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

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

Chapter4 Materials and Methods

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),

and penicillin–streptomycin,Chinese hamster ovary-Leukaemia Inhibitory Factor LIF) (1000U/ml) and 1µg/ml Doxycyclin (Clontech,UK.) Theexpression of the Nodal transgene was suppressed in the presenceof 1µg/ml doxycyclin and activated when the drug was removed from the medium.

(CHO-Feeder-free undifferentiated HuES9 human ES cells were maintained on 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

matrigel-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

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 37oC 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

pSuper (1µg) (600 ng/µl) 1.65

• Use 1µg DNA : 5-10 units enzymes

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

Temp ( o C) used Time (min)

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

Temp ( o C) Time (min)

Phosphorylation reaction composition Volume (µl)

PNK (New England Biolab) 1

Ligase buffer (New England Biolab) 2

The following ligation mix was prepared and incubated overnight at 16 oC

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

Sequencingreaction composition Volume (µl)

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 oC, 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’

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 × 106 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)

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 × 105cells 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

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 × 104 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,

Oakridge, TN) The pRL-SV40 plasmid served as an internal controlfor 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, 2X105 mouse EScells were seeded whereas 4X105 mouse EScells 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

4.8 Rescue experiment for Lefty2 RNAi

4.8.1 Lefty2 RNAi rescue experiment using shRNA resistant Lefty2 overexpression

construct

2X105 mouse EScells 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

4X105 mouse EScells 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

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

4X105 mouse EScells 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

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

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