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 temperature before use.</li>
	</ol>
	</li>
	<li>Weigh 0.0176 g of Vc into 1 mL of a centrifuge tube and reconstitute it with 1 mL of sterile water to a final concentration of 100 mM prior to use.</li>
	<li>Inactivate FBS: incubate 500 mL of FBS in a water bath at 56 &#176;C for 30 min.</li>
	<li>Prepare 600 mL of basic medium for culturing mouse ES cells.
	<ol>
		<li>Use a bottle of DMEM/high glucose medium (500 mL) and remove 40 mL.</li>
		<li>Supplement 460 mL of DMEM/high glucose medium with 20% inactivated FBS (120 mL), 1,000 U/mL LIF (60 &#181;L of 107 U/mL stock solution), 0.1 mM non-essential amino acids (NEAA) (6 mL of 10 mM stock solution), 1 mM sodium pyruvate (6 mL of 100 mM stock solution), 2 mM L-glutamine (6 mL of 200 mM stock solution), 0.1 mM &#946;-mercaptoethanol (600 &#181;L of 100 mM stock solution), and 33 IU/mL penicillin and 33 &#181;g/mL streptomycin (2 mL of penicillin-streptomycin mixed solution (10,000 IU/mL penicillin and 10,000 &#181;g/mL streptomycin)). Define the basic medium as serum medium.</li>
		<li>Pipette 50 &#181;L of stock solution of PD0325901 (1.0 mM) and Vc (100 mM), respectively, into 50 mL of the serum medium (final concentration: 1.0 &#181;M PD0325901 and 100 &#181;M Vc), and define the medium as Vc/PD0325901 medium. 
		NOTE: Prepare the Vc/PD0325901 medium fresh before use due to the instability of Vc.</li>
		<li>Using a pipette, transfer 50 &#181;L of stock solution of PD0325901 (1.0 mM) and CHIR99021 (3.0 mM), respectively, into 50 mL of the serum medium (final concentration: PD0325901 1.0 &#181;M and CHIR99021 3.0 &#181;M), and define the medium as 2i medium.</li>
	</ol>
	</li>
	<li>Prepare the gelatin-coated dishes.
	<ol>
		<li>Prepare the 0.1% gelatin solution: dissolve 0.3 g of gelatin into 300 mL of ultrapure water in a glass reagent bottle with a cap and sterilize in an autoclave at 121 &#176;C for 20 min. Store the sterile solution at room temperature.</li>
		<li>Incubate the cell culture dishes (6 cm diameter) with 2 mL of 0.1% gelatin solution for 15 min at room temperature and then aspirate the gelatin. Dry the dishes in air and use them within 12 h.</li>
	</ol>
	</li>
	<li>Prepare 10 mL of mouse ES cell freezing medium in a 15 mL centrifuge tube: 90% FBS (9 mL) and 10% DMSO (1 mL). Use the medium within 1 day.</li>
	<li>Prepare a freezing container: add 100% isopropyl alcohol to the fill line of the freezing container, and discard the old isopropyl alcohol. Add new isopropyl alcohol directly after every fifth use.</li>
	<li>Make the enzymatic digestion buffer: weigh 1.2114 g of Tris powder into 100 mL of ultrapure water to prepare 100 mM Tris solution and add concentrated HCl solution (about 12 M) into the Tris solution to adjust the pH to 7.6 (about 0.6 mL of concentrated HCl solution).</li>
	<li>Prepare 50 mL of radio immunoprecipitation assay buffer (RIPA buffer): 20 mM Tris, pH 7.4, 1 mM MgCl2, 150 mM NaCl, 20% glycerol, 0.5% NP40, 1 mM EDTA, 1 mM EGTA. Supplement with 1 mM DTT, 1X protease inhibitor cocktail, and 1 mM PMSF prior to use. Once the PMSF is added, use the buffer within 30 min.</li>
</ol>
2. Grow Mouse ES Cells on Gelatin-coated Dishes
<ol>
	<li>Pre-warm mouse ES cells (wild type, 129 SvEv) culture medium, trypsin, and phosphate buffered solution (PBS) in a water bath at 37 &#176;C.</li>
	<li>Passage the cells. Aspirate the medium completely from the dish and pipette 2 mL of PBS to the dishes to rinse the cells.
	<ol>
		<li>Remove the PBS with a pipette and wash the cells again with 2 mL of PBS.</li>
		<li>Remove the PBS and add 0.3 mL of trypsin-EDTA (0.25%) to each dish.</li>
		<li>Cover the whole dish with the trypsin quickly and then remove it immediately.</li>
		<li>Put the dish in an incubator at 37 &#176;C for 1 min to detach the cells from the dish. Take out the dishes from the incubator and add 2 mL of pre-warmed serum medium to terminate the action of trypsin.</li>
		<li>Dissociate the cell colonies into a single cell suspension by resuspending with a pipette (5 mL pipette tip) 10 times.</li>
	</ol>
	</li>
	<li>Transfer 150 &#181;L of 2 mL of the cell solution (about 190,000 cells by counting with a hemocytometer) into a new gelatin-coated dish, and supplement with 3 mL of freshly made Vc/PD0325901 medium per 6 cm culture dish. Culture the cells in a 37 &#176;C incubator with 5% CO2.</li>
	<li>Every 24 h while incubating, remove the old culture medium and add 3 mL of fresh Vc/PD0325901-medium into the culture dish due to the instability of Vc. Expect 70&#8211;80% confluency after 2&#8211;3 days.</li>
	<li>After reaching 70-80% confluency, passage the cells and continue to culture them as described in steps 2.2&#8211;2.4.</li>
</ol>
3. Freeze and Thaw Mouse ES Cells
<ol>
	<li>Freeze mouse ES cells.
	<ol>
		<li>Detach the cells from the dishes with trypsin by following step 2.2.</li>
		<li>Transfer the cells into a 15 mL plastic tube and centrifuge at 800 x g and room temperature for 3 min.</li>
		<li>Dump the supernatant gently into a beaker and resuspend the cell pellet with 1 mL of freshly made freezing medium. Prepare the freezing medium 30 min prior to this step to ensure that it is room temperature before use.</li>
		<li>Transfer the cells in freezing medium to a 1.8 mL microcentrifuge tube with a 1 mL pipette and place the microcentrifuge tube into a freezing container. Add isopropyl alcohol to the fill line of the freezing container and keep it at room temperature before using.</li>
		<li>Leave the freezing containers overnight in a -80 &#176;C freezer.</li>
		<li>Transfer the microcentrifuge tubes to a liquid nitrogen container for up to 2&#8211;3 years. 
		NOTE: Do not keep cells in the freezing medium for a long time and quickly transfer the cells to a -80 &#176;C freezer due to the toxicity of DMSO.</li>
	</ol>
	</li>
	<li>Thaw mouse ES cells.
	<ol>
		<li>Pre-warm 50 mL of serum medium in a 37 &#176;C water bath.</li>
		<li>Take out a cell-containing vial from the liquid nitrogen container and immerse the capped vial in a 37 &#176;C water bath. Shake it quickly in the water bath to thaw. Transfer the cells into a 15 mL tube with a 1 mL pipette. Add 2 mL of pre-warmed serum culture medium into the tube to dilute the freezing medium and then centrifuge at 800 x g and room temperature for 3 min.</li>
		<li>Remove the supernatant and gently resuspend the cell pellets with 3 mL of fresh Vc/PD0325901 medium using a 5 mL pipette.</li>
		<li>Transfer the cells from the 15 mL tube to a gelatin-coated dish and culture the cells for 24 h. Culture the cells again as described in Step 2.</li>
	</ol>
	</li>
</ol>
4. Extract Total Protein from Cells
<ol>
	<li>Rinse the collected cells with 1 mL of PBS and then centrifuge at 800 x g and room temperature for 3 min.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet thoroughly with RIPA buffer supplemented with DTT, protease inhibitor cocktail, and PMSF by pipetting gently. The volume of RIPA buffer is about 5x that of the cell pellet. 
	NOTE: Carry out the cell resuspension on ice.</li>
	<li>Keep the cells in RIPA buffer for 30 min on ice and centrifuge at 13,000 x g and 4 &#176;C for 5 min.</li>
	<li>Transfer the supernatant to a new tube and store aliquots at -80 &#176;C.</li>
	<li>Determine the concentration of the protein with a Bradford protein assay kit.</li>
</ol>
5. Extract DNA from Cells and Digest DNA into a Single Nucleoside Using Enzymes
<ol>
	<li>Collect the cells with trypsin as described in steps 3.1.1 and 3.1.2.</li>
	<li>Perform the DNA extraction with a genomic DNA purification kit following the manufacturer&#39;s instructions.</li>
	<li>Store the extracted DNA in 1-mL centrifuge tubes. Add 100 &#181;L of ultrapure water into the centrifuge tube, and dissolve the DNA by pipetting approximately 15 times. Determine the concentration and evaluate the quality of DNA by the measurement of the absorbance at 260 nm and 280 nm15. The DNA concentration is about 500 ng/&#181;L.</li>
	<li>Digest 5 &#181;g of DNA in a 50 &#181;L solution system: add 5 &#181;L of 100 mM Tris-HCl solution, pH 7.6 (final concentration: 10 mM), 2 U calf intestinal phosphatase, 1 U DNase I, 0.005 U snake venom phosphodiesterase I, and 5 &#181;g of DNA (calculate the added volume according to the measured DNA concentration, ~10 &#181;L) into a centrifuge tube and increase the volume of solution to 50 &#181;L with ultrapure water. Incubate the mixture at 37 &#176;C overnight. Centrifuge the digested DNA solution at 1,000 g and room temperature for 1 min to collect the solution to the bottom of tubes.</li>
	<li>Transfer the digested DNA solution from the centrifuge tubes into ultra-filtration tubes (a molecule weight cutoff: 3 kDa) and centrifuge at 13,000 g and 4 &#176;C for 30 min to remove the digestion enzymes.</li>
	<li>Prepare the samples for 5mC and 5hmC analysis.
	<ol>
		<li>For 5hmC analysis: pipet 36 &#181;L of filter solution to a new centrifuge tube (1 mL) and add 4 &#181;L of 5&#39;-(hydroxymethyl-d3)-2&#39;-deoxycytidine ([D3]-5hmC) with the final concentration of 3 nM.</li>
		<li>For 5mC analysis: add 196 &#181;L of ultrapure water into a new centrifuge tube and pipet 4 &#181;L of filter solution to the tube (50-fold dilution), due to the high density of 5mC in genomic DNA. Transfer 36 &#181;L of the diluted solution to a new centrifuge tube and add 4 &#181;L of (5&#39;-(methyl-d3)-2&#39;-deoxycytidine ([D3]-5mC) with the final concentration of 50 nM. Use [D3]-5hmC and [D3]-5mC as the internal standard to calibrate 5hmC and 5mC, respectively.</li>
	</ol>
	</li>
	<li>Transfer the sample solution to measuring tubes and store at 4 &#176;C. Analyze 5mC and 5hmC with ultra-high-performance liquid chromatography-triple quadrupole mass spectrometry with multiple-reaction monitoring (UHPLC-MS/MS (MRM)) as described in a previous report15.</li>
</ol>
6. Analyze 5mC and 5hmC Using UHPLC-MS/MS15
<ol>
	<li>Set up various parameters for analyzing 5mC and 5hmC using the UHPLC-MS software.
	<ol>
		<li>Establish a new method. Open the software and click &#34;File | New | Method&#34;. Exhibit the message box of &#34;Method Editor&#34; with the content &#34;Do you want to save the changes for the current method&#34; and click &#34;No&#34;.</li>
		<li>Build the parameters of the sampler. Click &#34;Sampler | Setup | Injection&#34;. Select &#34;Standard injection&#34; and input &#34;5 &#181;L at Injection volume&#34;.</li>
		<li>Build the parameters of the pump in UHPLC.
		<ol>
			<li>Click &#34;BinPump2 | Setup&#34; to build the parameters of the pump. Set up &#34;Flow&#34; at &#34;0.25 mL/min&#34; and &#34;Stop time&#34; at &#34;15 min&#34;.</li>
			<li>Set up &#34;Solvent B at 5%&#34; and &#34;Pressure limits&#34; at &#34;Max 600 bar&#34;.</li>
			<li>Click &#34;BinPump2 | Timetable&#34; to build the isocratic elution parameters for 5mC analysis. Click &#34;Append&#34; to add a row. Input &#34;0.00&#34; at &#34;Time&#34; and &#34;5.0&#34; at &#34;B%&#34;. Click &#34;Append&#34; once more to add a new row. Input &#34;15.00&#34; at &#34;Time&#34; and &#34;5.0&#34; at &#34;B%&#34;.</li>
			<li>Click &#34;BinPump2 | Timetable&#34; to build the gradient elution parameters for 5hmC analysis. Click &#34;Append to add a row. Input 0.00 at Time and 5.0 at &#34;B%&#34;. Click &#34;Append&#34; once more to add a new row. Input &#34;3.00&#34; at &#34;Time&#34; and &#34;5.0&#34; at &#34;B%&#34;. Click &#34;Append&#34; for another 6 times to add 6 rows. Input &#34;3.01&#34; at &#34;Time&#34; and &#34;15.0&#34; at &#34;B%&#34;, &#34;6.00&#34; at &#34;Time&#34; and &#34;15.0&#34; at &#34;B%&#34;, &#34;6.01&#34; at &#34;Time&#34; and &#34;100&#34; at &#34;B%&#34;, &#34;10.00&#34; at &#34;Time&#34; and &#34;100.0&#34; at &#34;B%&#34;, &#34;10.01&#34; at &#34;Time&#34; and &#34;5.0&#34; at &#34;B%&#34;, &#34;15.00&#34; at &#34;Time&#34; and &#34;5.0&#34; at &#34;B%&#34; in sequence. 
			NOTE: Perform the analysis of 5mC and 5hmC individually due to the different separation conditions of UHPLC.</li>
		</ol>
		</li>
		<li>Build the parameters of the temperature of the column compartment (TCC) in UHPLC. 
		Click &#34;TCC | Setup | temperature (Left)&#34; and input &#34;30 &#176;C&#34;.</li>
		<li>Establish the parameters of the QQQ-MS/MS.
		<ol>
			<li>Click &#34;MS QQQ | Stop time | No limit/As pump&#34;, &#34;Ion Source | ESI&#34;, &#34;Time filtering | Peak width: 0.07 min&#34;. Set up &#34;Time segments: Start time: 0&#34;; &#34;Scan Type: MRM&#34;; &#34;Div Value: To MS&#34;; &#34;Delta EMV (+): 200&#34; and &#34;Delta EMV (-): 0&#34;.</li>
			<li>Build the parameters of monitoring the transitions of 5mC, D3-5mC, dC, 5hmC, and D3-5hmC
			<ol>
				<li>Click &#34;MS QQQ | Acquisition | Scan segments: Dwell: 90&#34;; &#34;Fragmentor: 90&#34;; &#34;Collision Energy: 5&#34;; &#34;Cell Accelerator Voltage: 4&#34; and &#34;Polarity: Positive&#34;.</li>
				<li>Input &#34;5mC&#34; at &#34;Compound Name&#34;, &#34;242&#34; at &#34;Precursor Ion&#34;, &#34;126&#34; at &#34;Product Ion&#34; for 5mC analysis. Click the right button and select &#34;Add row&#34; to add a new row. Input &#34;D3-5mC&#34; at &#34;Compound Name&#34;, &#34;245&#34; at &#34;Precursor Ion&#34;, &#34;129&#34; at &#34;Product Ion&#34; for D3-5mC analysis.</li>
				<li>Supplement another three rows using the same mode. Input &#34;dC&#34; at &#34;Compound Name&#34;, &#34;228&#34; at &#34;Precursor Ion&#34;, &#34;112&#34; at &#34;Product Ion&#34; for dC analysis. Input &#34;5hmC&#34; at &#34;Compound Name&#34;, &#34;258&#34; at &#34;Precursor Ion&#34;, &#34;142&#34; at &#34;Product Ion&#34; for 5hmC analysis. Input D3-5hmC at Compound Name, &#34;261&#34; at &#34;Precursor Ion&#34;, &#34;145&#34; at &#34;Product Ion&#34; for D3-5hmC analysis.</li>
			</ol>
			</li>
			<li>Build the parameters of the gas source. Click &#34;MS QQQ | Source | Source parameters: Gas Temp: 300 &#176;C&#34;; &#34;Gas Flow: 11 L/min&#34;; &#34;Nebulizer: 15 psi&#34;; &#34;Positive-Capillary: 3500 V&#34;.</li>
			<li>Save the proposed method. Click &#34;MS QQQ | Instrument | Save method As&#34;. Select a pathway for the proposed method by &#34;Directory&#34; and give a name for the method by inputting the content in &#34;Method&#34;, for example: 5mC and 5hmC analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>UHPLC separation and MS/MS analysis of mononucleosides
	<ol>
		<li>Prepare the mobile phase for 5mC and cytosine (C) analysis: form the mobile phase of solution A and B. Solution A: 500 mL of ultrapure water with 500 &#181;L of formic acid (final concentration: 0.1%), and solution B: 500 mL of 100% methanol. Mix solution A and B at 95:5 (v:v) as the mobile phase to elute 5mC and C (set in step 6.1.3.3). Set the flow rate at 0.25 mL/min (set in step 6.1.3.1).</li>
		<li>Prepare the mobile phase by solvent A and B for 5hmC analysis. Solvent A is 2.0 mM NH4HCO3 aqueous solution (pH 9.0) (500 mL), and solvent B is 100% methanol (500 mL). Separate 5hmC by an optimized gradient elution: 0-3 min, 5.0% B and 95% A (v:v); 3-6 min, 15.0% B and 85% A; 6-10 min, 100% B; 10-15 min, 5.0% B and 95% A (set in step 6.1.3.4). Set the flow rate at 0.25 mL/min (set in step 6.1.3.1).</li>
	</ol>
	</li>
	<li>Utilize electrospray MS/MS with MRM mode (set in step 6.1.5.1) to analyze the elution from the column and adopt the positive ion mode (set in step 6.1.5.2.1).
	<ol>
		<li>Monitor the transitions: 5mC, m/z 242&#8594;126 (collision energy, 5 eV); [D3]-5mC, m/z 245&#8594;129 (5 eV); dC, m/z 228&#8594;112 (5 eV); 5hmC, m/z 258&#8594;142 (5 eV); [D3]-5hmC, m/z 261&#8594;145 (5 eV) (set in step 6.1.5.2.2).</li>
		<li>Set the capillary and fragment voltages at +3,500 V (set in step 6.1.5.3) and 90 V (set in step 6.1.5.2.1), respectively.</li>
		<li>Set the injection volume at 5 &#181;L and analyze each sample 3 times (set in step 6.1.2). Employ the corresponding standard curves to evaluate the amount of 5mC and 5hmC.
		<ol>
			<li>Establish the standard curve of 5mC: transfer 1&#8211;50 nM (1, 5, 10, 20, 50 nM, final concentration) standard solution of 5mC into centrifuge tubes and add 50 nM [D3]-5mC to tubes. Supplement the total volume to 50 &#181;L. Perform the analysis of standard 5mC following the instructions for the 5mC sample (step 6).</li>
			<li>Build the standard curve of 5hmC: transfer 1&#8211;20 nM (1, 2, 5, 10, 20 nM, final concentration) standard solution of 5hmC to centrifuge tubes and add 3 nM [D3]-5hmC to the tubes. Supplement the total volume to 50 &#181;L. Perform the analysis of standard 5hmC following the instructions for the 5hmC sample (step 6).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
7. Analyze the Statistical Significance
<ol>
	<li>Perform the statistical analysis using software.
	<ol>
		<li>Open the software and select &#34;Column&#34; in &#34;New table &#38; graph&#34;. Set the parameters as follows: &#34;Sample data | Start with an empty data table&#34;; &#34;Choose a graph-the first mode&#34;; &#34;Graphing replicates or error bars | Plot | Mean with SEM&#34;.</li>
		<li>Click &#34;Create&#34; to input the three replicates of Control and Vc/PD0325901-treated group in the &#34;A&#34; and &#34;B&#34; line, respectively. Select the six data and click &#34;Analyze&#34; in the &#34;Analysis&#34;.</li>
		<li>Select &#34;t tests (and nonparametric tests)&#34; in &#34;Column Analyses&#34; and click &#34;OK&#34; to enter the next interface.</li>
		<li>Set the parameters as follows: &#34;Choose test | Test name | Unpaired t test; Options | Two-tailed&#34;; &#34;Confidence Intervals: 95%&#34;; &#34;Significant digits | Show 4 significant digits&#34;. Click &#34;OK&#34; to acquire the p value between the Control and Vc/PD0325901 treatment.</li>
	</ol>
	</li>
	<li>Evaluate the statistical significance between the control and the experimental groups employing the two-tailed and unpaired Student&#39;s t-test16. 
	NOTE: p &#60;0.05 denotes the difference possessing statistical significance. *p &#60;0.05, **p &#60;0.01, ***p &#60;0.001.</li>
</ol>

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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>

The authors disclose no potential conflicts of interest.

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

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Research

JoVE Journal

Biochemistry

6.6K Views.  Chinese Academy of Sciences. Wij beschreven twee chemische-gebaseerde protocollen voor het kweken van muis embryonale stamcellen in detail. Deze nieuwe methode maakt gebruik van synergetische mechanismen ter bevordering van Tet-gemedieerde oxidatie (door vitamine C) en onderdrukken van DOVO synthese van 5-methylcytosine (door PD0325901) om DNA hypomethylation en pluripotent van muis embryonale stamcellen te onderhouden.

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

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

Developmental Biology

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

Genetics

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Biology

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

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

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