Name: an alternative culture method to maintain genomic hypomethylation of mouse embryonic stem cells using mek inhibitor pd0325901 and vitamin c
Uploaded: 2018-06-01T13:00:00
Description: 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

Dette arbejde blev støttet af National Natural Science Foundation of China (21435008 og 21327006 til H.W.), og den strategiske prioritet Research Program af den kinesiske Academy of Sciences (XDB14030200 til H.W.).

1. præparater
<ol><li>
Forberede en opløsning af 1.0 mM PD0325901 (MEK hæmmer) og 3,0 mM CHIR99021 (GSK3-hæmmer).
	<ol>
<li>2 mg af PD0325901 afvejes og tilføje 4,15 mL af DMSO i en rav hætteglasset.</li>
		<li>2 mg af CHIR99021 afvejes og tilføje 1,43 mL af DMSO i en rav hætteglasset.</li>
		<li>Følgende rekonstitution, gemme delprøver (50 µL/rør i 200 µL PCR rør) ved-20 ° C og beskyttet mod lys. Fjerne et rør, der indeholder den frosne lager fra en fryser og tø ved stuetemperatur fø...

<ol>
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I arbejdet viste vi en roman metode kombinerer Vc og PD0325901 for at opretholde mus ES celler på en udifferentierede og hypomethylated tilstand, som blev opnået ved en synergistiske virkning af at fremme DNA demetylering af Vc og undertrykke de novo DNA methylering af PD0325901. Derudover mus ES celler viste stor morfologi under Vc/PD0325901 kultur system.
For at opretholde bedre tilstand af musen ES celler i Vc/PD0325901 kultur system, er der nogle afgørende skridt. For det førs...

ES-celler stammede fra ICM af en blastocyst1. Cellerne er i tilstanden pluripotency og kan danne alle somatiske lineages og kønscelleoverførsel celler2. Oprettelsen af ES-celler giver mulighed for at undersøge udviklingen processer in vitro- og kan generere celler af medicinsk relevans for regenerativ medicin baseret på deres pluripotency3.
To grupper Ecrd etablerede cellelinier mus ES i 1981 og når celler, der stammer fra den tidlige mus Foster var kulturperler i føtal bovint serum (FBS)-indeholdende medium med musen embryonale fibroblaster (MEFs) som feeder lag1,4 . MEFs var inaktiveret mitotically og blev præ dyrket i retter inden dyrkning af ES-celler. MEFs yder støtte til musen ES celle attachment og producere vækstfaktorer for at fremme udbredelse og undertrykke differentiering5, mens FBS tilbyder væsentlige trofiske faktorer og hormoner for celledelingen. Efterfølgende undersøgelser viste, at LIF produceret af feeder celler var den centrale cytokin til selvfornyelse og vedligeholdelse af pluripotency i mus ES-celler og tilsætning af LIF i medium kunne erstatte feeder celler6. I øjeblikket, er opretholdelse af musen ES-celler i FBS/LIF medium på foderautomater stadig standard metode vedtaget af mange forskere. Imidlertid opstår nogle problemer med denne klassiske kultur tilgang. For det første feeder celler udskiller overskydende og ukontrolleret faktorer og kan forårsage sygdomsfremkaldende forurening7. At undgå denne indblanding, belægning overfladen af retter med gelatine og tilsætning af LIF i serum, der indeholder mediet er alternative metoder til at opretholde mus ES celler uden feeder-lag celler. Derudover udviser mus ES celler dyrkes serum/LIF betingelser morfologiske heterogenitet i cellepopulationer og endda i niveauet udtryk pluripotency-relaterede faktorer8. Nylige undersøgelser tyder på, at serum/LIF betingelser pluripotency-relaterede core transskriptionsfaktorer (herunder SOX2, Nanog og OCT4) kan opretholde pluripotency gennem LIF og WNT signalering; dog især aktivere de også en fibroblast vækstfaktor (FGF) signal til at udløse differentiering8. På grund af den ambivalent dual action af transkriptionsfaktorer core præsentere mus ES celler dyrkes i serum heterogenitet, bestående af to udskiftelige populationer, en lignende til ICM og en anden der ligner PRIMES epiblast stat8. Derudover mus ES celler i serum udviser ofte en global hypermethylation9, ICM og PGCs er i en global hypomethylated stat, som er tæt forbundet med deres pluripotency9,10.
Der er en betydelig efterspørgsel til at udvikle nye metoder til dyrkning mus ES celler. Flere forbedrede protokoller har været etableret siden 200311 men der fortsat er visse begrænsninger og mangler7. Siden 2008, den kombinerede udnyttelse af to små-molekyle kinase hæmmere, PD0325901 (hæmmer af mitogen-aktiveret protein kinase (MAPK) / ekstracellulær signal-reguleret kinase (ERK) (MEK)) og CHIR99021 (hæmmer af glykogen syntase kinase 3 (GSK3)), i N2B27 medium med LIF og uden serum har åbnet nye perspektiver for dyrkningsbaserede mus ES-celler12. Denne nye definerede medium er karakteriseret ved brugen af to hæmmere (2i). Musen ES celler dyrkes i 2i/LIF medium er mere homogen cellepopulationer og udtryk pluripotency faktorer. 2i/LIF-kulturperler mus ES celler udviser desuden DNA hypomethylation globalt, som er tættere på ICM-lignende celler9,13. Selv så, har 2i kultur sine ulemper. PD0325901 og CHIR99021 er uopløseligt i vand, og generelt er opløst i dimethylsulfoxid (DMSO)-baserede stock løsning for at tilføje dem i dyrkningsmediet. Undersøgelser har vist, at langvarig og lavdosis udsættelse af celler for DMSO kan føre til cytotoksicitet14.
Her, vi udnyttede to små-molekyle forbindelser og udviklet en ny kultur metode af musen ES celler. Roman kultur metoden kombinerer Vc og MEK hæmmer PD0325901 for at fremme DNA hypomethylation hurtigt og til en sammenlignelig PGC, benævnt effektivt nemlig Vc/PD0325901 dyrkningsbaserede protokol. Musen ES-celler i Vc/PD0325901-tilføjet serum-holdige medium udstille homogenitet i morfologi og er påført i en grundtilstand. I forhold til 2i kultur, mus ES celler kulturperler Vc/PD0325901 betingelser udviser hurtigere kinetik af DNA demetylering og kan nå det hypomethylation niveau sammenlignes med PGC. Desuden brug af en enkelt hæmmer (PD0325901) nedsætter input mængden af DMSO i medium i forhold til, bruges i 2i (PD0325901/CHIR99021) og reducerer skader på celler.

<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

Embryonale stamceller (ES) har potentiale til at differentiere i nogen af de tre Kim lag (endoderm, mesoderm eller ectoderm), og kan generere mange lineages for regenerativ medicin. ES celle kultur i vitro har længe været genstand for udbredt bekymring. Klassisk, mus ES celler bevares i serum og leukæmi hæmmende faktor (LIF)-indeholdende medium. Dog under serum/LIF betingelser, celler Vis heterogenitet i morfologi og profilen udtryk pluripotency-relaterede gener, og er for det meste i en metastabile tilstand. Desuden kulturperler ES-celler udstille globale hypermethylation, men naiv ES cellerne i den indre cellemasse (ICM) og primordiale kønsceller (PGCs) er i en tilstand af globale hypomethylation. Tilstanden hypomethylated af ICM og PGCs er tæt forbundet med deres pluripotency. For at forbedre mus ES celle kultur metoder, har vi for nylig udviklet en ny metode baseret på selektivt kombineret udnyttelse af to små-molekyle forbindelser til at opretholde den DNA hypomethylated og pluripotente tilstand. Her præsenterer vi at fælles behandling af C-vitamin (Vc) og PD0325901 kan slette omkring 90% af 5-methylcytosine (5mC) på 5 dage i mus ES-celler. Genererede 5mC indhold kan sammenlignes i PGCer. Den mekanistiske undersøgelse viser, at PD0325901 op-regulerer Prdm14 udtryk for at undertrykke Dnmt3b (de novo DNA methyltransferase) og Dnmt3l (cofaktor for Dnmt3b), ved at reducere de novo 5mC syntese. VC letter konvertering af 5mC til 5-hydroxymethylcytosine (5hmC) katalyseret hovedsagelig af Tet1 og Tet2, der angiver inddragelse af både passive og aktive DNA demethylations. Desuden under Vc/PD0325901 betingelser Vis mus ES celler homogene morfologi og pluripotente stat. Kollektivt, foreslår vi en roman og kemisk-synergy kultur metode til at opnå DNA hypomethylation og vedligeholdelse af pluripotency i mus ES celler. Små-molekyle kemisk-dependent metode overvinder serum kultur og holder løfte om at generere homogene ES-celler for yderligere kliniske applikationer og forsker store mangler.

VC/PD0325901 induceret synergistisk globale sletning af musen ES-celler. Musen ES-celler i serum udviser DNA hypermethylation, mens pluripotente ICM celler og PGCs vise global sletning af DNA methylering og hypomethylated staten er tæt forbundet med deres pluripotency9,10.
Tidligere, vi og andre konstateret, at Vc kan forbedre Tet-medieret 5mC demetylering<sup class="xr...

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

En alternativ kultur metode til at opretholde genomisk Hypomethylation af mus embryonale stamceller ved hjælp af MEK hæmmer PD0325901 og C-Vitamin

Vi beskrev i detaljer to kemiske-baserede protokoller for dyrkning mus embryonale stamceller. Denne nye metode udnytter synergistisk mekanismer til at fremme Tet-medierede oxidation (af C-vitamin) og undertrykke de novo syntesen af 5-methylcytosine (af PD0325901) for at opretholde DNA hypomethylation og pluripotency mus embryonale stamceller.

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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