Cell culture and differentiation
The mouse ES cell line R1 was adapted to feeder-free culture. R1 ES and all genetically modified cell lines derived from them were cultivated as described previously (22). Undifferentiated cells were cultivated in DMEM media supplemented with 15% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 1x non-essential amino acid (all from Gibco-Invitrogen) and 0.05 mM β-mercaptoethanol (Sigma), supplemented with 1 000 U/ml of leukemia inhibitory factor (LIF, Chemicon). Differentiation of the cells was induced by seeding them onto a non-adhesive surface in bacteriological dishes to form floating embryoid bodies or by seeding them in the form of hanging drops (400 cells per 0.03 ml drop), all in medium without LIF. After five days of the cultivation of embryoid bodies, they were transferred to adherent tissue culture dishes and cultivated in DMEM-F12 (1:1) supplemented with insulin, transferrin, selenium (ITS, Gibco-Invitrogene), and antibiotics (as above), referred to as ITS medium. Cells were cultivated for up to 21 days depending on individual experiments and the medium was changed every two days. In the case of experiments with genetically selected cardiomyocytes, on day 14 medium was supplemented with 0.5mg/ml of antibiotic G418. An ES cell line for genetically selected cardiomyocytes was prepared subsequently: R1_MHC-neor/pGK-hygro ES cells (referred to as HG8 cells), carrying the Myh6 promoter regulating the expression of neomycin phosphotransferase, were prepared by the transfection of R1 cells by MHC- neor/pGK-hygro plasmid (kindly provided by Dr. Loren J. Field, Krannert Institute of Cardiology, Indianapolis, US) (23).
Creating KO lines using CRISPR-Cas9
DUSP7-null mouse embryonic cell lines were prepared using the CRISPR-Cas9 system as previously described (24, 25). Guide RNA was designed using the online CHOPCHOP tool (26). Plasmid pSpCas9(BB)-2A-Puro (PX459) V2.0 (#62988, Addgene) with puromycin resistance was used as the target plasmid to carry the guide RNA. Plasmids were prepared as described previously (27). For transfection, 24h after passaging, cells were transfected in a serum-free medium using polyethyleneimine (PEI) in a stoichiometry of 6 µl of PEI per 1 µg of DNA for 8h. Then, the medium was changed for medium with puromycin (Invivogen, 10ug/ml). Selection lasted for 24h, after which the medium was changed for fresh medium without puromycin, and when formed, colonies of potentially KO cells were picked. Acquired KO cell lines were tested by PCR using the primers TGTTGTGTGAGTCCTGACCG and AGAGGTAGGGCAGGATCTGG (337bp product) for the amplification of genomic DNA, and Hpy166II restriction enzyme (R0616S, New England BioLabs) (240bp and 97bp products), then verified by next generation sequencing using the Illumina platform, as described previously (28).
Cell growth
Cells were seeded at a concentration of 1000 cells/cm2 and cultivated in full medium for up to 5 days. From day 3, cells were stained with crystal violet, as previously described (29). After the colonies had dried, 10% acetic acid was added to the wells and incubated with shaking for 30min. The absorbance of the obtained solution was then measured at 550nm on a Sunrise Tecan spectrophotometer.
For proliferation analysis using the WST-8 assay, cells were seeded on a culture-treated flat bottom 96-well plate at concentrations of 1000, 500, 250, 125 and 67 cells/well and cultivated in full medium for 3 days. Cells were incubated with WST-8/Methoxy-PMS (MedChem Expres HY-D0831 and HY-D0937, final concentration 0,25mg/ml and 2,5ug/ml respectively) for 5 hours and absorbance was measured at 650 nm and 450 nm on Multiscan GO (Thermo Scientific).
For proliferation analysis using the EdU assay, cells were seeded at a concentration of 5000cells/cm2 and cultivated in full medium for 3 days. Cell proliferation was measured using the Click-iT™ Plus EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit (Thermo Fisher, C10632). Cells were treated with 10 µM EdU (5-ethynyl-2´-deoxyuridine) for one hour prior to harvesting and processed according to the kit manufacturer’s instructions. The untreated cells of each analyzed line were used as a control. Cells were analyzed using Cytek® Northern Lights spectrum flow cytometry. 20,000 events were acquired per each sample the percentage of EdU positive cells was analyzed using SpectroFlo software (Cytek). Single cells were identified and gated by pulse-code processing of the area and the width of the signal. Cell debris was excluded by using the forward scatter threshold.
Analysis of gene expression by qRT-PCR
Total RNA was extracted by RNeasy Mini Kit (Qiagen). Complementary DNA was synthesized according to the manufacturer’s instructions for RevertAid Reverse Transcriptase (200 U/µL) (EP0442, Thermo Fisher). qRT-PCR was performed in a Roche Light-cycler using the protocols for SyberGreen (Roche) or TaqMan (Roche). The protocol for primers using SyberGreen was as follows: an initial activation step at 95°C for 5 min, followed by 40 cycles at 95°C for 10 s, an annealing temperature (Table 1) for 10 s, and a temperature of 72°C for 10 s, followed by melting curve genotyping and cooling. The protocol for primers using TaqMan was as follows: an initial activation step at 95°C for 10 min followed by 45 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s with data acquisition. Primer sequences, annealing temperatures, and the probes used are listed in Table 1. The gene expression of each sample was expressed in terms of the threshold cycle normalized to the average of at least two so-called house-keeping genes. These were Actb and Tbp in the case of the SybrGreen protocol, and Rpl13a and Hprt in the case of the TaqMan protocol.
Counting of cardiomyocytes
The relative number of cardiomyocytes in differentiating ES cell cultures was determined. For these experiments, cells that were initially differentiated in the form of hanging drops were used. After 5 days of differentiation, embryoid bodies were transferred to ITS medium in 24-well plates and cultivated for a total of 20 days. Before analyses, cells were washed with phosphate buffered saline (PBS), incubated in a 0.3% solution of Collagenase II (Gibco) in DMEM media without serum for 20 minutes, and then incubated in trypsin (0.25% in PBS-EDTA, Gibco) for 5 minutes. Trypsin was inactivated by adding DMEM media with FBS, and cells in this medium were transferred to a new 24-well plate and cultivated for a further 24h. Cells were then washed with PBS, fixed for 20min with 4% formaldehyde, permeabilized by 0.1% TWEEN 20 solution in PBS, and stained using anti-cardiomyocyte heavy myosin antibody (anti-MHC, clone MF20, kindly provided by Dr. Donald Fischman, Developmental Studies Hybridoma Bank, Iowa City, IA, USA). They were then visualized using anti-mouse IgG conjugate Alexa568 (Invitrogen). Nuclei were counterstained with DAPI (1 mg/l). Images were acquired using an Olympus IX-51 inverted fluorescence microscope (Olympus) or Leica TCS SP8 (Leica) confocal microscope. In each repetition at least five images were taken from at least two wells for each line and the ratio between red and blue signals was analyzed using ImageJ software. The analysis of whole embryoid body staining was performed on cells cultivated in the same manner, but cells were seeded on day 5 on cover slips and on day 20 cells were not disassociated, but the whole embryoid body was fixed and stained as described above. Representative images were acquired using Leica TCS SP8 (Leica) confocal microscope.
The relative number of cardiomyocytes was also determined using R1_MHC-neor/pGK-hygro ES cells (HG8 cells) and their DUSP7 KO clones (MHC-neo/DUSP7 KO; analysis of frame shift mutations in both DUSP7 alleles in these cell lines by NGS shown Fig. 2A), as described previously (22). Estimation of the relative number of viable cells after antibiotic selection was performed by ATP quantification in whole cell lysates. Cells were lysed in Somatic cell ATP releasing reagent for ATP determination (FLSAR-1VL, Sigma-Aldrich). Each cell lysate was mixed with Cellular ATP Kit HTS (155-050, BioThema) in the ratio of 1:1 and luminescence was analyzed using Microlite™ 1+ strips (Thermo Scientific) and Chameleon V (Hindex).
Small Interfering RNA (siRNA) Transfection
Cells were transfected by commercially available siRNA DUSP6 (sc-39001) and DUSP7 (sc-61428) to knock down gene expression, or by related non-silencing control (all Santa Cruz Biotechnology, USA) using Lipofectamine RNAiMAX Reagents (Thermo Fisher Scientific Inc., USA) according to the manufacturer's instructions. Cells were harvested 24h after transfection and the expressions of selected proteins and posttranslational modification were analyzed by western blot (30).
Western-blot analysis
Cells were directly lysed in Laemmli buffer (100 mM Tris/HCl (pH 6.8), 20% glycerol, 1% SDS, 0.01% bromophenol blue, and 1% 2-mercaptoethanol). Western blotting was performed according to the manufacturer's instructions with minor modifications (SDS-PAGE run at 110 V, transfer onto PVDF membrane for 1 h at 110 V (BIO-RAD)). Membranes were blocked in 5% non-fat dry milk solution in TBS-T for 30 min and subsequently incubated overnight at 4°C with primary antibodies listed in Table 2. Next, membranes were washed in TBS-T and incubated with HRP-conjugated secondary antibodies (Sigma-Aldrich). Immunoreactive bands were detected using ECL detection reagent kit (Merck-Millipore) and the FusionSL chemiluminescence documentation system (Vilber-Lourmat). Results were quantified by the densitometric analysis of Western blot bands using the Fiji distribution of ImageJ.
Isolation of mouse hearts
CD1 mice were maintained and bred under standard conditions and were used in accordance with European Community Guidelines on accepted principles for the use of experimental animals. Mouse hearts were isolated according to an experimental protocol that was approved by the National and Institutional Ethics Committee (protocol MSMT-18110/2017-5). Individual heart samples were prepared as described previously (22).
Table 1
Probes and sequences of primers and temperature used in quantitative RT-PCR.
Gene of interest
|
Forward primer 5′ → 3′
|
Reverse primer 5′ → 3′
|
UPL probe no
|
Ta (°C)
|
Hprt
|
tcctcctcagaccgctttt
|
cctggttcatcatcgctaatc
|
#95
|
60
|
Rpl13a
|
catgaggtcgggtggaagta
|
gcctgtttccgtaacctcaa
|
#25
|
60
|
Nkx2.5
|
gacgtagcctggtgtctcg
|
gtgtggaatccgtcgaaagt
|
#53
|
60
|
Myh6
|
cgcatcaaggagctcacc
|
cctgcagccgcattaagt
|
#6
|
60
|
Myh7
|
cgcatcaaggagctcacc
|
ctgcagccgcagtaggtt
|
#6
|
60
|
Mef2c
|
accccaatcttctgccact
|
gatctccgcccatcagac
|
#6
|
60
|
Gata4
|
ggaagacaccccaatctcg
|
catggccccacaattgac
|
#13
|
60
|
T
|
actggtctagcctcggagtg
|
ttgctcacagaccagagactg
|
#27
|
60
|
Mesp1
|
acccatcgttcctgtacgc
|
gcatgtcgctgctgaagag
|
#89
|
60
|
Sox1
|
ccagcctccagagcccgact
|
ggcatcgcctcgctgggttt
|
|
61
|
Actb
|
gatcaagatcattgctcctcct
|
taaaacgcagctcagtaacag
|
|
60
|
Tbp
|
accgtgaatcttggctgtaaac
|
gcagcaaatcgcttgggatta
|
|
60
|
Oct4
|
agaggatcaccttggggtaca
|
cgaagcgacagatggtggtc
|
|
61
|
Nanog
|
aggacaggtttcagaagcaga
|
ccattgctagtcttcaaccactg
|
|
60
|
Zfp42
|
gcacacagaagaaagcagga
|
cactgatccgcaaacacct
|
|
59
|
Fgf5
|
aagtagcgcgacgttttcttc
|
ctggaaactgctatgttccgag
|
|
61
|
Klf4
|
gactaaccgttggcgtgag
|
gggttagcgagttcgaaagg
|
|
60
|
Dusp7
|
gcccatccgctccatcattccc
|
cagccgtcgtctcgcagcttc
|
|
62
|
Pax6
|
cgggaaagactagcagccaa
|
gtgaaggaggagacaggtgtg
|
|
62
|
Afp
|
tggttacacgaggaaagccc
|
aatgtcggccattccctcac
|
|
60
|
Gata1
|
gaagcgaatgattgtcagca
|
cagcagaggtccaggaaaag
|
|
61
|
Gata2
|
gggagtgtgtcaactgtggt
|
gcctgttaacattgtgcagc
|
|
61
|
Mash1
|
ttctccggtctcgtcctactc
|
ccagttggtaaagtccagcag
|
|
62
|
Tubb3
|
tgaggcctcctctcacaagta
|
gtcgggcctgaataggtgtc
|
|
62
|
Dusp6
|
acctggaaggtggcttcagt
|
tccgttgcactattggggtc
|
|
62
|
Table 2
Primary antibodies used for western-blot analysis.
Antibody
|
Catalog number
|
Company
|
p-ERK1/2
|
CS-4370S
|
Cell Signaling
|
ERK1/2
|
CS-4695S
|
Cell Signaling
|
PARP
|
9532
|
Cell Signaling
|
JNK
|
sc-571
|
Santa Cruz Biotechnology
|
pJNK
|
sc-6254
|
Santa Cruz Biotechnology
|
p-38
|
9212
|
Cell Signaling
|
pp-38
|
9211
|
Cell Signaling
|
MHC
|
anti-MHC, clone MF20
|
Developmental Studies Hybridoma Bank
|
βIIItubulin
|
Ab7751
|
Abcam
|
DUSP6
|
sc-377070
|
Santa Cruz Biotechnology
|
DUSP6
|
3058
|
Cell Signaling
|
Vinculin
|
V9264
|
Sigma
|
β-Actin
|
sc-47778
|
Santa Cruz Biotechnology
|
Statistical analysis
Data analysis was performed by GraphPad Prism. Data are expressed as mean ± standard deviation (SD). Statistical analysis was assessed by t-test and by one- or two-way ANOVA, and by Bonferroni’s Multiple Comparison post hoc test. Values of P < 0.05 were considered to be statistically significant (∗ p < 0.05).