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

Summary

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.

Abstract

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 naïve 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.

Introduction

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....

Protocol

1. Preparations

1. Prepare a solution of 1.0 mM PD0325901 (MEK inhibitor) and 3.0 mM CHIR99021 (GSK3 inhibitor).
   1. Weigh 2 mg of PD0325901 and add 4.15 mL of DMSO in an amber glass vial.
   2. Weigh 2 mg of CHIR99021 and add 1.43 mL of DMSO in an amber glass vial.
   3. Following reconstitution, store aliquots (50 µL/tube in 200 µL PCR tubes) at -20 °C and protected from light. Remove a tube containing the frozen stock from a freezer and thaw at room temperature before use.
2. 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 co....

Results

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 pluripotency^9,10.

Previously, we and others found that Vc may enhance Tet-mediated 5mC demethylation

Discussion

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.......

Materials

List of materials used in this article

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