SUPPLEMENT TO
Induction of stem cell gene expression in MRPS18-2 immortalized rat
embryonic fibroblasts
Materials and Methods
Methylation of gene promoter region
DNA extraction and sodium bisulfite treatment
Genomic DNA was obtained from rat embryo fibroblasts (REFs) and 18IM cells (PureLinkTM,
Genomic DNA Mini Kit, Invitrogen, Carlsbad, CA, USA). The quality of DNA was checked by
the gel electrophoresis and spectophotometry. 1µg of the genomic DNA from both cell types
was treated by sodium bisulfite (EZ DNA Methylation Kit, Zymo Research, Irvine, CA, USA).
Modified DNA was quantified by the spectrophotometer.
PCR
From the bisulfite treated genomic DNA, CpG island rich regions from the promoter of the
genes Sox-2, Nanog, Oct-4, C-myc and Lin28 were amplified. Primer sequences used were:
Sox-2
For
Rev
5′-TTTATGTATTTAAGAGAGAGTTAATATTT-3′,
5′-
CAAAAAATTTTACATAAAAAAAA-3′; Nanog For 5′-TTTGGTAGGAGGGATTA ATTGTG-3′,
Rev
5′-TCAACCTATCTAAAAACCAACAACTC-3′;
Oct-4
For
5′-AGTTAG
GTGTTTAGTTATGGGGAAG-3′, Rev 5′-AATCT TCCAAACTTCAAACAAATTT-3′; Lin-28 For
5′-TATTTGTTAAGGGAAAAATGGATGA-3′, Rev 5′-CTTCCTAATATCCAAAAC TCCAAATC3′;
C-Myc
For
5’-TTGTTTTTTCGATTTTAGAGAG-3’,
Rev
5’-ATCCTTTC
CCTTTCTATACAAT-3’.
GeneAmp, PCR System 2700 (Applied Biosystem, Foster City, CA, USA) instrument was
used for the PCR. For each PCR reaction 50 ng of sodium bisulfite treated DNA template, 1U
of DNA polymerase (DreamTaqTM Green DNA polymerase, Fermentas, St. Leon-Rot,
Germany), and 1 µM of each primer were used. For all PCR, initial denaturation was run at
940C for 10 minutes and then 35 cycles with denaturation at 940C for 30 sec, annealing at
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different specific temperature for each gene for 30 sec. and extension at 720C for 40 sec.
was run. Final extension was allowed for 10 min. at 720C. Annealing temperature for each
amplicon, amplicon size and genomic position of the amplicon for each gene are shown in
Table S2.
Cloning of the amplified products
PCR products were separated by 2% agarose gel electrophoresis. Bands of the correct size
were extracted and purified (GeneJetTM, Gel Extraction Kit, Fermentas). The fragments were
cloned in the TOPO vector (TOPO TA cloning kit for sequencing, Invitrogen). Eight of the
colonies of the correct insert size were selected for every gene and cell type. The plasmid
DNA was purified, using Miniprep kit (GeneJETTM, Fermentas). Plasmids were sequenced,
using a capillary sequence machine from (Applied Biosystems).
Cell culture, immunostaining, and imaging
Prior to immunostaining, the cells were grown on coverslips and fixed in a 1:1 mixture of cold
methanol and acetone (-20ºC). After re-hydration in phosphate buffer saline, cells were
stained with antibodies. Exposure to mouse antibodies against the stage specific embryonic
antigen SSEA-1 (R&D System, Minneapolis, MN, USA) or -catenin (BD Pharmingen) was
followed by staining with rabbit anti-mouse FITC-conjugated antibody (DAKO). Hoechst
33258 (Sigma-Aldrich) was added at a concentration of 0.4 g/ml to the secondary antibody
for DNA staining when necessary. The images were captured using DAS microscope Leitz
DM RB with a dual mode cooled charged coupled device (CCD) camera C4880
(Hamamatsu, Japan).
Biochemical assays
Rat embryo fibroblasts and 18IM cells were assayed for L-lactate, lactate dehydrogenase
and pyruvate concentration. 2 million cells were cultured for 48 hours. Colorimetric assays
were performed, according to the manufacturer’s protocols (Bioassay System, Hayward, CA)
for the lactate (L-Lactate Assay Kit, ECLC-100), lactate dehydrogenase (Lactate
Dehydrogenase Kit, DLDH-100) and pyruvate (Pyruvate Assay Kit, EPYR-100). To assess
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the lactate concentration, the cell culture medium was collected. For lactate dehydrogenase
and pyruvate estimation, 2 million cells were sonicated in 300 µl of buffer containing 100 mM
potassium phosphate (pH 7.0) and 2 mM EDTA. Spectra Max Plus- Molecular Device
microplate reader was used to read the absorbance at 565 nm.
Results
18IM cells produce elevated levels of puryvate
As we have mentioned already, a significant set of genes that are involved in glycolysis and
red-ox reactions were differentially expressed in 18IM cells compared to REFs (Table S1).
Biochemical assays were performed to measure the concentration of lactate, pyruvate and
lactate degydrogenase activity (supplementary Figure S1). 18IM cells produced at least twice
more pyruvate, suggesting an increased level of ATP production, associated with a higher
proliferation rate.
Promoter methylation
The methylation status of the promoter region of 5 genes, Oct-4, Sox2, Nanog, Lin28, and Cmyc was compared in 18IM cells and REFs. Genomic DNA was treated with sodium bisulfite.
After PCR amplification, the DNA fragments were cloned in TOPO vector and sequenced to
investigate the methylation status. No substantial changes of the methylation status were
detected (Table S3).
Total genomic DNA methylation
To compare the overall methylation status of the genome of rat embryonic fibroblasts (REF)
and 18IM cells genomic DNA was digested with the isoschizomeric restriction enzymes, MspI
and HpaII. Digested DNA was run on the native polyacrylamide sequence gel to separate the
restricted DNA fragments. The comparison of the band pattern between REFs and 18IM cells
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confirmed that the global methylation status of the genome was not changed essentially
during the immortalization (Figure S2).
Expression of stem cell markers on 18IM cells
The expression of embryonic stem cell marker of the 18IM cells that were grown in a glass
bottle over one year, an immunostaining was assessed, using anti-SSEA1 antibody.
Approximately 30% of 18IM cells showed expression of SSEA1, like a young culture (8
month old, described in {Kashuba, 2009 #40}) (supplementary Figure S3).
Figure legends
Figure S1
Biochemical analysis of L-lactate, lactate dehydrogenase, and pyruvate concentration in
18IM cells compared to REFs. 18IM cells produced the similar amount of lactate and showed
similar level of lactate dehydrogenase activity, compared with REFs. Notice, that the level of
puryvate was much higher in 18IM cells, however.
Figure S2
Analysis of the overall methylation status of the genome 18IM cells and REF. Genomic DNA
was digested with the MspI and HpaII. Digested DNA was run on the native polyacrylamide
sequence gel. Notice, that the band pattern was quite similar between 18IM cells and REFs.
Figure S3
Immunostaining of SSEA1 and beta-catenin in 18IM cells that were kept in culture for more
than two years. A - SSEA1 staining on 18IM cells. DNA was stained in blue (a, c), SSEA1- in
green (a-d). B - Staining of beta-catenin on 18IM cells. DNA was stained in blue (a, c), betacatenin (a-d) – in green.
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Table S2. Annealing temperature, amplicon size and genomic position for some differentially
expressed genes
Name
gene
of Annealing
temperature
Amplicon
(bp)
(0C)
Sox-2
Nanog
Oct-4
Lin-28
C-myc
size Genomic
position
(Start - End)
REF
- 55
18IM
- 52
REF
- 59
18IM
- 52
REF
- 59
18IM
- 59
REF
- 60
18IM
- 59
REF
- 55
18IM
- 55
256
662 – 917
183
831 – 1013
212
3 – 214
175
524 – 698
180
429 - 609
Table S3. CpG islands methylation status
Gene
CpG island methylation status*
REFs
18IM cells
Sox-2
1 (±0.5)%
2 (±1)%
Oct-4
12.5 (±5.0)%
10 (±5.0)%
Nanog
5 (±2)%
4 (±3)%
Lin-28
No methylation
No methylation
C-myc
No methylation
No methylation
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* number of methylated CpG islands as a % of their total number in the studied region
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