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>
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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 ...

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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

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

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...