Name: differentiation and characterization of neural progenitors and neurons from mouse embryonic stem cells
Uploaded: 2020-05-15T15:00:00
Description: Watch this Scientific Journal Video about Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells at JoVE.com

Detta arbete stöddes av ett bidrag från NIH (1R35GM133496-01) till Z. Gao. Vi vill tacka dr Ryan Hobbs för hjälpen med sektionsavsnittet. Vi tackar Penn State College of Medicine kärnanläggningar, inklusive genomvetenskap och bioinformatik, Advanced Light Microscopy Imaging och Flow Cytometry. Vi tackar också Dr. Yuka Imamura för hjälpen i RNA-seq-analysen.

1. mESC-kultur
<ol><li>Täck en 10 cm vävnadskulturbehandlad tallrik med 0,1% gelatin och låt gelatinet sättas i minst 15-30 minuter innan du aspirer ut det.</li>
	<li>Frö γ bestrålade mus embryonala fibroblaster (MEFs) en dag innan odling av mESC i det förvärmda mESC-mediet (Dulbeccos modifierade Eagle medium (DMEM) med 15% fetala nötkreaturserum (FBS), icke-essentiella aminosyror, β-mercaptoethanol, L-glutamin, penicillin/streptomycin, natrium pyruvat, LIF, PD0325901(PD032590).</li>
	<li>Låt de γ bestrål...

<ol>
	<li>Kaufman, M. H., Evans, M. J. <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, 154-156 (1981).</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> Proceedings of the National Academy of Sciences of the United States of America. 78, 7634-7638 (1981).</li><li>Czechanski, 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=Derivation+and+characterization+of+mouse+embryonic+stem+cells+from+permissive+and+nonpermissive+strains.">Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains.</a> Nature Protocols. 9 (3), 559-574 (2014).</li><li>Sugaya, K., Vaidya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Therapies+for+Neurodegenerative+Diseases.">Stem Cell Therapies for Neurodegenerative Diseases.</a> Exosomes, Stem Cells and MicroRNA: Aging, Cancer and Age Related Disorders. , 61-84 (2018).</li><li>Dang, S. M., Kyba, M., Perlingeiro, R., Daley, G. Q., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+embryoid+body+formation+and+hematopoietic+development+from+embryonic+stem+cells+in+different+culture+systems.">Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems.</a> Biotechnology and Bioengineering. 78 (4), 442-453 (2002).</li><li>McKee, C., Chaudhry, 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=Advances+and+challenges+in+stem+cell+culture.">Advances and challenges in stem cell culture.</a> Colloids and Surfaces B: Biointerfaces. 159, 62-77 (2017).</li><li>Bibel, 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=Differentiation+of+mouse+embryonic+stem+cells+into+a+defined+neuronal+lineage.">Differentiation of mouse embryonic stem cells into a defined neuronal lineage.</a> Nature Neuroscience. 7 (9), 1003-1009 (2004).</li><li>Wang, Q., 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=WDR68+is+essential+for+the+transcriptional+activation+of+the+PRC1-AUTS2+complex+and+neuronal+differentiation+of+mouse+embryonic+stem+cells.">WDR68 is essential for the transcriptional activation of the PRC1-AUTS2 complex and neuronal differentiation of mouse embryonic stem cells.</a> Stem Cell Research. 33, 206-214 (2018).</li><li>Ying, Q. L., Stavridis, M., Griffiths, D., Li, M., 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=Conversion+of+embryonic+stem+cells+into+neuroectodermal+precursors+in+adherent+monoculture.">Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture.</a> Nature Biotechnology. 21 (2), 183-186 (2003).</li><li>Visan, 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=Neural+differentiation+of+mouse+embryonic+stem+cells+as+a+tool+to+assess+developmental+neurotoxicity+in+vitro.">Neural differentiation of mouse embryonic stem cells as a tool to assess developmental neurotoxicity in vitro.</a> NeuroToxicology. 33 (5), 1135-1146 (2012).</li><li>Fraichard, 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=In+vitro+differentiation+of+embryonic+stem+cells+into+glial+cells+and+functional+neurons.">In vitro differentiation of embryonic stem cells into glial cells and functional neurons.</a> Journal of Cell Science. 108 (10), 3181-3188 (1995).</li><li>Stavridis, M. P., 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=Neural+differentiation+of+mouse+embryonic+stem+cells.">Neural differentiation of mouse embryonic stem cells.</a> Biochemical So. 31, 45-49 (2003).</li><li>Park, Y. -. 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=Effects+of+Feeder+Cell+Types+on+Culture+of+Mouse+Embryonic+Stem+Cell+In+vitro.">Effects of Feeder Cell Types on Culture of Mouse Embryonic Stem Cell In vitro.</a> Development &amp; Reproduction. 19 (3), 119-126 (2015).</li><li>Lee, J. H., Lee, E. J., Lee, C. H., Park, J. H., Han, J. Y., Lim, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+leukemia+inhibitory+factor+for+establishing+and+maintaining+embryonic+stem+cells+in+mice.">Requirement of leukemia inhibitory factor for establishing and maintaining embryonic stem cells in mice.</a> Fertility and Sterility. 92 (3), 1133-1140 (2009).</li><li>Onishi, K., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIF+signaling+in+stem+cells+and+development.">LIF signaling in stem cells and development.</a> Development (Cambridge). 142 (13), 2230-2236 (2015).</li><li>Smith, A. 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=Inhibition+of+pluripotential+embryonic+stem+cell+differentiation+by+purified+polypeptides.">Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides.</a> Nature. 336, 688-690 (1988).</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+leukemia+inhibitory+factor+maintains+the+developmental+potential+of+embryonic+stem+cells.">Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stem cells.</a> Nature. 336, 684-687 (1988).</li><li>Ghimire, S., 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+analysis+of+naive,+primed+and+ground+state+pluripotency+in+mouse+embryonic+stem+cells+originating+from+the+same+genetic+background.">Comparative analysis of naive, primed and ground state pluripotency in mouse embryonic stem cells originating from the same genetic background.</a> Scientific Reports. 8 (1), 1-11 (2018).</li><li>Kurosawa, H., Imamura, T., Koike, M., Sasaki, K., Amano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Method+for+Forming+Embryoid+Body+from+Mouse+Embryonic+Stem+Cells.">A Simple Method for Forming Embryoid Body from Mouse Embryonic Stem Cells.</a> Journal of Bioscience and Bioengineering. 96 (4), 409-411 (2003).</li><li>Wang, X., Yang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+mouse+embryonic+stem+(mES)+cells+using+the+hanging+drop+method.">In vitro differentiation of mouse embryonic stem (mES) cells using the hanging drop method.</a> Journal of Visualized Experiments. (17), 2-3 (2008).</li><li>Soprano, D. R., Teets, B. W., Soprano, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Retinoic+Acid+in+the+Differentiation+of+Embryonal+Carcinoma+and+Embryonic+Stem+Cells.">Role of Retinoic Acid in the Differentiation of Embryonal Carcinoma and Embryonic Stem Cells.</a> Vitamins and Hormones. 75 (06), 69-95 (2007).</li><li>Venere, M., Han, Y. G., Bell, R., Song, J. S., Alvarez-Buylla, A., Blelloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox1+marks+an+activated+neural+stem/progenitor+cell+in+the+hippocampus.">Sox1 marks an activated neural stem/progenitor cell in the hippocampus.</a> Development (Cambridge). 139 (21), 3938-3949 (2012).</li><li>Chen, Y., 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=NS21:+Re-defined+and+modified+supplement+B27+for+neuronal+cultures.">NS21: Re-defined and modified supplement B27 for neuronal cultures.</a> Journal of Neuroscience Methods. 171 (2), 239-247 (2008).</li><li>Bahmad, H. 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=The+Akt/mTOR+pathway+in+cancer+stem/progenitor+cells+is+a+potential+therapeutic+target+for+glioblastoma+and+neuroblastoma.">The Akt/mTOR pathway in cancer stem/progenitor cells is a potential therapeutic target for glioblastoma and neuroblastoma.</a> Oncotarget. 9 (71), 33549-33561 (2018).</li><li>Bastiaens, A. J., 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=Advancing+a+MEMS-Based+3D+Cell+Culture+System+for+in+vitro+Neuro-Electrophysiological+Recordings.">Advancing a MEMS-Based 3D Cell Culture System for in vitro Neuro-Electrophysiological Recordings.</a> Frontiers in Mechanical Engineering. 4, 1-10 (2018).</li><li>Antill-O'Brien, N., Bourke, J., O'Connell, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer-by-layer:+The+case+for+3D+bioprinting+neurons+to+create+patient-specific+epilepsy+models.">Layer-by-layer: The case for 3D bioprinting neurons to create patient-specific epilepsy models.</a> Materials. 12 (19), (2019).</li><li>Duval, 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=Modeling+physiological+events+in+2D+vs.+3D+cell+culture.">Modeling physiological events in 2D vs. 3D cell culture.</a> Physiology. 32 (4), 266-277 (2017).</li><li>Joshi, P., Lee, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+imaging+(HCI)+on+miniaturized+three-dimensional+(3D)+cell+cultures.">High content imaging (HCI) on miniaturized three-dimensional (3D) cell cultures.</a> Biosensors. 5 (4), 768-790 (2015).</li></ol>

Metoden för neural differentiering av mus embryonala stamceller har fastställts i årtionden och forskare har fortsatt att modifiera de tidigare protokollen eller skapa nya för olika ändamål7,10,11. Vi använde en serie analyser för att omfattande analysera effektiviteten och framstegen av differentiering stadier av mESCs till nervceller, som kan användas i analys av andra härstamning differentiering av mus eller mänskl...

Sedan deras första härledning från den inre cellmassan hos de framväxande mus blastocysts1,2, mus embryonala stamceller (mESC) har använts som kraftfulla verktyg för att studera stamceller självförnyelse och differentiering3. Att studera mESC-differentiering leder dessutom till en enorm förståelse för molekylära mekanismer som kan förbättra effektiviteten och säkerheten vid stamcellsbaserad terapi vid behandling av sjukdomar som neurodegenerativa sjukdomar4. Jämfört med djurmodeller ger detta in vitro-system många fördelar, inklusive enkelhet i praktiken och bedömning, låg kostnad för att upprätthålla cellinjer i motsats till djur och relativ lätthet i genetiska manipuleringar. Effektiviteten och kvaliteten hos differentierade celltyper påverkas dock ofta av olika linjer av mESC samt differentieringsmetoderna5,6. De traditionella analyserna för att utvärdera differentieringseffektiviteten bygger också på kvalitativ undersökning av utvalda markörgener som saknar robusthet och de därför inte förstår globala förändringar i genuttryck.
Här strävar vi efter att använda ett batteri av analyser för systematisk bedömning av neuronal differentiering. Med hjälp av både traditionella in vitro-analyser på utvalda markörer och RNA-seq etablerar vi en plattform för mätning av differentieringseffektiviteten samt transkriptomiska förändringar under denna process. Baserat på ett tidigare etablerat protokoll7genererade vi embryoidkroppar (EBs) genom hängande droppteknik, följt av induktion med suprafysiologic mängd retinsyra (RA) för att generera neurala stamceller (NPCs), som därefter differentierades till nervceller med neuralt induktionsmedium. För att undersöka effektiviteten i differentieringen, förutom traditionella RT-qPCR och immunofluorescens (IF) analyser, utförde vi RNA-seq och flöde cytometri. Dessa analyser ger omfattande mätning av utvecklingen av den stegspecifika differentieringen.

<table border="0" cellpadding="0" cellspacing="0" style="width:805px;" width="804">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">0.05% Trypsin + 0.53mM EDTA 1X</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">25-052-CV</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">0.1% Gelatin</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">G1890-100G</td>
			<td>Prepared in de-ionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">16% Paraformaldehyde</td>
			<td style="width:148px;">Thermo Scientific</td>
			<td style="width:127px;">28908</td>
			<td>Diluted in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">40-&#956;m cell strainer</td>
			<td style="width:148px;">Falcon</td>
			<td style="width:127px;">352340</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Albumax</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">11020021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">AlexaFluor 488 goat anti-mouse IgG (H+L)</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:127px;">A11001</td>
			<td>Antibody was diluted at 1:500 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Alkaline Phosphatase Staining Kit II</td>
			<td style="width:148px;">Stemgent</td>
			<td style="width:127px;">00-0055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">AzuraQuant Green Fast qPCR Mix LoRox</td>
			<td style="width:148px;">Azura Genomics</td>
			<td style="width:127px;">AZ-2105</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">B27 supplement</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">BD FACSCanto</td>
			<td style="width:148px;">BD</td>
			<td style="width:127px;">657338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">bFGF</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">11123149001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">BioAnalyzer High Sensitivity DNA Kit</td>
			<td style="width:148px;">Agilent</td>
			<td style="width:127px;">5067-4626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Chir99021</td>
			<td style="width:148px;">Cayman Chemicals</td>
			<td style="width:127px;">13122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Chloroform</td>
			<td style="width:148px;">C298-500</td>
			<td style="width:127px;">Fisher Chemical</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">DAPI</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:127px;">R37606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">DMEM</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">DMEM/F12 medium</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">11320033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">EB buffer</td>
			<td style="width:148px;">Qiagen</td>
			<td style="width:127px;">19086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Ethanol</td>
			<td style="width:148px;">111000200</td>
			<td style="width:127px;">Pharmco</td>
			<td>Diluted in de-ionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Fetal bovine serum</td>
			<td style="width:148px;">Atlanta Biologicals</td>
			<td style="width:127px;">S10250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Fisherbrand Superfrost Plus Microscope Slides</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:127px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">HiSeq 2500 Sequencing System</td>
			<td style="width:148px;">Illumina</td>
			<td style="width:127px;">SY-401-2501</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Isopropanol</td>
			<td style="width:148px;">BDH1133-4LG</td>
			<td style="width:127px;">BDH VWR Analytical</td>
			<td>Diluted in de-ionized water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">L-glutamine</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">25030024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">LIF</td>
			<td style="width:148px;">N/A</td>
			<td style="width:127px;">N/A</td>
			<td>Collected from MEF supernatant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">m18srRNA primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-GCAATTATTCCCCATGAACG-3&#39; 
			5&#39;-GGCCTCACTAAACCATCCAA-3&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">MEM Non-essential amino acids</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">25-025-Cl</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mNanog primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-AGGCTTTGGAGACAGTGAGGTG-3&#39; 
			5&#39;-TGGGTAAGGGTGTTCAAGCACT-3&#39;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">mNes primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-AGTGCCCAGTTCTAGTGGTGTCC-3&#39; 
			5&#39;-CCTCTAAAATAGAGTGGTGAGGGTTG-3&#39;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">mNeuroD1 primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-CGAGTCATGAGTGCCCAGCTTA-3&#39; 
			5&#39;-CCGGGAATAGTGAAACTGACGTG-3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mOct4 primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-AGATCACTCACATCGCCAATCA-3&#39; 
			5&#39;-CGCCGGTTACAGAACCATACTC-3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mPax6 primers</td>
			<td style="width:148px;">IDTDNA</td>
			<td style="width:127px;">N/A</td>
			<td style="width:255px;">5&#39;-CTTGGGAAATCCGAGACAGA-3&#39; 
			5&#39;-CTAGCCAGGTTGCGAAGAAC-3&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">N2 supplement</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">17502048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Nestin primary antibody</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:127px;">MAB5326</td>
			<td>Antibody was diluted at 1:200 for IF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">Neural basal</td>
			<td style="width:148px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">21103049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Neurofilament primary antibody</td>
			<td style="width:148px;">DSHB</td>
			<td style="width:127px;">2H3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">NEXTflex Illumina Rapid Directional RNA-Seq Library Prep Kit</td>
			<td style="width:148px;">BioO Scientific</td>
			<td style="width:127px;">NOVA-5138-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">PD0325901</td>
			<td style="width:148px;">Cayman Chemicals</td>
			<td style="width:127px;">13034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Penicillin/streptomycin</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Phosphate-buffered saline (PBS)</td>
			<td style="width:148px;">N/A</td>
			<td style="width:127px;">N/A</td>
			<td>Prepared in de-ionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">- Potassium chloride</td>
			<td style="width:148px;">P217-500G</td>
			<td style="width:127px;">VWR</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">- Potassium phosphate monobasic anhydrous</td>
			<td style="width:148px;">0781-500G</td>
			<td style="width:127px;">VWR</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">- Sodium chloride</td>
			<td style="width:148px;">BP358-10</td>
			<td style="width:127px;">Fisher Bioreagents</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:276px;">- Sodium phosphate, dibasic, heptahydrate</td>
			<td style="width:148px;">SX0715-1</td>
			<td style="width:127px;">Milipore</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Random hexamer primer</td>
			<td style="width:148px;">Thermo Scientific</td>
			<td style="width:127px;">SO142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Retinoic acid</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">R2625</td>
			<td>Prepared in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Sodium pyruvate</td>
			<td style="width:148px;">Corning</td>
			<td style="width:127px;">25-000-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Sucrose</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:127px;">84097</td>
			<td>Diluted in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">SuperScript III Reverse Transcriptase</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:127px;">18064022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:148px;">Sakura</td>
			<td style="width:127px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">TriPure Isolation Reagent</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:127px;">11667165001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">TruSeq Rapid</td>
			<td style="width:148px;">Illumina</td>
			<td style="width:127px;">20020616</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">&#946;-mercaptoethanol</td>
			<td style="width:148px;">Fisher BioReagents</td>
			<td style="width:127px;">BP176-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation and characterization of neural progenitors and neurons from mouse embryonic stem cells

Vi beskriver steg-för-steg förfarandet för odling och differentiering mus embryonala stamceller i neuronal härstamningar, följt av en serie analyser för att karakterisera de differentierade cellerna. E14 mus embryonala stamceller användes för att bilda embryoid kroppar genom hängande dropp metod, och sedan induceras att differentiera i neurala stamceller genom retinsyra, och slutligen differentieras i nervceller. Kvantitativ omvänd transkription polymeras kedjereaktion (RT-qPCR) och immunofluorescens experiment visade att neurala stamceller och nervceller uppvisar motsvarande markörer (nestin för neurala stamceller och neurofilament för nervceller) dag 8 och 12 post-differentiering, respektive. Flöde cytometri experiment på en E14 linje uttrycker en Sox1 promotor-driven GFP reporter visade att cirka 60% av cellerna på dag 8 är GFP positiva, vilket indikerar den framgångsrika differentiering av neurala stamceller i detta skede. Slutligen användes RNA-seq-analys för att profilera de globala transkriptomiska förändringarna. Dessa metoder är användbara för att analysera inblandning av specifika gener och vägar i regleringen av cellidentitetsövergången under neuronal differentiering.

Som en representation av vår metod utförde vi ett EB, NPC och neuron differentiering experiment på E14 celler. E14-celler odlades på γ bestrålade MEF(figur 1A) tills den γ bestrålade MEF-populationen utspädd. Vi bekräftade pluripotency av E14 celler genom att utföra alkaliska fosfatas (AP) färgning (figur 1B) och senare RT-qPCR (se nedan) för Nanog och Oct4 markörer. De γ bestrålade MEF-fria E14-cellerna inducerades sedan för d...

Watch this Scientific Journal Video about Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells at JoVE.com

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

Vi beskriver förfarandet för in vitro differentiering av mus embryonala stamceller i neuronala celler med hjälp av hängande dropp metod. Dessutom utför vi en omfattande fenotypisk analys genom RT-qPCR, immunofluorescens, RNA-seq och flöde cytometri.

Differentiering och karakterisering av neurala stamceller och nervceller från mus embryonala stamceller

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

In addition to the protocol for mouse embryonic stem cell differentiation into neuron cells, we also show a set of experiments that can comprehensively analyze the differentiation process. These techniques are pertinent to early ectodermal embryonic development, providing researchers with options to analyze neuron cell differentiation. When attempting this procedure, starting with stem cells in optimal condition is essential.Hence it is important that the EBs, MPCs and neurons are cultured in fresh and optimum media. Visual demonstration of this protocol is important, because it contains minute detail that can affect its success and effectiveness, especially the differentiation part. Begin by setting up a hanging drop cell culture.For a 10-centimeter cell culture plate, adjust the concentration to 500 cells per 20-microliters of differentiation medium. Prepare enough cell suspension for 50 20-microliter droplets. Use a micro-pipette or a repeater pipette to place 20-microliter droplets of cell suspension onto the lid of a tissue culture plate, making sure that the droplets aren't too close together, to prevent them from merging.Fill the plate with five to 10-milliliters of PBS and carefully put the lid back on the plate. Incubate the culture in the 37 degrees Celsius incubator. After two days, collect the droplets from the lid, and place them in a 10-centimeter cell culture suspension plate filled with 10-milliliters of differentiation medium, then place the plate on an orbital shaker set to low speed in the incubator.After another two days, centrifuge the cells at 200 times G for three minutes, and remove the supernatant. To induce embryoid body differentiation into neural progenitor cells, re-suspend the cells in differentiation medium with five-micromolar retinoic acid. Incubate the plate for two days, then replace the least half of the medium with fresh medium, by tilting the plate and pipetting it out.Collect the cells two days later by centrifuging them at 200 times G for three minutes, and removing the supernatant. Fix the embryoid bodies and neural progenitor cells with 4%PFA in PBS for 30 minutes at room temperature. Then remove the PFA and wash the cells in PBS for five minutes.Incubate the cells serially in PBS 10, 20 and 30%sucrose solutions, at 25 to 28 degrees Celsius, with each incubation lasting 30 minutes or longer until the cells settle at the bottom of the tube. And use it to transfer the sample to the center of a Cryomold, pipetting out the excess liquid. Carefully add optimum cutting temperature or OCT solution to the mold, without re-suspending the sample, then remove excessive bubbles with a pipette.Placed the mold with the sample on a laboratory mixer at low speed for 15 minutes, which will settle the embryoid bodies and neural progenitor cells to the bottom of the OCT solution. Then quickly freeze the sample by placing the mold in liquid nitrogen. Block OCT sections or culture chambers containing neurons in 10%normal donkey serum, and 0.1%Triton X-100 in PBS for one hour at room temperature.Then incubate the samples in primary antibody, diluted in 5%normal donkey serum, and 0.05%Triton X-100 in PBS, at four degrees Celsius overnight. On the next day, wash samples in PBS with 0.1%Triton X-100 three times for five minutes per wash. Incubate them with secondary antibody, diluted in 5%normal donkey serum, and 0.05%Triton X-100 in PBS, for one hour at room temperature.Repeat the washes in Triton X-100 and PBS, then incubate the sample in one microgram-per-milliliter DAPI. Mount the sample with a cover slip and some mounting medium, allow it to dry, and image it with a fluorescence microscope. To perform neuron differentiation, E14 cells were cultured on gamma-irradiated mouse embryonic fibroblasts until the fibroblast population diluted out.The pluripotency of the E14 cells was confirmed with alkaline phosphatase staining. After differentiation, embryonic bodies had a round shape that continuously increased in size. By day 10, neural progenitor cells differentiating into neurons, had an elongated cell shape.Immunofluorescence experiments were performed on neural progenitor cells at day eight, and on neurons at day 12. Positive staining was observed for nesting in neural progenitor cells, and for neurofilament in neurons. A mouse embryonic stem cell line expressing a Sox1 promoter-driven GFP reporter was differentiated, and flow cytometry was performed on the embryonic stem cells and neural progenitor cells.It was found that 58.7%of total cells at the NPC stage are GFP positive, while 0%are GFP positive at the ESC stage. RNA sequence experiments were performed on E14 embryonic stem cells, embryoid bodies, and neural progenitor cells. The genes in the RNA sequence heat map was sorted based on their expression levels.Gene ontology analysis of the four gene clusters showed that they correspond to distinct cellular functions or pathways. When performing this protocol, EBS should appear as spherical, blackened masses of cells. To ensure that EBS grow with such, change the medium on day one, if the medium starts to become yellowish.The same approach is important for MPC culture, as both EBs and MPCs are in a critical stage of early embryonic development. Other functional genomic assays, such as ChIPseek and chromatin confirmation capture assays from the different stages of neuron differentiation, can be performed to understand the underlying biological processes. Integrating by chemical and functional genomics assays in the neuron differentiation process, allows for researchers to better delineate the molecular mechanisms of early embryonic development.

Research

JoVE Journal

Developmental Biology

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

Neural differentiering av Mouse embryonala stamceller i Serumfri enskiktskultur

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Epigenetisk reglering av hjärt Differentiering av embryonala stamceller och vävnader

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Regisserad differentiering av primitiv och slutgiltig hematopoetiska progenitorceller från mänskliga pluripotenta stamceller

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Effektiv Differentiering av pluripotenta stamceller till NKX6-1<sup&gt; +</sup&gt; Pankreasstamfäder

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

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

Analys av retinsyra-inducerade Neural differentiering av Mouse embryonala stamceller i två och tre-dimensionella Embryoid organ

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 ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Snabb, regisserad differentiering av Retinal Pigment epitelial celler från mänskliga embryonala eller inducerade pluripotenta stamceller

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Isolering, kultur och differentiering av benmärgens stromaceller och Osteoclast stamfäder från möss

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Generering av inducerade neurala stamceller från perifera mononukleära celler och differentiering mot dopaminerga neuron prekursorer för transplantations studier

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Effektiv neurala differentiering med hjälp av Single-cell kultur av mänskliga embryonala stamceller

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

Beredning av små RNA-bibliotek för sekvensering från tidiga musembryon

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...