
in medium containing DMEM/15% knockout serum replacement (Invitrogen,Carlsbad,
CA), 0.1 mM MEM nonessential amino acids (Invitrogen), 2mM L-glutamine
(Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), and penicillin–streptomycin. No
Chinese hamster ovary-Leukaemia Inhibitory Factor was added.

4.14.1 Enhancement of Nodal/Activin signaling

E14, TAG1 or tet-Nodal E14tg2a cells were seeded in ES cell medium at a
density of 676 cells/cm
2
without gelatin. The next day, the seeded cells were subjected to
the following three treatments to enhance Nodal/Activin signaling in chemically defined,
LIF free medium.

4.14.1.1 Induction of Alk4* overexpression

To induce upregulation of ALK4* expression, the growth medium was replaced
with chemically defined medium containing 1µg/ml Doxycyclin (Clontech,UK.). The
TAG1 cells were fed daily with fresh medium with Doxycyclin for the entire duration of

the experiment.


178

4.14.1.2 Induction of Nodal overexpression

The

expression of the Nodal transgene was suppressed in the presence

of 1µg/ml
doxycyclin (Clontech,UK.). Hence to activate Nodal induction, the drug was removed
from the cells by changing the culture medium.

The E14tg2a cells were fed daily with
fresh medium containing no Doxycyclin.

4.14.1.3 Treatment with Activin

E14 cells were simulated with 25ng/ml Activin A (R&D Systems) in chemically
defined, LIF free medium for the entire duration of the experiment. The E14 cells were
fed daily with medium containing fresh Activin A.

4.14.2 Inhibition of Nodal/Activin signaling

To abolish/diminish Nodal/Activin signaling, the cells were treated with 10µM
SB431542 (Tocris Bioscience, Bristol, U.K.) or 100ng/ml of recombinant Lefty protein
(R&D Systems) in chemically defined, LIF free medium.

SB431542 is solubilized using DMSO, which is a potent chemical solvent and
may induce differentiation when used at concentration higher than 0.1%. Hence, to
minimize effect of DMSO on ES cell fate decisions if any, the SB431542 was first
dissolved to a very high stock concentration of 50mM prior to diluting the SB431542
5000 times to achieve the working concentration of 10µM for all the experiment
179

described below. At such a high dilution, it is unlikely that DMSO will affect the
response of ES cells to SB431542. An equivalent amount of DMSO (1:5000 dilution)
used to dissolve SB431542 was added to cultures without SB431542 to control for the
effect of DMSO

4.14.3 Inhibition of SMAD3 signaling

To abolish/diminish specifically Smad3 signaling, the cells were treated with
40µM SIS3 (Sigma Aldrich) in chemically defined, LIF free medium

4.15 Chromatin immunoprecipitation assay

4.15.1 Manipulation of SMAD2 phosphorylation level and sample preparation for
chIP

5x10
7
TAG1 cells were seeded on each 150mm plate in ES cell medium and
incubated for 18 hours. The next day, the level of phosphorylated SMAD2 of TAG1 ES
cells was manipulated using two treatments. To upregulate the level of pSMAD2, the
TAG1 ES cells were induced for ALK4* overexpression with 1µg/ml Doxycyclin in
chemically defined medium containing DMEM/20% knockout serum replacement
(Invitrogen,Carlsbad, CA), 0.1 mM MEM nonessential amino acids (Invitrogen), 2mM

L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), and penicillin–
streptomycin. To abolish SMAD2 phosphorylation, the TAG1 cells were treated with
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10µM SB431542 in the same chemically defined medium. To control for the effect of
DMSO which is used to dissolve SB431542 and to look at baseline signaling, a set of
cells were treated with the equivalent amount of DMSO (1:5000 dilution in medium)
used to dissolve 10µM SB431542. The treatment was carried out for 15 hours and then
the cells were processed for chIP as stated below.

4.15.2 Phosphorylated SMAD2 (Ser465/467) Chromatin immunoprecipitation

Cells were fixed in 1% formaldehyde for 10 min at room temperature, with
occasional swirling. Glycine was added to a final concentration of 0.125 M and the
incubation was continued for an additional 5 min. Cells were harvested with a cell
scraper and washed with ice-cold PBS three times. Cells were then resuspended and
centrifuged in lysis buffer as described in the Agilent Mammalian chIP-on-chip protocol
version 9.1. Sonication was for 15 cycles at 40% amplitude, 30s on, 60s off. Magnetic
dynal beads were prepapred according to the Agilent protocol with 10ng of P-SMAD2
antibodies (Abcam). DNA product from chIP was then washed, reverse cross-linked
proteinase K and RNaseA treated as described in the Agilent protocol. The products from
reverse cross linking were then purified with phenol/chloroform/isomyl alcohol, and the
DNA pellet was then resuspended in 300µl of 100mM Tris-HCl, pH8.0. Quantitative
PCR analyses were performed using the ABI PRISM 7900 Sequence Detection System
and SYBR Green Master Mix for all ChIP experiments. Relative occupancy values were
calculated by determining the apparent immunoprecipitation efficiency (ratios of the
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amount of immunoprecipitated DNA to that of the input sample) and normalized to the
level observed. Primer sequences are available upon request.

4.16 ES cell differentiation assay

4.16.1 Trophoblast stem cell derivation and giant cell differentiation assay

Mouse feeder conditioned medium is required as part of the culture condition for
TS cells. Briefly, the MEF conditioned medium was prepared as follow: 2X10
6

mitomycin treated MEF cells were plated in 100mm dishes and cultured in 10ml TS
medium containing RPMI 1640/20% FBS, 0.1 mM MEM nonessential amino acids
(Invitrogen), 2mM L-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen),
1mM sodium pyruvate and 50µg/ml penicillin–streptomycin for 72 hours, prior to harvest.
The collected medium was then spun to remove floating cells and debris, filtered with
0.45µM filter and stored at -20 degree Celsius until further use. The mitomycin treated
MEFs were used to prepare two more batches of conditioned medium and then discarded.

For trophoblast stem cell derivation, E14 cells were first seeded on 12 well plates
at a density of 3000 per well and incubated in growth medium overnight. The culture
dishes were not gelatinized. The next day, the growth medium was removed and replaced
with a medium composed of 70% feeder conditioned medium and 30% TS medium
supplemented with 25ng/ml FGF4 and 1.0µg/ml Heparin. The cells were fed every two
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days. To induce giant cell differentiation, FGF4 was withdrawn from the culture medium.
Similarly, the cells were fed with fresh medium with no FGF4 every two days.

To compare the trophoblastic potential of SB431542 treated cells vs non treated
cells,10µM of SB431542 was added to one culture while the controls were supplemented

with the same amount of DMSO (Sigma) used to dissolve the equivalent amount of
SB431542. On day 6, a set of cells was fixed and analyzed for differential CDX2
expression using immunofluorescence microscopy. FGF4 was withdrawn from the
remaining cultures to induce differentiation into trophoblast giant cells. On day 10, the
cells were stained and compared for P-Cadherin expression. Observation was carried out
using a Zeiss microscope.

4.17 Statistical analysis.

A Student’s non-paired t-test was used to determine the statistical significance,
where indicated.