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>
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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...

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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.

neurogenesis-using-p19-embryonal-carcinoma-cells

Research

JoVE Journal

Developmental Biology

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological problems after stroke and is shown to impair neurogenic processes. Interestingly, the adult zebrafish has recently emerged as a relevant and useful model to mimic hyperglycemia/diabetes and to investigate constitutive and regenerative neurogenesis. This work provides methods to develop zebrafish models of hyperglycemia to explore the impact of hyperglycemia on brain cell proliferation under homeostatic and brain repair conditions. Acute hyperglycemia is established using the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) into adult zebrafish. Chronic hyperglycemia is induced by immersing adult zebrafish in D-glucose (111 mM) containing water for 14 days. Blood-glucose-level measurements are described for these different approaches. Methods to investigate the impact of hyperglycemia on constitutive and regenerative neurogenesis, by describing the mechanical injury of the telencephalon, dissecting the brain, paraffin embedding and sectioning with a microtome, and performing immunohistochemistry procedures, are demonstrated. Finally, the method of using zebrafish as a relevant model for studying the biodistribution of radiolabeled molecules (here,[18F]-FDG) using PET/CT is also described.

This work describes methods to establish acute and chronic hyperglycemia models in zebrafish. The aim is to investigate the impact of hyperglycemia on physiological processes, such as constitutive and injury-induced neurogenesis. The work also highlights the use of zebrafish to follow radiolabeled molecules (here, [18F]-FDG) using PET/CT.

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...