This work was supported by the National Natural Science Foundation of China (21435008 and 21327006 to H.W.), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030200 to H.W.).

1. Preparations
<ol>
	<li>Prepare a solution of 1.0 mM PD0325901 (MEK inhibitor) and 3.0 mM CHIR99021 (GSK3 inhibitor).
	<ol>
		<li>Weigh 2 mg of PD0325901 and add 4.15 mL of DMSO in an amber glass vial.</li>
		<li>Weigh 2 mg of CHIR99021 and add 1.43 mL of DMSO in an amber glass vial.</li>
		<li>Following reconstitution, store aliquots (50 &#181;L/tube in 200 &#181;L PCR tubes) at -20 &#176;C and protected from light. Remove a tube containing the frozen stock from a freezer and thaw at room temperatur...

<ol>
	<li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292 (5819), 154-156 (1981).</li><li>Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo-derived+stem+cells:+of+mice+and+men.">Embryo-derived stem cells: of mice and men.</a> Annu. Rev. Cell Dev. Biol. 17, 435-462 (2001).</li><li>Murry, C. E., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+embryonic+stem+cells+to+clinically+relevant+populations:+lessons+from+embryonic+development.">Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.</a> Cell. 132 (4), 661-680 (2008).</li><li>Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+pluripotent+cell+line+from+early+mouse+embryos+cultured+in+medium+conditioned+by+teratocarcinoma+stem+cells.">Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.</a> Proc. Natl. Acad. Sci. USA. 78 (12), 7634-7638 (1981).</li><li>Eiselleova, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+mouse+and+human+feeder+cells+for+human+embryonic+stem+cells.">Comparative study of mouse and human feeder cells for human embryonic stem cells.</a> Int. J. Dev.Biol. 52 (4), 353-363 (2008).</li><li>Williams, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+leukaemia+inhibitory+factor+maintains+the+developmental+potential+of+embryonic+stem+cells.">Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.</a> Nature. 336 (6200), 684-687 (1988).</li><li>Tamm, C., Galit&#243;, S. P., Anner&#233;n, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+of+protocols+for+mouse+embryonic+stem+cell+culturing.">A comparative study of protocols for mouse embryonic stem cell culturing.</a> Plos One. 8 (12), e81156 (2013).</li><li>Yamaji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRDM14+ensures+naive+pluripotency+through+dual+regulation+of+signaling+and+epigenetic+pathways+in+mouse+embryonic+stem+cells.">PRDM14 ensures naive pluripotency through dual regulation of signaling and epigenetic pathways in mouse embryonic stem cells.</a> Cell Stem Cell. 12 (3), 368-382 (2013).</li><li>Ficz, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+signaling+inhibition+in+ESCs+drives+rapid+genome-wide+demethylation+to+the+epigenetic+ground+state+of+pluripotency.">FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency.</a> Cell Stem Cell. 13 (3), 351-359 (2013).</li><li>Smith, Z. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+regulatory+phase+of+DNA+methylation+in+the+early+mammalian+embryo.">A unique regulatory phase of DNA methylation in the early mammalian embryo.</a> Nature. 484 (7394), 339-344 (2012).</li><li>Ying, Q. L., Nichols, J., Chambers, I., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+induction+of+Id+proteins+suppresses+differentiation+and+sustains+embryonic+stem+cell+self-renewal+in+collaboration+with+STAT3.">BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.</a> Cell. 115 (3), 281-292 (2003).</li><li>Ying, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ground+state+of+embryonic+stem+cell+self-renewal.">The ground state of embryonic stem cell self-renewal.</a> Nature. 453 (7194), 519-523 (2008).</li><li>Leitch, H. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+pluripotency+is+associated+with+global+DNA+hypomethylation.">Naive pluripotency is associated with global DNA hypomethylation.</a> Nat. Struct. Mol. Biol. 20 (3), 311-316 (2013).</li><li>Galvao, J., Davis, B., Tilley, M., Normando, E., Duchen, M. R., Cordeiro, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+low-dose+toxicity+of+the+universal+solvent+DMSO.">Unexpected low-dose toxicity of the universal solvent DMSO.</a> FASEB J. 28 (3), 1317-1330 (2014).</li><li>Yin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbic+acid+enhances+Tet-mediated+5-methylcytosine+oxidation+and+promotes+DNA+demethylation+in+mammals.">Ascorbic acid enhances Tet-mediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals.</a> J. Am. Chem. Soc. 135 (28), 10396-10403 (2013).</li><li>Guerra, A. D., Rose, W. E., Hematti, P., Kao, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minocycline+modulates+NF&#954;B+phosphorylation+and+enhances+antimicrobial+activity+against+Staphylococcus+aureus+in+mesenchymal+stromal/stem+cells.">Minocycline modulates NF&#954;B phosphorylation and enhances antimicrobial activity against Staphylococcus aureus in mesenchymal stromal/stem cells.</a> Stem Cell Res. Ther. 8 (1), 171 (2017).</li><li>Blaschke, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+C+induces+Tet-dependent+DNA+demethylation+and+a+blastocyst-like+state+in+ES+cells.">Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells.</a> Nature. 500 (7461), 222-226 (2013).</li><li>Tahiliani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+5-methylcytosine+to+5-hydroxymethylcytosine+in+mammalian+DNA+by+MLL+partner+TET1.">Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.</a> Science. 324 (5929), 930-935 (2009).</li><li>Wu, H., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functions+of+Tet+protein-mediated+5-methylcytosine+oxidation.">Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation.</a> Genes Dev. 25 (23), 2436-2452 (2011).</li><li>Inoue, A., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-dependent+loss+of+5-hydroxymethylcytosine+in+mouse+preimplantation+embryos.">Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos.</a> Science. 334 (6053), 194 (2011).</li><li>He, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet-mediated+formation+of+5-carboxylcytosine+and+its+excision+by+TDG+in+mammalian+DNA.">Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.</a> Science. 333 (6047), 1303-1307 (2011).</li><li>Maiti, A., Drohat, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymine+DNA+glycosylase+can+rapidly+excise+5-formylcytosine+and+5-carboxylcytosine:+Potential+implications+for+active+demethylation+of+CpG+sites.">Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: Potential implications for active demethylation of CpG sites.</a> J. Biol. Chem. 286 (41), 35334-35338 (2011).</li><li>Habibi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-genome+bisulfite+sequencing+of+two+distinct+interconvertible+DNA+methylomes+of+mouse+embryonic+stem+cells.">Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells.</a> Cell Stem Cell. 13 (3), 360-369 (2013).</li><li>von Meyenn, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+DNA+methylation+maintenance+is+the+main+cause+of+global+demethylation+in+naive+embryonic+stem+cells.">Impairment of DNA methylation maintenance is the main cause of global demethylation in naive embryonic stem cells.</a> Mol. Cell. 62, 848-861 (2016).</li><li>Li, C., Liu, B., Zhong, S., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEK+inhibitor+PD0325901+and+vitamin+C+synergistically+induce+hypomethylation+of+mouse+embryonic+stem+cells.">MEK inhibitor PD0325901 and vitamin C synergistically induce hypomethylation of mouse embryonic stem cells.</a> Oncotarget. 7 (26), 39730-39739 (2016).</li><li>Hackett, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+mechanisms+of+DNA+demethylation+during+transition+to+ground-state+pluripotency.">Synergistic mechanisms of DNA demethylation during transition to ground-state pluripotency.</a> Stem Cell Reports. 1 (6), 518-531 (2013).</li><li>Nakaki, F., Saitou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRDM14:+a+unique+regulator+for+pluripotency+and+epigenetic+reprogramming.">PRDM14: a unique regulator for pluripotency and epigenetic reprogramming.</a> Trends Biochem. Sci. 39 (6), 289-298 (2014).</li><li>Nichols, J., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotency+in+the+embryo+and+in+culture.">Pluripotency in the embryo and in culture.</a> Cold Spring Harb. Perspect. Biol. 4 (8), (2012).</li></ol>

In the work, we demonstrated a novel method of combining Vc and PD0325901 to sustain mouse ES cells at an undifferentiated and hypomethylated state, which was achieved by a synergistic action of promoting DNA demethylation by Vc and suppressing de novo DNA methylation by PD0325901. Moreover, mouse ES cells showed great morphology under the Vc/PD0325901 culture system.
To better sustain the state of mouse ES cells in the Vc/PD0325901 culture system, there are some critical steps. First...

ES cells are originated from the ICM of a blastocyst1. The cells are in a pluripotency state and can form all somatic lineages and germline cells2. Establishment of ES cells provides the opportunity to investigate the development processes in vitro and can generate cells of medical relevance for regenerative medicine based on their pluripotency3.
Two groups seminally established the mouse ES cell lines in 1981 and when cells derived from the early mouse embryo were cultured in fetal bovine serum (FBS)-containing medium with mouse embryonic fibroblasts (MEFs) as the feeder layer1,4. MEFs were inactivated mitotically and were pre-grown in dishes prior to culturing ES cells. MEFs provide support for mouse ES cell attachment and produce growth factors to promote propagation and repress the differentiation5, while FBS offers essential trophic factors and hormones for cell proliferation. Subsequent studies indicated that LIF produced by feeder cells was the key cytokine for self-renewal and maintenance of pluripotency in mouse ES cells, and the addition of LIF into the medium could substitute for feeder cells6. Currently, the sustainment of mouse ES cells in FBS/LIF medium on feeders is still the standard method adopted by many researchers. However, some problems arise with this classical culture approach. Firstly, feeder cells secrete excess and uncontrolled factors and may cause pathogenic contamination7. To avoid this interference, coating the surface of dishes with gelatin and the addition of LIF in serum-containing medium are alternative methods for maintaining mouse ES cells without feeder-layer cells. Additionally, mouse ES cells grown under serum/LIF conditions exhibit morphological heterogeneity in cell populations and even in the expression level of pluripotency-related factors8. Recent studies suggest that under serum/LIF conditions, the pluripotency-related core transcription factors (including SOX2, Nanog, and OCT4) can sustain the pluripotency through LIF and WNT signaling; however, notably, they also activate a fibroblast growth factor (FGF) signal to trigger differentiation8. Due to the ambivalent dual action of the core transcription factors, mouse ES cells cultured in serum present heterogeneity, consisting of two interchangeable populations, one similar to ICM and another resembling the primed epiblast state8. Moreover, mouse ES cells in serum often exhibit global hypermethylation9, whereas ICM and PGCs are in a global hypomethylated state, which is closely linked with their pluripotency9,10.
There is a considerable demand to develop new methods for culturing mouse ES cells. Several improved protocols have been established since 200311 but there continues to be some limitations and shortcomings7. Since 2008, the combined utilization of two small-molecule kinase inhibitors, PD0325901 (the inhibitor of the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) (MEK)) and CHIR99021 (the inhibitor of glycogen synthase kinase 3 (GSK3)), in N2B27 medium with LIF and without serum has opened new perspectives for culturing mouse ES cells12. This new defined medium is characterized by the use of two inhibitors (2i). Mouse ES cells cultured in 2i/LIF medium are more homogeneous in cell populations and the expression of pluripotency factors. In addition, 2i/LIF-cultured mouse ES cells exhibit DNA hypomethylation globally, which is closer to ICM-like cells9,13. Even so, 2i culture has its disadvantages. PD0325901 and CHIR99021 are insoluble in water and generally are dissolved in dimethyl sulfoxide (DMSO)-based stock solution to add them in culture medium. Studies have showed that long-term and low-dose exposure of cells to DMSO can lead to cytotoxicity14.
Here, we utilized two small-molecule compounds and developed a new culture method of mouse ES cells. The novel culture method combines Vc and MEK inhibitor PD0325901 to promote DNA hypomethylation rapidly and effectively to a comparable level of PGC, named as the Vc/PD0325901 culturing protocol. Mouse ES cells in Vc/PD0325901-added serum-containing medium exhibit homogeneity in morphology and are sustained in a ground state. Compared to 2i culture, mouse ES cells cultured under Vc/PD0325901 conditions exhibit faster kinetics of DNA demethylation and can reach the hypomethylation level comparable to that of PGC. In addition, the use of a single inhibitor (PD0325901) decreases the input amount of DMSO into medium in comparison to that used in 2i (PD0325901/CHIR99021) and reduces the damage to cells.

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fetal bovine serum Australia source</td>
			<td style="width:205px;">Corning</td>
			<td style="width:119px;">35-076-CV</td>
			<td style="width:251px;">Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/high glucose</td>
			<td style="width:205px;">Hyclone</td>
			<td style="width:119px;">SH30022</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIF</td>
			<td>Millipore</td>
			<td style="width:119px;">ESG1107</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">non-essential amino acids (NEAA)</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:119px;">11140050</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium pyruvate</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:119px;">11360070</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:119px;">25030081</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">penicillin streptomycin solution</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:119px;">15140122</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">P5493</td>
			<td>Cells rinse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trypsin</td>
			<td style="width:205px;">Hyclone</td>
			<td style="width:119px;">SH30042</td>
			<td>Cell dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gelatin</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">48722</td>
			<td>Dishes coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PD0325901</td>
			<td>Stemolecule</td>
			<td>04-0006-10</td>
			<td>small-molecule chemical for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CHIR99021</td>
			<td>Stemolecule</td>
			<td>04-0004</td>
			<td>small-molecule chemical for cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">vitamin C</td>
			<td style="width:205px;">Sinopharm Chemical Reagent Co.,Ltd&#160;</td>
			<td style="width:119px;">XW00508171</td>
			<td>small-molecule chemical for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Bioscience water purification systemr</td>
			<td style="width:205px;">Purelab</td>
			<td style="width:119px;">Ultra</td>
			<td>Ultra water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td>Cell freezing medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuger</td>
			<td style="width:205px;">Eppendorf</td>
			<td style="width:119px;">5427R</td>
			<td>DNA and protein extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">protease inhibitor cocktail</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td>Component of RIPA buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PMSF Protease Inhibitor</td>
			<td style="width:205px;">ThermoFisher Scientific</td>
			<td style="width:119px;">36978</td>
			<td>Component of RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">43815</td>
			<td>Component of RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">E3889</td>
			<td>Component of RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Genomic DNA Purification Kit&#160;</td>
			<td style="width:205px;">Promega</td>
			<td style="width:119px;">A1125</td>
			<td>Genomic DNA extraction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">NanoDrop 2000</td>
			<td style="width:205px;">ThermoFisher Scientific</td>
			<td style="width:119px;">NanoDrop 2000</td>
			<td>Determination of DNA concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calf intestinal phosphatase</td>
			<td>New England Biolabs</td>
			<td style="width:119px;">M0290</td>
			<td>DNA digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>New England Biolabs</td>
			<td style="width:119px;">M0303</td>
			<td>DNA digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">snake venom phosphodiesterase I</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">P4506</td>
			<td>DNA digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanosep 3K Omega</td>
			<td>Pall</td>
			<td style="width:119px;">OD003C35</td>
			<td>filtration of digested DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1290 UHPLC system</td>
			<td style="width:205px;">Agilent&#160;</td>
			<td style="width:119px;">1290</td>
			<td>UHPLC separation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G6410B triple quadrupole mass spectrometer</td>
			<td style="width:205px;">Agilent&#160;</td>
			<td style="width:119px;">G6410B</td>
			<td>MS/MS analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Zorbax Eclipse Plus C18 column</td>
			<td style="width:205px;">Agilent&#160;</td>
			<td style="width:119px;">Zorbax Eclipse Plus</td>
			<td>colume for UHPLC separation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5-methylcytosine</td>
			<td>Solarbio Life Sciences</td>
			<td style="width:119px;">SM8900&#160;</td>
			<td>standard 5mC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5-hydroxymethylcytosine (standard)</td>
			<td>Toronto Research Chemicals</td>
			<td style="width:119px;">M295900</td>
			<td>standard 5hmC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prdm14 antibody</td>
			<td>bioworld</td>
			<td>BS7634</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnmt3a antibody</td>
			<td>bioworld</td>
			<td>BS6587</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnmt3b antibody</td>
			<td>abcam</td>
			<td>ab13604</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnmt3l antibody</td>
			<td>abcam</td>
			<td>ab3493</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnmt1 antibody</td>
			<td>abcam</td>
			<td>ab13537</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-tubulin antibody</td>
			<td>bioworld</td>
			<td>BS1482MH</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-rabbit IgG</td>
			<td>abcam</td>
			<td style="width:119px;">ab6721</td>
			<td>Western blot analysis-secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse IgG</td>
			<td>abcam</td>
			<td style="width:119px;">ab6789</td>
			<td>Western blot analysis-secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol</td>
			<td style="width:205px;">Invitrogen</td>
			<td>15596-026</td>
			<td>RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">reverse transcription system</td>
			<td style="width:205px;">Promega</td>
			<td style="width:119px;">A3500</td>
			<td>RNA reverse transcription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoTaq qPCR Master Mix</td>
			<td style="width:205px;">Promega</td>
			<td style="width:119px;">A6001</td>
			<td>RT-PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StemTAG alkaline phosphatase staining and activity assay kit</td>
			<td style="width:205px;">Cells Biolabs, Inc.</td>
			<td>CBA-302</td>
			<td>&#160;alkaline phosphatase staining analysis</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">mouse ES cells: WT, 129 SvEv</td>
			<td style="width:205px;">Provided by Professor Guoliang Xu (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China)</td>
			<td style="width:119px;"></td>
			<td>cell strain of mouse ES cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microscope</td>
			<td style="width:205px;">Zeiss</td>
			<td style="width:119px;">LSM510</td>
			<td>cells observation</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">GraphPad Prism 5.0</td>
			<td style="width:205px;">GraphPad Prism Software Inc.</td>
			<td style="width:119px;">5.0</td>
			<td>statistical analysis</td>
		</tr>
	</tbody>
</table>

an alternative culture method to maintain genomic hypomethylation of mouse embryonic stem cells using mek inhibitor pd0325901 and vitamin c

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many lineages for regenerative medicine. ES cell culture in vitro has long been the subject of widespread concerns. Classically, mouse ES cells are maintained in serum and leukemia inhibitory factor (LIF)-containing medium. However, under serum/LIF conditions, cells show heterogeneity in morphology and the expression profile of pluripotency-related genes, and are mostly in a metastable state. Moreover, cultured ES cells exhibit global hypermethylation, but na&#239;ve ES cells of the inner cell mass (ICM) and primordial germ cells (PGCs) are in a state of global hypomethylation. The hypomethylated state of ICM and PGCs is closely associated with their pluripotency. To improve mouse ES cell culture methods, we have recently developed a new method based on the selectively combined utilization of two small-molecule compounds to maintain the DNA hypomethylated and pluripotent state. Here, we present that the co-treatment of vitamin C (Vc) and PD0325901 can erase about 90% of 5-methylcytosine (5mC) at 5 days in mouse ES cells. The generated 5mC content is comparable to that in PGCs. The mechanistic investigation shows that PD0325901 up-regulates Prdm14 expression to suppress Dnmt3b (de novo DNA methyltransferase) and Dnmt3l (the cofactor of Dnmt3b), by reducing de novo 5mC synthesis. Vc facilitates the conversion of 5mC to 5-hydroxymethylcytosine (5hmC) catalyzed mainly by Tet1 and Tet2, indicating the involvement of both passive and active DNA demethylations. Moreover, under Vc/PD0325901 conditions, mouse ES cells show homogeneous morphology and pluripotent state. Collectively, we propose a novel and chemical-synergy culture method for achieving DNA hypomethylation and maintenance of pluripotency in mouse ES cells. The small-molecule chemical-dependent method overcomes the major shortcomings of serum culture, and holds promise to generate homogeneous ES cells for further clinical applications and researches.

Vc/PD0325901 synergistically induced global erasure of mouse ES cells. Mouse ES cells in serum exhibit DNA hypermethylation, while pluripotent ICM cells and PGCs show global erasure of DNA methylation and the hypomethylated state is closely associated with their pluripotency9,10.
Previously, we and others found that Vc may enhance Tet-mediated 5mC demethylation<sup class...

Watch this Scientific Journal Video about An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C at JoVE.com

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

The overall goal of this protocol is to use the small molecule compounds PD0325901 and Vitamin C to maintain a hypomethylated and pluripotent state in mouse embryonic stem cells. This method can help answer key questions in the field of FBS cultured mouse embryonic stem cells, such as heterogenerity in morphology and the dehypermethylation. So the main advantage of this technique is that the mouse embryonic stem cells are able to sustain excellent morphology and the hypomethylated and the undifferentiated state.After preparing plates and buffers, according to the text protocol, prewarm the mouse ES cells, culture medium, trypsin, and PBS in a water bath at 37 degrees Celsius. To passage the cells, aspirate the medium from the dish and use two milliliters of PBS to rinse the cells two times. Then remove the PBS and add 0.3 milliliters of trypson EDTA to the cells and quickly tilt the dish to cover the cells in the solution.Immediately remove the trypsin and incubate the plate at 37 degrees Celsius for one minute to detach the cells. Add two milliliters of pre-warmed serum medium to inactivate the trypsin and use a five milliliter pipet to re-suspend the cell colonies 10 times into a single cell suspension. Plate 150 microliters of the cell solution into a new six centimeter, gelatin-coated dish and supplement with three milliliters of freshly made VCPD0325901 medium.Culture the cells at 37 degrees Celsius and five percent CO2. Due to the instability of Vc, every 24 hours while incubating, remove the old culture medium and add three milliliters of fresh Vcpd0325901 medium. After reaching 70 to 80 percent confluency, passage the cells and continue to culture them as just demonstrated.To carry out DNA extraction, collect the cells with trypsin as demonstrated earlier and use a genomic DNA purification kit following the manufacturers instructions. Add 100 microliters of ultra pure water to the tube of DNA and dissolve the DNA by pipetting approximately 15 times. Then, use a spectrophotometer to measure the Od260 to 280 ratio to determine the concentration and quality of DNA.The DNA concentration should be about 500 nonograms per microliter. Next, digest five micrograms of DNA by combining it with five microliters of 100 millimolar tris hydrochloride PH7.6, two units of calf intestinal phosphatase, one unit of Dnase one, and 0.005 units of snake venom, phosphodyesterase one. Then, use ultra pure water to bring up the volume to 50 microliters.Incubate the reaction at 37 degrees Celsius over night. The following morning, collect the sample by centrifugation at 1, 000xg at room temperature for one minute. Then transfer the solution into ultra filtration tubes and spin the samples at 13, 000xg and 4 degrees Celsius for 30 minutes to remove the digestion enzymes.To prepare the samples for 5HMC analysis, transfer 36 microliters of the filtrate to a new one milliliter tube and add four microliters of D35HMC for a final concentration of three nanomolar. For 5MC analysis, pipet 196 microliters of ultra pure water into a new centrifuge tube and add four microliters of filtrate due to the high density of 5MC in genomic DNA. Transfer 36 microliters of the diluted 5MC solution to a new centrifuge tube and add four microliters of D35MC for a final concentration of 50 nanomolar.Use D35HMC and D35MC as internal standards to calibrate 5HMC and 5MC respectively. After setting up the parameters to analyze 5MC and 5HMC in the UHPLCMS software, according to the text protocol, establish the parameters for QQQMSMS by clicking MS QQQ. For stop time, choose No limit/As Pump.For ion source, choose ESI. For time filtering, check Peak width and enter 0.07 minutes. To set up the time segments, for start time, choose zero.To set scan mode in MRM to analyze the allusion from the column, for scan type, choose MRM. For Div Value, choose To MS.For Delta EMV plus, enter 200 and for Delta EMV minus, enter zero. Build the parameters for monitoring the transitions by first clicking MSQQQ one acquisition.To set scan segments, for Dwell, enter 90. To set the fragment voltages for the Fragmentor, enter 90. For Collision Energy, enter five.For the Cell Accelerator Voltage, enter four. To set the positive ion mode, for Polarity, choose positive. To carry out UHPLC separation of mononucleosides, prepare solutions a and b for the allusion of 5MC and cytosine by mixing 500 milliliters of ultra pure water with 500 microliters of formic acid for solution a.Then measure 500 milliliters of 100 percent methanol and solution b. Mix solution a and solution b at a 95 to five volume to volume ratio as the mobile phase to elute 5MC and C.Then set the flow rate to 0.25 milliliters per minute. Separate 5HMC using an optimized gradient allusion.Then set the flow rate at 0.25 milliliters per minute. Monitor the transitions for 5MC, D35MC, DC, 5HMC, and D35HMC. Set the capillary voltages at 3, 500 volts.Then set the injection volume to 5 microliters and analyze each sample three times. Employ the corresponding standard curves to evaluate the amount of 5MC and 5HMC according to the text protocol. As seen in this analysis of genomic DNA in mouse ES cells by UHPLC MSMS, VcPD0325901 treatment resulted in a more rapid decline in DNA methylation and reached steady five MC levels after five days, while Vc 2i treatment resulted in a comparable level at day 11.This graph shows that Vc containing treatment dramatically increased 5HMC frequency after one day and thereafter, 5HMC levels gradually declined due to a progressive reduction in 5MC substrate. Western blot analysis showed that Prdm14 was significantly elevated, while Dnmt3b and its co-factor, Dnmt3l, were considerably down regulated when cells retreated with PD0325901, indicating the action of PD0325901 in erasing 5MC. In this alkaline phosphatase assay, mouse ES cells were all stained purple with a ball-like morphology when cultivated for 26 days in VcPD032590, indicating an undifferentiated state.In contrast, cells grown in FBS containing medium alone appeared light in color with partial outspreading indicating partial differentiation. Once mastered, this technique can be done in five hours if it is performed properly. While attempting this procedure, it's important to remember that FBS need to be pre-screened.Also, avoid over-pipetting cells through the passage and changing the VcPDO325901 culture medium daily. After this development, this technique paves the way for researchers in the field of culturing mouse ES cells in vitro to explore the development and processes of embryos in vitro and the generated cells of medical relevance for regenerative medicine. After watching this video, you should have a greater understanding of how to maintain growth morphology and hypermethylated and plural potent state for mouse ES cells by using two small molecular components, the MEK inhibitor, PD0325901.Don't forget that working with Trisol, DMS or PMS and the DTT can be extremely hazardous and the precautions such as wearing gloves, a mask, and the goggles should always be taken while performing this procedure.

an-alternative-culture-method-to-maintain-genomic-hypomethylation

Research

JoVE Journal

Biochemistry

6.6K visualizações.  Chinese Academy of Sciences. The overall goal of this protocol is to use the small molecule compounds PD0325901 and Vitamin C to maintain a hypomethylated and pluripotent state in mouse embryonic stem cells. This method can help answer key questions in the field of FBS cultured mouse embryonic stem cells, such as heterogenerity in morphology and the dehypermethylation. So the main advantage of this technique is that the mouse embryonic stem cells are able to sustain excellent morphology and the hypomethylated and the und...