<p class="jove_content">We thank Evan Dysart and Marc L&eacute;onard for expert technical assistance in video production and editing, Matt Cooke for assistance in data collection, and Sarah Gelbard for expert editorial assistance.  The work was funded by operating grants from the Canadian Institute of Health Research (CIHR, MOP 62626) to SALB, a Strategic Training Initiative in Health Research program grant to SALB and SF (CIHR Training Program in Neurodegenerative Lipidomics, TGF 9121), and infrastructure support from the Canadian Foundation Innovation and Ontario Innovation Trust to SF.  SI received an Ontario Graduate Scholarship in Science and Technology with contributions from the Parkinson Research Consortium.  NV receives an Institute of Aging and CIHR Training Program in Neurodegenerative Lipidomics post-professional fellowship.  We gratefully acknowledge the educational software and technical support provided by AutoDesk Research.</p>

<ol>
	<li>Kempermann, G., Jessberger, S., Steiner, B., Kronenberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milestones+of+neuronal+development+in+the+adult+hippocampus.">Milestones of neuronal development in the adult hippocampus.</a> <em style='font-style: italic'>Trends Neurosci</em>. <b>27</b>, 447-452 (2004).</li><li>Campos, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurospheres:+insights+into+neural+stem+cell+biology.">Neurospheres: insights into neural stem cell biology.</a> <em style='font-style: italic'>J Neurosci Res</em>. <b>78</b>, 761-769 (2004).</li><li>Imbeault, 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+extracellular+matrix+controls+gap+junction+protein+expression+and+function+in+postnatal+hippocampal+neural+progenitor+cells.">The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells.</a> <em style='font-style: italic'>BMC Neurosci</em>. <b>10</b>, 13-13 (2009).</li></ol>

<p class="jove_content">All experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Ottawa Animal Care Committee and the Canadian Council on Animal Care.</p>

<p class="jove_content">This protocol describes a procedure to culture and serially cryosection post-natal hippocampal NPCs, localize protein expression by immunodetection, and finally reconstruct and analyze the topographical position of immunopositive cells within the entire 3D neurosphere.  By combining cell biology culture and processing techniques, microscopy imaging (OpenLab, Improvision and other image analysis softwares), graphic editing software capacities (Adobe Photoshop), and 3D animation and compositing software capacities (Autodesk Maya), we present a methodology to reconstruct the entire cellular composition of 3D cultures from serial 2D images allowing for the faithful reconstruction of cellular position and protein expression throughout a neurosphere culture.  This protocol can be combined with equal effectiveness to the registration and reconstruction of epifluorescent thin serial sections or confocal Z-stacks through thicker serial sections.  Because clonal expansion of single NPCs in neurosphere culture recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain, the impact of its 3D structure represents an important NPC fate determinant in progeny exposed to the same experimental milieu<sup>1</sup>. Divergence in lineage and protein expression at the core and periphery of serial neurosphere sections suggests that multiple physical factors including cell position, mechanical stress, and shear force play important roles in regulating NPC biology<sup>2</sup>. Moreover, connexin protein expression, analyzed here, has been shown to change depending upon when NPCs are cultured as 3D neurospheres in suspension or plated on a laminin substrate with functional connexin-mediated communication altered whether cells are cultured on plastic and substrate or in 3D free-floating cultures<sup>3</sup>. The impact of cellular position on NPC fate remains unclear.  It is technically challenging to analyze impact of spatial location within 3D culture on NPC biology given that antigenic assessment of lineage and signalling proteins requires disruption of the 3D architecture to enable cell permeabilization and antibody access to all cells.  Here, we show that a hybrid visualization methodology provides a means of determining whether certain proteins exhibit unique positional localizations in 3D culture, can be used to infer and test protein function and regulation with respect to regional localization within the neurosphere, and, perhaps most importantly, provides the positional data required to study the impact of 3D topography and cellular positioning on NPC fate in 3D culture.</p>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Euthansol</td>
<td>Reagent</td>
<td>Schering-Plough Canada Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Krazy Glue</td>
<td>Reagent</td>
<td>Home Hardware</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>VT1000S vibratome</td>
<td>Equipment</td>
<td>Leica Microsystems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Neural protease</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6141</td>
<td>Stock solutions stored at -20&deg;C</td>
</tr>
<tr>
<td>Papain</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P4762</td>
<td>Stock solutions stored at -20&deg;C</td>
</tr>
<tr>
<td>DNAse I</td>
<td>Reagent</td>
<td>Roche</td>
<td>11 284 932 001</td>
<td>Stock solutions stored at -20&deg;C</td>
</tr>
<tr>
<td>MZ6 Dissecting Microscope</td>
<td>Equipment</td>
<td>Leica Microsytems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ProBlot 6 Hybridization Oven</td>
<td>Equipment</td>
<td>Labnet via Mandel Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5702</td>
<td>Equipment</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>kPBS</td>
<td>Reagent</td>
<td>BioShop</td>
<td>PBS404</td>
<td>&nbsp;</td>
<tr>
<td>B27 Supplement</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Ultra Low Binding Tissue Culture Dishes</td>
<td>Disposable</td>
<td>Corning</td>
<td>3261</td>
<td>&nbsp;</td>
<tr>
<td>Human Epidermal growth Factor</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>13247-051</td>
<td>Stock solutions stored at -20&deg;C</td>
</tr>
<tr>
<td>Fibroblast growth factor-2</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>13256-029</td>
<td>Also known as basic fibroblast growth factor (bFGF)<br />Stock solutions stored at -20&deg;C</td>
</tr>
<tr>
<td>37% formaldehyde (molecular grade)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F8775</td>
<td>&nbsp;</td>
<tr>
<td>Belly Dancer Shaker</td>
<td>Equipment</td>
<td>Stovall</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tissue- Tek Cryomold</td>
<td>Disposable</td>
<td>Sakura</td>
<td>4566</td>
<td>&nbsp;</td>
<tr>
<td>Tissue-Tek OCT compound</td>
<td>Reagent</td>
<td>Sakura</td>
<td>4583</td>
<td>&nbsp;</td>
<tr>
<td>Whatman Grade 703 Blotting Paper</td>
<td>Disposable</td>
<td>VWR</td>
<td>28298-020</td>
<td>&nbsp;</td>
<tr>
<td>CM1900 Cryostat</td>
<td>Equipment</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Polyclonal Anti-Cx29 Primary Antibody</td>
<td>Reagent</td>
<td>Kindly provided by Dr David Paul, Harvard Medical School</td>
<td>&nbsp;</td>
<td>Stored 1:1 in glycerol at -20&deg;C</td>
</tr>
<tr>
<td>Cy3-conjugated donkey anti-rabbit IgG</td>
<td>Reagent</td>
<td>Jackson ImmunoResearch</td>
<td>&nbsp;</td>
<td>Stored 1:1 in glycerol at -20&deg;C</td>
</tr>
<tr>
<td>Hoechst 33258</td>
<td>Reagent</td>
<td>Sigma</td>
<td>861405</td>
<td>&nbsp;</td>
<tr>
<td>DMRA2 epiflourescent microscope, Orca ER camera, OpenLab v3.56</td>
<td>Equipment/ Software</td>
<td>Leica Hamamatsu Improvision/Perkin Elmer</td>
<td>&nbsp;</td>
<td>This protocol can be performed with any epifluorescent or confocal microscope.</td>
</tr>
<tr>
<td>Photoshop CS4</td>
<td>Software</td>
<td>Adobe</td>
<td>&nbsp;</td>
<td>Graphic software</td>
</tr>
<tr>
<td>Maya v10</td>
<td>Software</td>
<td>Autodesk</td>
<td>&nbsp;</td>
<td>Modeling software</td>
</tr>
<tr>
<td>Computer</td>
<td>Equipment</td>
<td>Processor:<br />Intel Corei7 920<br />Memory:<br />12GB DDR3<br />Video card:<br />Nvidia GTX 285 (1GB RAM)</td>
<td>&nbsp;</td>
<td>Modeling hardware</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
			<p class="jove_content">* All other standard chemical reagents listed in the protocol are from Sigma-Aldrich.  All other standard tissue culture reagents are from Invitrogen.</p>		</div>
		

localizing protein in 3d neural stem cell culture: a hybrid visualization methodology

<p class="jove_content">The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study.  When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres.  Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu.  This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment.  Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult.  To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned.  This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere.  As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere.  Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins.  Connexin29 (Cx29) is analyzed as an example.  Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere.  This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis <em>in vitro</em>.</p>
<p class="jove_content">Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.</p>

Watch this Scientific Journal Video about Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology at JoVE.com

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

<p class="jove_content">Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture.  Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.</p>

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

localizing-protein-3d-neural-stem-cell-culture-hybrid-visualization

Research

JoVE Journal

Neuroscience

12.6K Views.  University of Ottawa. Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.