Name: an enzyme- and serum-free neural stem cell culture model for emt investigation suited for drug discovery
Uploaded: 2016-08-23T13:00:00
Description: Watch this Scientific Journal Video about An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery at JoVE.com

The study was supported by the University of Basel Science Foundation and the Swiss National Science Foundation by a grant to MHS and AG (SNF IZLIZ3_157230). We thank: Dr. Tania Rinaldi Burkat for generously providing infrastructure; all members of the Bettler group for discussions and comments. We thank Gerhard Dorne (Leica Microsystems, Switzerland) for professional and competent installation of the Full HD MC170 video camera (Leica Microsystems, Switzerland).

All animal procedures followed the &#39;Guide for the Care and Use of Laboratory Animals&#39; (NIH publication, 8th edition, 2011) and were approved by the Animal Welfare Committee of Basel (Swiss Guidelines for the Care and Use of Animals). By these guidelines the animal protocol is considered of &#34;lowest animal severity grade&#34;.
1. Preparation of Expansion Medium
Note: Work in aseptic conditions as standard for tissue culture.
<ol>
	<li>Take two 15 ml tubes, and add 5 ml of L-glutamine-...

<ol>
	<li>Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-Mesenchymal+Transitions+in+development+and+disease:+old+views+and+new+perspectives.">Epithelial-Mesenchymal Transitions in development and disease: old views and new perspectives.</a> Int J Dev Biol. 53, 1541-1547 (2009).</li><li>Sauka-Spengler, T., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+regulatory+network+orchestrates+neural+crest+formation.">A gene regulatory network orchestrates neural crest formation.</a> Nat Rev Mol Cell Biol. 9, 557-568 (2008).</li><li>Barriere, G., Fici, P., Gallerani, G., Rigaud, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Transition:+a+double-edged+sword.">Mesenchymal Transition: a double-edged sword.</a> Clin Transl Med. 4, 14 (2015).</li><li>Zawadzka, 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=CNS-resident+glial+progenitor/stem+cells+produce+Schwann+cells+as+well+as+oligodendrocytes+during+repair+of+CNS+demyelination.">CNS-resident glial progenitor/stem cells produce Schwann cells as well as oligodendrocytes during repair of CNS demyelination.</a> Cell Stem Cell. 6, 578-590 (2010).</li><li>Paxinos, G., Ashwell, K. W. S., To&#776;rk, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atlas of the developing rat nervous system. , (1994).</li><li>O'Leary, D. D., Chou, S. J., Sahara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Area+patterning+of+the+mammalian+cortex.">Area patterning of the mammalian cortex.</a> Neuron. 56, 252-269 (2007).</li><li>O'Leary, D. D., Sahara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+arealization+of+the+neocortex.">Genetic regulation of arealization of the neocortex.</a> Curr Opin Neurobiol. 18, 90-100 (2008).</li><li>Sailer, M. 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=Non-invasive+neural+stem+cells+become+invasive+in+vitro+by+combined+FGF2+and+BMP4+signaling.">Non-invasive neural stem cells become invasive in vitro by combined FGF2 and BMP4 signaling.</a> J Cell Sci. 126, 3533-3540 (2013).</li><li>Sailer, M. 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=BMP2+and+FGF2+cooperate+to+induce+neural-crest-like+fates+from+fetal+and+adult+CNS+stem+cells.">BMP2 and FGF2 cooperate to induce neural-crest-like fates from fetal and adult CNS stem cells.</a> J Cell Sci. 118, 5849-5860 (2005).</li><li>Sailer, M., Oppitz, M., Drews, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cellular+contractions+in+the+human+melanoma+cell+line+SK-mel+28+after+muscarinic+cholinergic+stimulation.">Induction of cellular contractions in the human melanoma cell line SK-mel 28 after muscarinic cholinergic stimulation.</a> Anat Embryol (Berl). 201, 27-37 (2000).</li><li>Wicki, A., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+role+of+podoplanin+in+tumour+invasion.">The potential role of podoplanin in tumour invasion.</a> Br J Cancer. 96, 1-5 (2007).</li><li>Wicki, 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=Tumor+invasion+in+the+absence+of+epithelial-mesenchymal+transition:+podoplanin-mediated+remodeling+of+the+actin+cytoskeleton.">Tumor invasion in the absence of epithelial-mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton.</a> Cancer Cell. 9, 261-272 (2006).</li><li>Mishima, 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=Increased+expression+of+podoplanin+in+malignant+astrocytic+tumors+as+a+novel+molecular+marker+of+malignant+progression.">Increased expression of podoplanin in malignant astrocytic tumors as a novel molecular marker of malignant progression.</a> Acta Neuropathol. 111, 483-488 (2006).</li><li>Motomura, 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=Immunohistochemical+analysis-based+proteomic+subclassification+of+newly+diagnosed+glioblastomas.">Immunohistochemical analysis-based proteomic subclassification of newly diagnosed glioblastomas.</a> Cancer Science. 103, 1871-1879 (2012).</li><li>Kono, T., 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=Immunohistochemical+detection+of+the+lymphatic+marker+podoplanin+in+diverse+types+of+human+cancer+cells+using+a+novel+antibody.">Immunohistochemical detection of the lymphatic marker podoplanin in diverse types of human cancer cells using a novel antibody.</a> Int J Oncol. 31, 501-508 (2007).</li><li>Raica, M., Cimpean, A. M., Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+podoplanin+in+tumor+progression+and+metastasis.">The role of podoplanin in tumor progression and metastasis.</a> Anticancer Res. 28, 2997-3006 (2008).</li><li>Panchision, D. M., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+neural+stem+cells+by+morphogenic+signals.">The control of neural stem cells by morphogenic signals.</a> Curr Opin Genet Dev. 12, 478-487 (2002).</li><li>Lamouille, S., Xu, J., Derynck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+epithelial-mesenchymal+transition.">Molecular mechanisms of epithelial-mesenchymal transition.</a> Nat Rev Mol Cell Biol. 15, 178-196 (2014).</li><li>Gonzalez-Perez, O., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrogenesis+in+the+subventricular+zone+and+the+role+of+epidermal+growth+factor.">Oligodendrogenesis in the subventricular zone and the role of epidermal growth factor.</a> Brain Res Rev. 67, 147-156 (2011).</li><li>Hu, J. 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=PDGF-AA+mediates+B104CM-induced+oligodendrocyte+precursor+cell+differentiation+of+embryonic+neural+stem+cells+through+Erk,+PI3K,+and+p38+signaling.">PDGF-AA mediates B104CM-induced oligodendrocyte precursor cell differentiation of embryonic neural stem cells through Erk, PI3K, and p38 signaling.</a> J Mol Neurosci. 46, 644-653 (2012).</li><li>Galvao, R. P., 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=Transformation+of+quiescent+adult+oligodendrocyte+precursor+cells+into+malignant+glioma+through+a+multistep+reactivation+process.">Transformation of quiescent adult oligodendrocyte precursor cells into malignant glioma through a multistep reactivation process.</a> Proc Natl Acad Sci U S A. 111, E4214-E4223 (2014).</li><li>Liu, C., 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=Mosaic+analysis+with+double+markers+reveals+tumor+cell+of+origin+in+glioma.">Mosaic analysis with double markers reveals tumor cell of origin in glioma.</a> Cell. 146, 209-221 (2011).</li><li>Das, S., Srikanth, M., Kessler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+and+glioma.">Cancer stem cells and glioma.</a> Nat Clin Pract Neurol. 4, 427-435 (2008).</li><li>Modrek, A. S., Bayin, N. S., Placantonakis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+stem+cells+as+the+cell+of+origin+in+glioma.">Brain stem cells as the cell of origin in glioma.</a> World J Stem Cells. 6, 43-52 (2014).</li><li>Sampetrean, O., 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=Invasion+precedes+tumor+mass+formation+in+a+malignant+brain+tumor+model+of+genetically+modified+neural+stem+cells.">Invasion precedes tumor mass formation in a malignant brain tumor model of genetically modified neural stem cells.</a> Neoplasia. 13, 784-791 (2011).</li><li>McNeill, R. 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=Modeling+astrocytoma+pathogenesis+in+vitro+and+in+vivo+using+cortical+astrocytes+or+neural+stem+cells+from+conditional,+genetically+engineered+mice.">Modeling astrocytoma pathogenesis in vitro and in vivo using cortical astrocytes or neural stem cells from conditional, genetically engineered mice.</a> JoVE. , e51763 (2014).</li><li>Lara-Padilla, E., Caceres-Cortes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+nature+of+the+tumor-initiating+cell.">On the nature of the tumor-initiating cell.</a> Curr Stem Cell Res Ther. 7, 26-35 (2012).</li><li>Busch, C., 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=BMP-2-dependent+integration+of+adult+mouse+subventricular+stem+cells+into+the+neural+crest+of+chick+and+quail+embryos.">BMP-2-dependent integration of adult mouse subventricular stem cells into the neural crest of chick and quail embryos.</a> J Cell Sci. 119, 4467-4474 (2006).</li><li>Thiery, J. P., Acloque, H., Huang, R. Y., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transitions+in+development+and+disease.">Epithelial-mesenchymal transitions in development and disease.</a> Cell. 139, 871-890 (2009).</li><li>McDonald, O. G., Wu, H., Timp, W., Doi, A., Feinberg, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+epigenetic+reprogramming+during+epithelial-to-mesenchymal+transition.">Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition.</a> Nat Struct Mol Biol. 18, 867-874 (2011).</li><li>Rahimi, R. A., Leof, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+signaling:+a+tale+of+two+responses.">TGF-beta signaling: a tale of two responses.</a> J Cell Biochem. 102, 593-608 (2007).</li><li>Xu, J., Lamouille, S., Derynck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta-induced+epithelial+to+mesenchymal+transition.">TGF-beta-induced epithelial to mesenchymal transition.</a> Cell Res. 19, 156-172 (2009).</li><li>Suva, M. L., Riggi, N., Bernstein, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+reprogramming+in+cancer.">Epigenetic reprogramming in cancer.</a> Science. 339, 1567-1570 (2013).</li><li>Sullivan, R., 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+possible+new+focus+for+stroke+treatment+-+migrating+stem+cells.">A possible new focus for stroke treatment - migrating stem cells.</a> Expert Opin Biol Ther. , 1-10 (2015).</li></ol>

In this study a standardized system for EMT analysis utilizing NSCs is described (summarized in Supplementary Figure 3). The standardization ensures reproducibility (Table 1 and 2). The NSCs are derived from the developing cortex, a tissue that normally does not undergo EMT. This is of advantage for the analysis of early steps in EMT. Initial steps in EMT cannot be adequately studied in tumor cells that have accumulated genetic changes and may have already adopted EMT features. Moreover,...

During several stages of embryonic development, epithelial cells lose their strong adherence to each other (e.g., tight junctions) and acquire a migratory phenotype in a process called epithelial to mesenchymal transition (EMT)1. EMT is required for the formation of additional cell types, such as the mesenchymal neural crest cells, a population that segregates from the neuroepithelium2. EMT is not only essential during embryonic stages but also required at later stages of adult life to maintain physiological processes in the adult organism, such as wound healing3and central nervous system (CNS) regeneration in demyelinating lesions4.
Epithelial tumors are known to reactivate EMT as an initiation step for migration, invasion and metastasis, ultimately leading to cancer progression1,3. EMT is indeed centrally linked to strong migration1,3. The cellular steps of conditioning, initiating, undergoing and maintaining EMT are not fully understood and need further investigation.
Here, a standardized in vitro EMT model system based on NSCs, with defined growth factors and media (no serum and no enzyme usage) is presented. This model system is of relevance for scientists working on EMT. The Snail, Zeb and Twist protein families have been shown to be critical for EMT both in development and disease1. The Snail, Zeb and Twist families are also involved in the presented system. The system is based on a specific region of the forebrain that normally does not undergo EMT providing a particular advantage for the study of initial events during EMT induction.
The model system could potentially be applied to study EMT in epithelia outside the CNS, since key EMT inducers, such as the Snail, Zeb and Twist proteins, are also found during EMT in tissue systems outside the CNS. This model system allows the standardized isolation of NSCs from the developing cortex to study stem cell features in general and EMT in particular. Using this system, we isolated NSCs, induced EMT and studied the subsequent migration under the effect of FGF2 and BMP4. We observed that FGF- and BMP-signaling interacts with TGF&#946;-signaling to promote cell migration, thus validating the model system.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BMP4, rhBMP4</td>
			<td>RnD Systems&#160;</td>
			<td>314-BP-01M</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine pancreas insulin</td>
			<td>Sigma&#160;</td>
			<td>I1882</td>
			<td></td>
		</tr>
		<tr>
			<td>Boyden chamber, CytoSelect cell invasion assay</td>
			<td>Cell Biolabs</td>
			<td>CBA-110</td>
			<td>24 well plate system</td>
		</tr>
		<tr>
			<td>Cell culture dish with grid</td>
			<td>Ibidi 500 mm dish, 35 mm</td>
			<td>80156</td>
			<td></td>
		</tr>
		<tr>
			<td>CellMask Orange</td>
			<td>Life Technologies</td>
			<td>C10045</td>
			<td>Plasma membrane dye, use at 1:1000 .</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>LifeTechnologies</td>
			<td>D1306</td>
			<td>Stock at 5mg/ml. Use at 1:10000. Cancerogenic. Appropriate protection (gloves, coat, goggles) required.</td>
		</tr>
		<tr>
			<td>DMEM/F12 1:1 medium bottle</td>
			<td>Gibco Invitrogen</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2, rhFGF2</td>
			<td>RnD Systems</td>
			<td>233-FB-01M</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectine, bovine</td>
			<td>Sigma&#160;</td>
			<td>F4759</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax supplement&#160;</td>
			<td>Gibco Invitrogen&#160;</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphics software with pixel measurement feature</td>
			<td>Fiji</td>
			<td>fiji.sc/Fiji</td>
			<td>version 2.0.0-rc-30/1.49s</td>
		</tr>
		<tr>
			<td>HBSS media</td>
			<td>Sigma&#160;</td>
			<td>H9394</td>
			<td></td>
		</tr>
		<tr>
			<td>Human apo-Transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td>Possible lung irritant. Avoid inhalation. Use appropriate protection.</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco Invitrogen&#160;</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin, Mouse anti Nestin antibody</td>
			<td>Genetex</td>
			<td>GTX26142</td>
			<td>Use at 1:100, 4% PFA fixation, Triton X100 at 0.1%</td>
		</tr>
		<tr>
			<td>Olig2, Rabbit anti Olig2 antibody</td>
			<td>Provided by Hirohide Takebayash</td>
			<td>Personal stock</td>
			<td>Use at 1:2000, 4% PFA fixation, Triton X100 at 0.1%</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Fungizone</td>
			<td>Gibco Invitrogen&#160;</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Podoplanin, Mouse anti Podoplanin antibody</td>
			<td>Acris</td>
			<td>DM3614P</td>
			<td>Use at 1:250, 4% PFA fixation, avoid Triton X100</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>Sigma&#160;</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma&#160;</td>
			<td>P5780</td>
			<td>Skin and eye irritant. Appropriate protection required.</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma&#160;</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Sox10, Rabbit anti Sox10 antibody</td>
			<td>Millipore Chemicon</td>
			<td>AB5774</td>
			<td>Use at 1:200, 4% PFA fixation, Triton X100 at 0.1%</td>
		</tr>
		<tr>
			<td>TGFb1, rhTGFb1</td>
			<td>RnD Systems</td>
			<td>240-B-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Uncoated Petri dishes</td>
			<td>Falcon Corning</td>
			<td>351029</td>
			<td></td>
		</tr>
	</tbody>
</table>

an enzyme- and serum-free neural stem cell culture model for emt investigation suited for drug discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

This EMT model system is based on the standardized isolation of NSCs both as single cells or as explants from a specific region of the developing neural tube, the central cortex (Figures 1 and 2). For quantitative assessment, explants were seeded right at the center of a 500 &#181;m grid culture dish (Figure 3). Explants from the central cortex were first exposed to FGF2 for two days, followed by additional two days in different combinati...

Watch this Scientific Journal Video about An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery at JoVE.com

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

The overall goal of this procedure is to induce migration in typically non-migratory cells, to identify specific compounds that activate or block migration. This method can help to answer key questions in the field of cell migration and invasion, and may be of particular interest to scientists working with neural stem cells. The main advantage of this technique is that non-migratory and non-invasive cells can be analyzed during the process of becoming migratory and invasive.The implications of this technique extend toward the identification of novel compounds that block epithelial to mesenchymal transition, or EMT, which occurs in human high-grade gliomas believed to derive from neural stem cells. Though this method can provide insight into EMT and neural stem cells, it can also be used to study other epithelial cancers, such as breast, lung, or colon cancer. On embryonic day 14.5, confirm the correct age of the rat embryos by the presence of beginning digit formation in a rat forelimb.Next, transfer the embryos into individual, uncoated petri dishes filled with ice-cold PBS, which are placed under a stereo microscope. Use a pair of fine-tipped forceps to remove the embryonic hulls. Keep embryos in expansion medium on ice.Using two autoclave-sterilized fine forceps, and starting at the intersection of the developing telencephalon and diencephalon, pull simultaneously anteriorly and posteriorly, to remove the head skin and skull. Then, identify the midbrain-hindbrain boundary separating the mesencephalon from the rhombencephalon, and cut the rhombencephalon just at or below the midbrain-hindbrain boundary. Holding the block of the mesencephalon with a pair of forceps, sever the connection between the telencephalon and diencephalon at the skull base with the facial skeleton, and immerse the telencephalon, diencephalon, mesencephalon block in an uncoated petri dish of expansion medium on ice.When all of the neural blocks have been harvested in the same manner, transfer one telencephalon, diencephalon, mesencephalon block into fresh, ice-cold PBS under the microscope. To separate the mesencephalon from the diencephalon, first sever the tissue between the diencephalon and the telencephalon. Next, split the two telencephalic hemispheres, and identify the medial and lateral ganglionic eminences, which are visible through the future foramen interventriculare.Dissect along a straight line at the intersection between the medial and lateral ganglionic eminences and the anterior cortex, followed by a straight line through the cortex, hippocampus, and choroid plexus. Cut a second straight line through the cortex, hippocampus, and choroid plexus at the intersection of the caudal ganglionic eminence and the posterior cortex. Then, flatten out the telencephalon, identify the cortical hem containing the hippocampus and the choroid plexus.Separate the cortical hem from the cortex, leaving the size of the cortex identical to the size of the ganglionic eminences. Flip the cortex-ganglionic eminence block so that the ventricular side is touching the dish surface, and the meninges are facing up. Pinning the ganglionic eminences to the dish surface with one hand, use forceps in the other hand to peel off the meninges.Then, cut each cortex along the lateral ganglionic eminence, about half of the distance of the diameter of the lateral ganglionic eminence. To prepare the explant cultures, cut the cortices into explants of less than 400 microns in diameter, using a 400 micron grid below the dissection dish as a reference if necessary. Then, transfer all of the explants into a fresh petri dish with ice cold expansion medium.Next, remove the excess fibronectin from a previously-coated 35 millimeter tissue culture dish and allow the dish to dry. Then, place the fibronectin-coated dish over a 10 by 10 millimeter grid and add one milliliter of cold expansion medium to the center of the dish, allowing the medium to form a spherical drop. Place up to eight explants into the center of the drop with at least three millimeters of space between each piece of tissue.Then, carefully place the dish in a cell culture incubator without shaking to allow the explants to attach to the fibronectin-coated surface. After one hour, fill the dish to a final volume of two milliliters of expansion medium plus the growth factors or test substances of interest, and return the plate to the incubator. In this experiment, explants were seeded at the center of 500 micron-grid culture dishes and cultured in different growth factors.In the control and TGF Beta 1 alone treated explants, a strong proliferations, coupled with almost no migration was observed. BMP4 however, induced an intermediate cell migration and proliferation response. With the combination of BMP4 and TGF Beta 1 inducing the strongest migration and proliferation of all the groups.Once mastered, the neural stem cell can be isolated from 10 to 15 embryos in approximately two hours. While attempting this procedure, it is important to maintain all of the embryos and cell explants in ice-cold medium. This cost-efficient and highly-reproducible system provides the flexibility of using either single cells or explant cultures to study cell migration and invasion in vitro.Culturing the cells in large multi-well plates allows analysis of the effects of a variety of compounds on migration and invasion in parallel. Further, the system can be easily adapted for high throughput screening of novel compounds. After watching this video, you should have a good understanding of how to isolate neural stem cells with the potential to undergo EMT from the appropriate region of the developing cortex.

Research

JoVE Journal

Developmental Biology

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

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

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

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