Temporal Gene Expression and DNA Methylation during Embryonic
Stem Cell Derivation
The first two authors equally contributed to this article.
Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor β (TGFβ) type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells (ESC). In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition.
Materials and Methods
In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast (MEF) feeder cells in both R2i and serum conventional media. The isolated inner cell mass (ICM), ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real time- polymerase chain reaction (qRT-PCR) analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC.
qRT-PCR revealed a significantly higher expression of the pluripotency-related genes (
Our investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-β signaling pathways in the first 5 days of ESC derivation.
Mouse embryonic stem cells (ESCs) are pluripotent cells that were initially isolated from blastocysts and cultured on cell cycle arrested mouse embryonic fibroblast (MEF) feeder cells using fetal calf serum (FCS) (1). Then, MEF and FCS were later replaced with leukemia inhibitory factor (LIF) (2) and bone morphogenetic protein 4 (BMP4) respectively (3). Later, the ground-state hypothesis of pluripotency by Smith and colleagues suggested that the chemical inhibition of endogenous differentiation signals, fibroblast growth factor 4 (FGF4) using PD0325901 and glycogen synthase kinase 3 (GSK3), with CHIR99021, known as 2i, can maintain cultured ESCs in the pluripotent state (4).
In another approach to the preserve ground state, Hassani et al. (5, 6), reported that dual inhibition of mitogenactivated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor ß (TGFß) type I receptors with PD0325901 and SB431542, known as R2i, results in highly-efficient generation of mouse ESCs even from refractory strains and single blastomeres (7). This medium also supports the efficient establishment of embryonic germ cell (EGC) lines from the primordial germ cells of mice (8) and rats (9).
In contrast to when the multifunctional GSK3 protein is inhibited in 2i, under R2i, the ESCs show better homogeneity (i.e. cell-to-cell conformity in expression of pluripotency genes such as Nanog and Dppa3), genomic integrity, and ground-state pluripotency. In which a less complex condition is required for investigating the molecular mechanisms of ground-state pluripotency (5, 6, 10).
These advantages of R2i prompted us to further assess the molecular mechanisms that underlie the transition from inner cell mass (ICM) to ESC. Recently, Totonchi et al. (11), reported the key genes involved in the transition from ICM to ESC via temporal microarray gene expression analysis. They also used deep hairpin bisulfite sequencing (DHBS) to show the methylation of individual CpG sites for three classes of repetitive elements, micro satellites (mSats), the 5´ untranslated region of L1Md_Tf (L1), and a class of LTR-retrotransposons (IAP-LTR1) (11, 12). Their results indicated that DNA methyltransferases play a pivotal role in efficient ESC generation. However, the exact molecular mechanisms through which the derivation of ESCs takes place still needed to be clarified.
Here, we assessed the expression of key genes involved
in pluripotency, epigenetic and early differentiation
using quantitative real time-polymerase chain reaction
(qRT-PCR) Then, the DNA methylation status of
cytosine guanine dinucleotides (CpG) upstream of the
transcription starting site of two pluripotency-related
Materials and Methods
Mice, embryos and media
We collected E3.5 blastocysts by flushing the uteri of BALB/c (for qRT-PCR analysis) and NMRI (for R2i time point analysis) mouse strains after superovulation. Immunosurgery was performed to isolate ICMs from the blastocysts. Derivation of ESCs was done by plating the zona-free whole E3.5 blastocysts on NMRI strain-derived MEF feeder cells in R2i and serum conventional medium, as previously described (13). R2i medium was composed of DMEM/F12 (Invitrogen, USA) and neurobasal (Invitrogen, USA) in a 1:1 ratio, 1% N2 supplement (Invitrogen, USA), 1% B27 supplement (Invitrogen, USA), 1% nonessential amino acids (Invitrogen, USA), 2 mM L-glutamine (Invitrogen, USA), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, USA), 0.1 mM ß-mercaptoethanol (Sigma-Aldrich, USA), 5 mg/mL bovine serum albumin (Sigma-Aldrich, USA), 1000 U/mlLIF (Royan BioTech, Iran), 1 µM PD0325901 (Stemgent, USA) and 10 µM SB431542 (Sigma-Aldrich, USA). Serum medium consisted of knockout Dulbecco’s modified Eagle’s medium (Invitrogen), 15% fetal bovine serum (FBS, HyClone), 1% nonessential amino acids, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin (Invitrogen, USA), 0.1 mM ß-mercaptoethanol, and 1000 U/ml mouse LIF.
The isolated ICMs, ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture in three independent replicates and stored at -80oC. Each experimental group included 20 to 30 embryos/ outgrowths. All experiments were approved by the Ethical Committee of Royan Institute.
RNA isolation and quantitative real time-PCR
Total RNA was extracted from the three independent replicates using RNeasy micro kit (Qiagen, USA). The purity and concentration of the RNA was assessed and quantified by measuring the absorbance A260 nm/ A280 nm using a Biowave II spectrophotometer (WPA, Biochrom, UK). The quality and integrity of the total RNA was verified by electrophoresis. A total amount of 2 µg of total RNA was converted into cDNA using RevertAid cDNA synthesis kit and Random hexamer primers (Thermo Fisher Scientific, USA).
Quantitative real time PCR was carried out using SYBR
Green master mix (ABI, Step one plus, USA). Primers
(Table S1,) (See Supplementary Online Information
DNA methylation assay
Pure DNA (1 µg) was treated with EpiTect Bisulfite
Kit (Qiagen, USA). Semi-nested methylation specific
primers (MSP) were designed for 2 promoter regions of
two pluripotency-related genes (
R: 5´-CCACCCTCTAACCTTAACCTC TAAC-3´
The PCR cycling program started at 95oC for 5 minutes, then 32 cycles of 95oC for 35 seconds, 53-54oC for 40 seconds, and 72oC for 35 seconds, followed by 72oC for 10 minutes. Subsequently, 1 µl of bisulfite-treated DNA from each sample was amplified by AmpliTaq Gold kit (Life technology, USA). The PCR products were cloned using a TA-cloning kit (Invitrogen, USA). Next, 15 single white colonies were selected and the cloned fragments were amplified with M13 universal primers. The PCR product of each selected clone was analyzed by BiQ Analyzer software.
The data were analyzed by one-way analysis of variance (ANOVA) test, followed by a Tukey post-hoc test for determination of significant differences among groups and are presented as mean ± SD. Differences among groups were considered statistically significant at P<0.05.
Temporal expression of pluripotency and differentiation-specific genes during transition from inner cell mass to embryonic stem cells
Whole zona pellucida-free blastocysts were plated ontomitotically inactivated MEF feeder layer in R2i and serumconditions. The blastocyst-outgrowths in R2i culture have atypical compact morphology as opposed to those cultured in theserum medium. Also, it seems the number of trophectodermlike
cells around attached blastocyst outgrowths decreased inR2i compared to serum (Fig .1,). Next, to assess the temporalexpression of key pluripotency-related genes during theprocedure of mouse ESC establishment, we gently isolatedthe ICM outgrowths with a Pasture pipette on days 3, 5,
and 7 after seeding the blastocysts. Then, qRT-PCR wasperformedto measure the
expression of pluripotency markersnamely,
R2i caused a significantly higher expression of pluripotencyrelated
genes during ESC derivation, while in serum, theexpression of these genes in
outgrowths was not detected orwas at very low levels. We observed two distinct
expressionpatterns for the genes in R2i codition. In the first group, theexpression
continuously increased during derivation (
Hierarchical clustering and heatmap analysis showedthat the expression of most pluripotency-related genes wasincreased in R2i compared to the ICM and the highest level of gene expression was observed on day 5 (Fig .2B,).
DNA methylation status of Oct4 and Nanog promoters and the expression of epigenetic-associated genes during embryonic stem cells derivation
Bisulfite sequencing was used to evaluate the methylation
status of the twelfth and tenth CpGs in the promoter regions
of the pluripotency-associated genes,
On the other hand, relative expression of epigenetic-related
Efficient embryonic stem cells generation after 5 days of treatment with R2i
As early evidence of high expression levels of pluripotency-association genes and hypomethylated DNA was found in R2i, we sought to determine whether ESCs could be established if we cultured ICM in R2i for 5 days and then continued the remainder of the culture in serum (5 days-R2i/serum). Concordantly, 3 groups of zona-free blastocysts (NMRI strain) were cultured on feeder cells (15 embryos for each group); entirely in R2i culture medium, 5 days in R2i and then serum and also solely in the serum condition. On day 7, the individual ICM outgrowths were picked from the outgrown trophectoderm using a Pasteur pipette and subsequently trypsinized (trypsin/EDTA, 0.05% w/v) and replated on freshly seeded MEF in 24-well plates. After 5 days, typical packed domed ESC-like colonies could be identified. The efficiency in the generation of ESCs in R2i was 100% and ~ 94% for 5 days-R2i/serum while in the serum condition, ESC colonies did not appear (Fig .5A,). The ESCs passaged easily and showed dome-shaped colony morphologies (Fig .5B,), high nuclear/cytoplasmic ratios, the ability to propagate following trypsin digestion and clonal growth from single cells while also displaying high levels of alkaline phosphatase activity (Fig .5C,) and Oct4 expression. Therefore, 5 days treatment of whole blastocysts with R2i on MEF is sufficient for efficient generation of ESCs.
In this study, we reported the gene expression and DNA methylation of ICM during ESC generation under dual inhibition of MEK and TGFß signaling pathways with PD0325901 and SB431542 (known as R2i) that resulted in efficient generation of the ground-state pluripotency. R2i provides high genomic stability and an efficient transition from ICM to ESC (6). These advantages enable us to study the molecular mechanisms during ESC derivation. We analyzed the morphology of ICM-outgrowths on the MEF feeder layer in both, serum and R2i media. ICM-Outgrowths in serum had a larger fraction of trophoectoderm cells, while under R2i, the proliferation of these cells appeared to be inhibited. Therefore, we could conclude that ESC derivation on feeder cells in R2i medium reduces proliferation of trophoectoderm cells. In addition, the ICM-outgrowths were more compact and homogenous in comparison with the serum/LIF condition.
Next, we designed experiments to develop an
appropriate strategy to explore genetic and epigenetic
mechanisms that underlie ESC derivation. Here, we
found that R2i significantly promotes upregulation of
the pluripotency-related genes (
Moreover, we found that the promoter regions of pluripotent-associated genes, Oct4 and Nanog, of ICM-outgrowths were significantly hypomethylated under R2i compared to the serum condition during the early days of ESC derivation. Moreover, we found that the genome of ESCs was hypermethylated in selected regions compared to ICM cells. Our data showed that DNA methylation status in ESCs is similar in relation to in line with the indings of a comparison between 2i and R2i (20, 21). These patterns of DNA methylation have an essential role in the establishment of pluripotency under R2i (22, 23). We found that inhibition of DNA methylation by a methyltransferase inhibitor, RG-108 resulted in efficient generation of ESCs under R2i condition (11).
The expression of epigenetic-related genes
We demonstrate that establishment of ESCs requires upregulation of pluripotency-related genes and downregulation of differentiation-affiliated genes. Moreover, maintaining of DNA methylation at low levels established the ground-state pluripotency. In addition, we show the importance of the medium during the early days of ESC derivation which enables the capture of ESCs from blastocysts by maintaining the ground state of pluripotency.
We truly thank all members of the Department of Stem Cells and Developmental Biology labs for their help. This work was financially supported by a grant from Royan Institute as well as grants from the Iranian Council of Stem Cell Research and Technology, the Iran National Science Foundation (INSF), and Iran Science Elites Federation. The authors declare no conflicts of interest.
A.S., M.H.; Performed and analyzed the data from experiments shown in all figures and wrote the paper. S.M.; Assisted in cell culture. B.A.; Performed the animal parts of this study. M.T., H.B.; Conceived and, coordinated the study, provided technical assistance and wrote the paper. All authors reviewed the results and approved the final version of the manuscript.