Name: neurogenesis using p19 embryonal carcinoma cells
Uploaded: 2019-04-27T13:00:00
Description: Watch this Scientific Journal Video about Neurogenesis Using P19 Embryonal Carcinoma Cells at JoVE.com

The study was financially supported by National Science Centre, Poland (grant no. UMO-2017/25/N/NZ3/01886) and KNOW (Leading National Research Centre) Scientific Consortium &#34;Healthy Animal - Safe Food&#34;, decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015

1. Culture Maintenance
<ol>
	<li>Culture the P19 cell line in Maintenance Medium (Dulbecco&#39;s modified Eagle&#39;s medium with 4,500 mg/L of glucose supplemented with 10% FBS, 100 units/mL penicillin and 100 units/mL streptomycin). Incubate at 37 &#176;C and 5% CO2.</li>
</ol>
2. Sub-culturing Cells
<ol>
	<li>When cells reach approximately 80% confluence, remove the spent medium from the cell culture flasks (surface area 25 cm2).</li>
	<li>Wash the cells with 2 mL of p...

<ol>
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H., 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=MicroRNA+miR-124+controls+the+choice+between+neuronal+and+astrocyte+differentiation+by+fine-tuning+Ezh2+expression.">MicroRNA miR-124 controls the choice between neuronal and astrocyte differentiation by fine-tuning Ezh2 expression.</a> Journal of Biological Chemistry. 289 (30), 20788-20801 (2014).</li><li>Jones-Villeneuve, E., McBurney, M. W., Rogers, K. A., Kalnins, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+induces+embryonal+carcinoma+cells+to+differentiate+into+neurons+and+glial+cells.">Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells.</a> The Journal of Cell Biology. 94 (2), 253-262 (1982).</li><li>McBurney, M. W., Rogers, B. J. <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+male+embryonal+carcinoma+cells+and+their+chromosome+replication+patterns.">Isolation of male embryonal carcinoma cells and their chromosome replication patterns.</a> Developmental Biology. 89 (2), 503-508 (1982).</li><li>Jones-Villeneuve, E., Rudnicki, M. A., Harris, J. F., McBurney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid-induced+neural+differentiation+of+embryonal+carcinoma+cells.">Retinoic acid-induced neural differentiation of embryonal carcinoma cells.</a> Molecular and Cellular Biology. 3 (12), 2271-2279 (1983).</li><li>Jasmin, D. C., Spray, A. C., Campos de Carvalho, R., Mendez-Otero, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+induction+of+cardiac+differentiation+in+P19+embryonal+carcinoma+stem+cells.">Chemical induction of cardiac differentiation in P19 embryonal carcinoma stem cells.</a> Stem Cells and Development. 19 (3), 403-412 (2010).</li><li>Solari, M., Paquin, J., Ducharme, P., Boily, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P19+neuronal+differentiation+and+retinoic+acid+metabolism+as+criteria+to+investigate+atrazine,+nitrite,+and+nitrate+developmental+toxicity.">P19 neuronal differentiation and retinoic acid metabolism as criteria to investigate atrazine, nitrite, and nitrate developmental toxicity.</a> Toxicological Sciences. 113 (1), 116-126 (2010).</li><li>Babuska, V., 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=Characterization+of+P19+cells+during+retinoic+acid+induced+differentiation.">Characterization of P19 cells during retinoic acid induced differentiation.</a> Prague Medical Report. 111 (4), 289-299 (2010).</li><li>Monzo, H. 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=A+method+for+generating+high-yield+enriched+neuronal+cultures+from+P19+embryonal+carcinoma+cells.">A method for generating high-yield enriched neuronal cultures from P19 embryonal carcinoma cells.</a> Journal of Neuroscience Methods. 204 (1), 87-103 (2012).</li><li>Popova, D., Karlsson, J., Jacobsson, S. O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+neurons+derived+from+mouse+P19,+rat+PC12+and+human+SH-SY5Y+cells+in+the+assessment+of+chemical-+and+toxin-induced+neurotoxicity.">Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity.</a> BMC Pharmacology and Toxicology. 18 (1), 42 (2017).</li><li>Woodgate, A., MacGibbon, G., Walton, M., Dragunow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+toxicity+of+6-hydroxydopamine+on+PC12+and+P19+cells.">The toxicity of 6-hydroxydopamine on PC12 and P19 cells.</a> Molecular Brain Research. 69 (1), 84-92 (1999).</li><li>Tsukane, M., Yamauchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+/calmodulin-dependent+protein+kinase+II+mediates+apoptosis+of+P19+cells+expressing+human+tau+during+neural+differentiation+with+retinoic+acid+treatment.">Ca2+/calmodulin-dependent protein kinase II mediates apoptosis of P19 cells expressing human tau during neural differentiation with retinoic acid treatment.</a> Journal of Enzyme Inhibition and Medicinal Chemistry. 24 (2), 365-371 (2009).</li><li>Adler, S., Pellizzer, C., Paparella, M., Hartung, T., Bremer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+solvents+on+embryonic+stem+cell+differentiation.">The effects of solvents on embryonic stem cell differentiation.</a> Toxicology in Vitro. 20 (3), 265-271 (2006).</li><li>Jones-Villeneuve, E. M., McBurney, M. W., Rogers, K. A., Kalnins, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+induces+embryonal+carcinoma+cells+to+differentiate+into+neurons+and+glial+cells.">Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells.</a> The Journal of Cell Biology. 94 (2), 253-262 (1982).</li><li>Roy, B., Taneja, R., Chambon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+activation+of+retinoic+acid+(RA)-responsive+genes+and+induction+of+embryonal+carcinoma+cell+differentiation+by+an+RA+receptor+alpha+(RAR+alpha)-,+RAR+beta-,+or+RAR+gamma-selective+ligand+in+combination+with+a+retinoid+X+receptor-specific+ligand.">Synergistic activation of retinoic acid (RA)-responsive genes and induction of embryonal carcinoma cell differentiation by an RA receptor alpha (RAR alpha)-, RAR beta-, or RAR gamma-selective ligand in combination with a retinoid X receptor-specific ligand.</a> Molecular and Cellular Biology. 15 (12), 6481-6487 (1995).</li><li>Hamada-Kanazawa, 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=Sox6+overexpression+causes+cellular+aggregation+and+the+neuronal+differentiation+of+P19+embryonic+carcinoma+cells+in+the+absence+of+retinoic+acid.">Sox6 overexpression causes cellular aggregation and the neuronal differentiation of P19 embryonic carcinoma cells in the absence of retinoic acid.</a> FEBS Letters. 560 (1-3), 192-198 (2004).</li><li>Tangsaengvit, N., Kitphati, W., Tadtong, S., Bunyapraphatsara, N., Nukoolkarn, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+Outgrowth+and+Neuroprotective+Effects+of+Quercetin+from+Caesalpinia+mimosoides+Lamk+on+Cultured+P19-Derived+Neurons.">Neurite Outgrowth and Neuroprotective Effects of Quercetin from Caesalpinia mimosoides Lamk on Cultured P19-Derived Neurons.</a> Evidence-Based Complementary and Alternative. , 838051 (2013).</li><li>Magnuson, D. S., Morassutti, D. J., McBurney, M. W., Marshall, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+derived+from+P19+embryonal+carcinoma+cells+develop+responses+to+excitatory+and+inhibitory+neurotransmitters.">Neurons derived from P19 embryonal carcinoma cells develop responses to excitatory and inhibitory neurotransmitters.</a> Developmental Brain Research. 90 (1-2), 141-150 (1995).</li><li>MacPherson, P., Jones, S., Pawson, P., Marshall, K., McBurney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P19+cells+differentiate+into+glutamatergic+and+glutamate-responsive+neurons+in+vitro.">P19 cells differentiate into glutamatergic and glutamate-responsive neurons in vitro.</a> Neuroscience. 80 (2), 487-499 (1997).</li><li>Hong, 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=Methyltransferase-inhibition+interferes+with+neuronal+differentiation+of+P19+embryonal+carcinoma+cells.">Methyltransferase-inhibition interferes with neuronal differentiation of P19 embryonal carcinoma cells.</a> Biochemical and Biophysical Research Communications. 377 (3), 935-940 (2008).</li><li>Wenzel, 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=Identification+of+a+classic+nuclear+localization+signal+at+the+N+terminus+that+regulates+the+subcellular+localization+of+Rbfox2+isoforms+during+differentiation+of+NMuMG+and+P19+cells.">Identification of a classic nuclear localization signal at the N terminus that regulates the subcellular localization of Rbfox2 isoforms during differentiation of NMuMG and P19 cells.</a> FEBS Letters. 590 (24), 4453-4460 (2016).</li><li>Harada, 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=Overexpression+of+Cathepsin+E+Interferes+with+Neuronal+Differentiation+of+P19+Embryonal+Teratocarcinoma+Cells+by+Degradation+of+N-cadherin.">Overexpression of Cathepsin E Interferes with Neuronal Differentiation of P19 Embryonal Teratocarcinoma Cells by Degradation of N-cadherin.</a> Cellular and Molecular Neurobiology. 37 (3), 437-443 (2017).</li><li>Morassutti, D. J., Staines, W. A., Magnuson, D. S., Marshall, K. C., McBurney, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+embryonal+carcinoma-derived+neurons+survive+and+mature+following+transplantation+into+adult+rat+striatum.">Murine embryonal carcinoma-derived neurons survive and mature following transplantation into adult rat striatum.</a> Neuroscience. 58 (4), 753-763 (1994).</li><li>Magnuson, D. S., Morassutti, D. J., Staines, W. A., McBurney, M. W., Marshall, K. 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Here, we describe a simple protocol for neurogenesis using the P19 cell line. Although many reports have been published in this regard, a detailed methodology for neurogenesis induction using P19 cell line remains unclear. Moreover, we utilized a simple high glucose (4,500 mg/L) DMEM medium with 10% FBS for the entire experiment. This allowed us to perform the neurogenic experiment in a user-friendly manner and expand the usage of this method for the future.
The most critical points within thi...

During embryonal development, a single cell layer is transformed into three separate germ layers1,2,3. To increase the research possibilities of phenomena occurring in vivo, generation of three-dimensional aggregates (embryonic bodies) have been developed as a convenient model. Cellular aggregates formed in this way can be exposed to various conditions causing cell differentiation, which reflect development of the embryo4,5. The P19 murine embryonic carcinoma cell line (P19 cell line) is commonly used as a cellular model for neurogenesis studies in vitro6,7,8. The P19 cell line exhibits typical pluripotent stem cell features and can differentiate into neurons in the presence of retinoic acid (RA) during cell aggregation followed by neurite outgrowth under adherent conditions. Moreover, the undifferentiated P19 cell line is also capable of forming muscle- and cardiomyocyte-like cells under the influence of dimethyl sulfoxide (DMSO)9,10,11,12.
Many methods13,14,15,16 have been reported for neuronal differentiation, but the methodology is sometimes complicated and not easy to grasp by only reading the descriptions. For example, protocols sometimes require a combination of Dulbecco&#39;s Modified Eagle Medium (DMEM) medium supplemented with a mixture of calf serum (CS) and fetal bovine serum (FBS)13. Moreover, media used for neuronal development are often composed of Neurobasal and B27 supplements13,14,15,16. As such, existing methods contain complexity in their preparation and our goal here is to simplify the protocols. In this study, we demonstrated that DMEM with FBS can be utilized for maintaining the P19 cell line (DMEM + 10% FBS) as well as for neuronal development (DMEM + 5% FBS + RA). This simplified method for neurogenesis using the P19 cell line allows us to study the molecular mechanism of how neurons are developed. Moreover, research on neurodegenerative diseases such as Alzheimer&#39;s disease is also conducted using P19 cell line17,18, and we believe that the method developed in this study will play a part in elucidating the molecular mechanisms in neurodevelopmental abnormalities and neurodegenerative diseases.

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		<tr height="21">
			<td height="21" style="height:21px;width:347px;">6x DNA Loading Dye</td>
			<td style="width:103px;">EURx</td>
			<td style="width:155px;">E0260-01</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Agarose</td>
			<td style="width:103px;">Sigma- Aldrich</td>
			<td style="width:155px;">A9539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">cDNA synthesis kit</td>
			<td style="width:103px;">EURx</td>
			<td style="width:155px;">E0801-02</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:347px;">DAPI (4&#8242;,6-Diamidine-2&#8242;-phenylindole dihydrochloride)</td>
			<td style="width:103px;">Sigma- Aldrich</td>
			<td style="width:155px;">10236276001</td>
			<td style="width:215px;">Working concentration: 1 &#956;g/mL</td>
		</tr>
		<tr height="138">
			<td height="138" style="height:138px;width:347px;">DMEM high glucose (4.5 g/L) with L-glutamine</td>
			<td style="width:103px;">Lonza</td>
			<td style="width:155px;">BE12-604Q</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Ethanol 99.8%</td>
			<td style="width:103px;">Chempur</td>
			<td style="width:155px;">CHEM*613964202</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:103px;">EURx</td>
			<td style="width:155px;">E5050-03</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">MAP2 antibody</td>
			<td style="width:103px;">Thermo Fisher Scientific</td>
			<td style="width:155px;">PA517646</td>
			<td style="width:215px;">Dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">PCR reaction kit</td>
			<td style="width:103px;">EURx</td>
			<td style="width:155px;">E0411-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Penicillin/Streptomycin 10K/10K</td>
			<td style="width:103px;">Lonza</td>
			<td style="width:155px;">DE17-602E</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:347px;">Phosphate Buffered Saline (PBS), 1x concentrated without Ca2+, Mg2+</td>
			<td style="width:103px;">Lonza</td>
			<td style="width:155px;">BE17- 517Q</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Retinoic acid</td>
			<td style="width:103px;">Sigma- Aldrich</td>
			<td style="width:155px;">R2625-50MG</td>
			<td style="width:215px;">&#160;dissolved in 99.8% ethanol; store in -20 &#176;C up to 6 months</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:347px;">Secondary Antibody (Alexa Fluor 488)</td>
			<td style="width:103px;">Thermo Fisher Scientific</td>
			<td style="width:155px;">A11034</td>
			<td style="width:215px;">Dilution 1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Skim milk</td>
			<td style="width:103px;">Sigma- Aldrich</td>
			<td style="width:155px;">1153630500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">TBE Buffer</td>
			<td style="width:103px;">Thermo Fisher Scientific</td>
			<td style="width:155px;">B52</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Triton-X 100</td>
			<td style="width:103px;">Sigma- Aldrich</td>
			<td style="width:155px;">T8787-100ML</td>
			<td></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:347px;">Trypsin 0.25% - EDTA in HBSS, without&#160; Ca2+, Mg2+,with Phenol Red</td>
			<td style="width:103px;">biosera</td>
			<td style="width:155px;">LM-T1720/500</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:347px;">Cell Culture Plastics</td>
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			<td height="21" style="height:21px;width:347px;">1 mL Serological Pipettes</td>
			<td style="width:103px;">Profilab</td>
			<td style="width:155px;">515.01</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">10 mL Serological Pipettes</td>
			<td style="width:103px;">Profilab</td>
			<td style="width:155px;">515.10</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">100 mm dish dedicated for suspension culture</td>
			<td style="width:103px;">Corning</td>
			<td style="width:155px;">C351029</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">15 mL centrifuge tubes</td>
			<td style="width:103px;">Sigma- Aldrich</td>
			<td style="width:155px;">CLS430791-500EA</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">5 mL Serological Pipettes</td>
			<td style="width:103px;">Profilab</td>
			<td style="width:155px;">515.05</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:347px;">6-well plate</td>
			<td style="width:103px;">Corning</td>
			<td style="width:155px;">CLS3516</td>
			<td style="width:215px;"></td>
		</tr>
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			<td height="24" style="height:24px;width:347px;">Cell culture flasks, surface area 25 cm2</td>
			<td style="width:103px;">Sigma- Aldrich</td>
			<td style="width:155px;">CLS430639-200EA</td>
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neurogenesis using p19 embryonal carcinoma cells

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of retinoic acid (RA), the suspension cultured P19 cell line is induced to differentiate into neurons. This phenomenon is extensively investigated as a neurogenesis model in vitro. Therefore, the P19 cell line is very useful for molecular and cellular studies associated with neurogenesis. However, protocols for neuronal differentiation of P19 cell line described in the literature are very complex. The method developed in this study are simple and will play a part in elucidating the molecular mechanisms in neurodevelopmental abnormalities and neurodegenerative diseases.

The simplified scheme of protocol for neurogenesis induction in P19 cell line is presented in Figure 1. In order to define the character of the P19 cell line in an undifferentiated state and during neurogenesis, the RT-PCR (reverse transcription-polymerase chain reaction) method was used. The undifferentiated P19 cell line expressed the pluripotency genes such as organic cation/carnitine transporter4 (Oct4) and Nanog homeobox (Nanog). Neurog...

Watch this Scientific Journal Video about Neurogenesis Using P19 Embryonal Carcinoma Cells at JoVE.com

Neurogenesis Using P19 Embryonal Carcinoma Cells

The P19 mouse embryonic carcinoma cell line (P19 cell line) is widely used for studying the molecular mechanism of neurogenesis with great simplification compared to in vivo analysis. Here, we present a protocol for retinoic acid-induced neurogenesis in the P19 cell line.

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of ...

P19 cell line is known to differentiate into neurons. However, protocol for neuron differentiation of P19 cell line are sometimes complicated. This method allow us to perform the neurogenic experiment in a user-friendly manner and expand the possibility of the usage of P19 cell line for the future.To begin, maintain P19 cells in maintenance medium, comprised of DMEM, with 4.5 grams per liter of glucose, supplemented with 10%fetal bovine serum, 100 units per milliliter penicillin, and 100 units per milliliter streptomycin. Incubate the cell culture in a 37 degree Celsius and 5%carbon dioxide environment until the cells are approximately 80%confluent. Remove the spent medium from the cell culture flask and wash the cells with two milliliters of calcium and magnesium-free PBS.Add one milliliter of 0.25%trypsin-EDTA to completely cover the monolayer of the cells and incubate at 37 degrees Celsius and 5%carbon dioxide for two to three minutes. Under a microscope, check to see if all the cells are detached. Resuspend the cells in nine milliliters of maintenance medium in order to neutralize the trypsin.Transfer the cells into a 15-milliliter tube and centrifuge at 200 times G and room temperature for five minutes to pellet the cells. Then, aspirate the supernatant without disturbing the pellet. Resuspend the pellet in 10 milliliters of fresh maintenance medium and use a cell counter to determine the cell number according to the manufacturer's instructions.Seed 20, 000 cells per square centimeter in a new T-25 flask and add up to 10 milliliters of maintenance medium. Incubate at 37 degrees Celsius and 5%carbon dioxide for two to three days. To begin trypsin digestion, first slowly aspirate the supernatant and wash the cells with two milliliters of calcium and magnesium-free PBS.Then, add one milliliter of 0.25%trypsin EDTA to the cells and incubate them at 37 degrees Celsius and 5%carbon dioxide for two to three minutes. After the incubation, to neutralize the trypsin, resuspend the cells in nine milliliters of differentiation medium, comprised of DMEM with high glucose level and supplemented with 5%fetal bovine serum, 100 units per milliliter penicillin, and 100 units per milliliter streptomycin. Transfer the cells into a 15-milliliter tube and centrifuge at 200 times G and room temperature for five minutes to pellet the cells.Then, slowly aspirate the supernatant and resuspend the pellet in one milliliter of fresh differentiation medium without RA.Use a cell counter to determine the cell number according to the manufacturer's instructions. For aggregate generation, start by adding 10 milliliters of differentiation medium, supplemented with five microliters of RA to a 100-milliliter, non-treated culture dish. Seed one million cells in the culture dish and incubate at 37 degrees Celsius and 5%carbon dioxide for two days in order to promote aggregate formation.After the incubation, with a 10-milliliter pipette, aspirate the medium containing the aggregates and transfer it to a 15-milliliter tube at room temperature. Wait for 1.5 minutes for the aggregates to settle at the bottom and then discard the supernatant. Add 10 milliliters of fresh differentiation medium supplemented with 0.5 micromolar RA.Gently seed the aggregates in a new 100-millimeter, non-treated culture dish and incubate at 37 degrees Celsius and 5%carbon dioxide for two days.To begin, use a 10-milliliter pipette to transfer the cell aggregates to a 15-milliliter tube. Wait for 1.5 minutes at room temperature for the aggregates to settle at the bottom and then discard the supernatant. Wash the aggregates with serum and antibiotic-free DMEM.Wait for cell aggregates to settle at the bottom, discard the supernatant, and add two milliliters of 0.25%trypsin EDTA. Incubate the tube in a water bath at 37 degrees Celsius for 10 minutes, tapping every two minutes to keep the cells in suspension. Then, add four milliliters of maintenance medium in order to stop the trypsin activity and pipette up and down 20 times using a one-milliliter pipette.Finally, centrifuge the cells at 200 times G and room temperature for five minutes. Remove the supernatant and resuspend the cell pellet in five milliliters of maintenance medium. Use a cell counter to determine the cell number and proceed with plating the cells.To plate cells, first add three milliliters per well of maintenance medium to a six-well culture plate. Then, seed the cells at a density of 500, 000 per well. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide.Seed the cells on cover glasses in a six-well culture plate. When they reach 20%confluence, proceed to immunostaining with Anti-MAP2 antibody. Then, isolate RNA, and perform reverse transcription PCR for MAP2, NueN, OCT4, NANOG, and a GAPDH.In this study, a simplified method for neuronal differentiation is introduced. The P19 cell line is cultured in a non-treated culture dish with 5%FBS and 0.5 micromolar RA.After four days, the cell aggregates are dissociated with trypsin and seeded on an adherent cell culture plate for the next four days. The efficiency of this method is evaluated by the comparison between RTPCR analysis of the P19 cell line in an undifferentiated state and during neurogenesis.Results show a rapid decrease of expression of pluripotency genes, such as Oct4 and NANOG, and upregulation of neurnomal marker genes, such as MAP2 and NeuN. We believe that the method developed in this study is simple and will play a part in elucidating the molecular mechanisms of neurogenesis, as well as neurodegenerative disease.

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