This work was supported by a grant from the NIH (1R35GM133496-01) to Z. Gao. We would like to thank Dr. Ryan Hobbs for the assistance in sectioning. We thank&#160;Penn State College of Medicine&#39;s core facilities, including the Genome Sciences and Bioinformatics, the Advanced Light&#160;Microscopy Imaging, and the Flow Cytometry. We also thank Dr.&#160;Yuka Imamura&#160;for the assistance in RNA-seq analysis.&#160;

1. mESC culture
<ol>
	<li>Coat a 10 cm tissue-culture-treated plate with 0.1% gelatin and allow the gelatin to set for at least 15&#8211;30 min before aspirating it out.</li>
	<li>Seed &#947;-irradiated mouse embryonic fibroblasts (MEFs) one day before culturing the mESCs in the pre-warmed mESC medium (Dulbecco&#8217;s modified Eagle medium (DMEM) with 15% fetal bovine serum (FBS), non-essential amino acids, &#946;-mercaptoethanol, L-glutamine, penicillin/streptomycin, sodium pyruvate, LIF, PD0325901 (PD), and Chir9902...

<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>

Authors declare that there are no competing financial interests.

The method for neural differentiation of mouse embryonic stem cells has been established for decades and researchers have continued to modify the previous protocols or create new ones for various purposes7,10,11. We utilized a series of assays to comprehensively analyze the efficiency and progress of the differentiation stages of mESCs to neurons, which may be used in analysis of other lineage differentiation of mouse or human E...

Since their first derivation from the inner cell mass of the developing mouse blastocysts1,2, mouse embryonic stem cells (mESC) have been used as powerful tools to study stem cell self-renewal and differentiation3. Furthermore, studying mESC differentiation leads to tremendous understanding of molecular mechanisms that may improve efficiency and safety in stem cell-based therapy in treating diseases such as neurodegenerative disorders4. Compared to animal models, this in vitro system provides many advantages including simplicity in practice and assessment, low cost in maintaining cell lines in contrast to animals, and relative ease in genetic manipulations. However, the efficiency and quality of differentiated cell types are often affected by different lines of mESCs as well as the differentiation methods5,6. Also, the traditional assays to evaluate differentiation efficiency rely on qualitative examination of selected marker genes which lack robustness and they therefore fail to grasp global changes in gene expression.
Here we aim to use a battery of assays for systematic assessment of the neuronal differentiation. Using both traditional in vitro analyses on selected markers and RNA-seq, we establish a platform for measurement of the differentiation efficiency as well as the transcriptomic changes during this process. Based on a previously established protocol7, we generated embryoid bodies (EBs) through the hanging drop technique, followed by induction using supraphysiologic amount of retinoic acid (RA) to generate neural progenitor cells (NPCs), which were subsequently differentiated to neurons with neural induction medium. To examine the efficiency of the differentiation, in addition to traditional RT-qPCR and immunofluorescence (IF) assays, we performed RNA-seq and flow cytometry. These analyses provide comprehensive measurement of the progression of the stage-specific differentiation.

<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

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

As a representation of our method, we performed an EB, NPC, and neuron differentiation experiment on E14 cells. E14 cells were cultured on &#947;-irradiated MEFs (Figure 1A) until the &#947;-irradiated MEF population diluted out. We confirmed the pluripotency of the E14 cells by performing Alkaline Phosphatase (AP) staining (Figure 1B) and later RT-qPCR (see below) for Nanog and Oct4 markers. The &#947;-irradiated MEF-free E14 cells were then i...

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

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

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.

differentiation-characterization-neural-progenitors-neurons-from

Research

JoVE Journal

Developmental Biology

4.6K Views.  Penn State Hershey Cancer Institute. 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 cult...