This work was supported by Merit Review grant (NEUD-004-07F) from the Veterans Administration (to S.P).

1. Expansion of Human NPCs (Figure 1)
<ol>
	<li>Revive the frozen stock of human NPCs from fetal brain (Lonza, Walkersville, MD, USA) and culture them in suspension in T-75 flasks as neurospheres (Figure 1A) in neurobasal medium containing proliferation supplements, EGF (10 ng/ml) and FGF (10 ng/ml).</li>
	<li>After 3 days in culture, transfer the neurospheres to a 15 ml tube and centrifuge at 500 rpm for 5 min.</li>
	<li>Discard the supernatant leaving behind ~100 &#956;l medium above the cell pellet and transfer into an Eppendorf tube. A 100 &#956;l cell pellet is enough to split into 2 T-75 flasks. If less, reduce the number of flasks as neuroprogenitor cells fail to proliferate if split thin.</li>
	<li>Triturate the pellet with a 200 &#956;l tip 50x while keeping the 200 &#956;l tip against the bottom of the tube. Divide the cell suspension equally into two parts and add them to 2 T-75 flasks (1-2 split), one for continued expansion and another for differentiation.</li>
	<li>Continue the culture of the neuroprogenitor cells for expansion (first flask) by changing the medium every 4-5 days until the neurospheres reach their original size of 300-500 &#956;m. Change medium by centrifugation (500 rpm; 5 min), discard the old medium and add new medium.</li>
</ol>
2. Differentiation of Human NPCs into a Neuron-rich Culture (Figure 2)
<ol>
	<li>For differentiation of neuroprogenitor cells into a neuron rich culture, place a single coverslip into each well of a 24-well plate and coat with 100 &#956;g/ml of poly-L-lysine by adding 500 &#956;l into each well. Incubate the dishes for 30 min at RT.</li>
	<li>Aspirate the poly-L-lysine solution and rinse the plate with sterile water.</li>
	<li>Next, coat the wells of the plate with 500 &#956;l of 5 &#956;g/ml mouse laminin and incubate for 30 min. Following incubation, wash the wells with PBS.</li>
	<li>4 days after splitting the cells (step 1.4), transfer the small neurospheres in the flask labeled &#39;For differentiation&#39; into the coated dishes at a density of ~500 neurospheres per well of a 24-well plate.</li>
	<li>After 6 hr, when the neurospheres attach to the dish, remove the proliferation medium and add differentiation medium consisting of neurobasal medium, B27 supplement, NGF (20 ng/ml), BDNF (10 ng/ml), DBC (100 mM), and retinoic acid (2 &#956;M).</li>
</ol>
3. Immunofluorescence Staining of Differentiated NPCs (Figure 3)
<ol>
	<li>Following culture of neuroprogenitor cells in neuronal differentiation medium for two weeks, a neuron-rich culture is obtained. Rinse the neurons once in PBS and then fix them in 4% paraformaldehyde for 30 min. Once fixed, rinse the cells three times with PBS.</li>
	<li>Incubate the cells with permeabilization buffer (5% BSA and 0.2% Triton X-100 in PBS) at RT for 60 min.</li>
	<li>Incubate the dishes O/N at 4 &#176;C in a shaker with the following combination of polyclonal and monoclonal antibodies in 3% BSA in PBS: NeuN (1:250) and synapsin (1:250); acetyl cholinesterase (1:500) and synaptophysin (1:250); BDNF (1:500) and GAP43 (1:250); STAT3 (1:500) and GFAP (1:1,000).</li>
	<li>Wash the coverslips three times with PBS and then incubate them with anti-rabbit-Cy3 and anti-mouse-FITC secondary antibodies at RT in dark for 90 min. Following incubation, wash the coverslips three times with PBS.</li>
	<li>Place 10 &#956;l of mounting medium onto a glass slide, take out the coverslip from the culture dish with forceps, and place it upside down on the mounting medium. Gently, wipe away any excess mounting medium and seal the edges with nail polish.</li>
	<li>Examine the immunostained neurons in a fluorescence microscope.</li>
</ol>
4. Laser Capture Microdissection of Neurons (Figure 4)
<ol>
	<li>Differentiate NPCs into a neuron-rich culture on PEN membrane slides coated with poly-L-lysine and mouse laminin following the procedures described for Figure 2.</li>
	<li>Perform all subsequent steps under RNAse-free conditions.</li>
	<li>Stain the cultures using HistoGene Stain (Arcturus) to visualize the cells.</li>
	<li>Find the areas of pure neuronal populations devoid of astrocytes using the road map image of the entire slide with the Veritas LCM system. Mark the neurons using the drawing tools.</li>
	<li>Perform laser capture of these areas using computer-controlled precision and automation in a two-step process. First, IR (Infrared) is fired at multiple spots with a laser power setting of 70 mW and a pulse of 2,500 &#956;sec to get the membrane attach to the cap. Next, the marked area is excised using an UV cutting tool at a low level setting of 10 mV.</li>
	<li>Combination of these processes selectively captures the marked areas from the PEN membrane onto CapSure LCM macro caps. When the cap is lifted, the membrane with the neurons adheres to the cap.</li>
	<li>Isolate total RNA from LCM samples using a PicoPure RNA isolation kit (Arcturus) and treat with DNase.</li>
	<li>Amplify the isolated RNA using RiboAMP RNA amplification kit following instructions from the kit.</li>
	<li>Perform Real time RT-PCR analysis using TaqMan probes to detect human neurofilament heavy chain (hNFHc).</li>
</ol>
Abbreviations:
AD, Alzheimer's disease; BDNF, brain-derived neurotrophic factor; CREB, cyclic AMP response element binding protein; DBC, Dibutyryl cyclic AMP; EGF, epidermal growth factor; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; LCM, laser capture microdissection; NGF, Nerve growth factor; NPC, neuroprogenitor cell; PEN, polyethylene naphthalate.

<ol>
	<li>Doetsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+identity+of+neural+stem+cells.">The glial identity of neural stem cells.</a> Nat Neurosci. 6, 1127-1134 (2003).</li><li>Galli, R., Gritti, A., Bonfanti, L., Vescovi, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells:+an+overview.">Neural stem cells: an overview.</a> Circ. Res. 92, 598-608 (2003).</li><li>Kim, S. U., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+cell+therapy+in+neurological+diseases:+a+review.">Stem cell-based cell therapy in neurological diseases: a review.</a> J. Neurosci. Res. 87, 2183-2200 (2009).</li><li>Breier, J. 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=Neural+progenitor+cells+as+models+for+high-throughput+screens+of+developmental+neurotoxicity:+state+of+the+science.">Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: state of the science.</a> Neurotoxicol. Teratol. 32, 4-15 (2010).</li><li>Christie, V. B., 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=Retinoid+supplementation+of+differentiating+human+neural+progenitors+and+embryonic+stem+cells+leads+to+enhanced+neurogenesis+in+vitro.">Retinoid supplementation of differentiating human neural progenitors and embryonic stem cells leads to enhanced neurogenesis in vitro.</a> J Neurosci Methods. 193, 239-245 (2010).</li><li>Bonner, R. F., 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=Laser+capture+microdissection:+molecular+analysis+of+tissue.">Laser capture microdissection: molecular analysis of tissue.</a> Science. 278, 1481-1483 (1997).</li><li>Vincent, V. A., DeVoss, J. J., Ryan, H. S., Murphy, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+neuronal+gene+expression+with+laser+capture+microdissection.">Analysis of neuronal gene expression with laser capture microdissection.</a> J. Neurosci. Res. 69, 578-586 (2002).</li><li>Robles, 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=Hippocampal+gene+expression+profiling+in+spatial+discrimination+learning.">Hippocampal gene expression profiling in spatial discrimination learning.</a> Neurobiol. Learn Mem. 80, 80-95 (2003).</li><li>Su, J. 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=Comparison+of+ethanol+versus+formalin+fixation+on+preservation+of+histology+and+RNA+in+laser+capture+microdissected+brain+tissues.">Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues.</a> Brain Pathol. 14, 175-182 (2004).</li><li>Shimamura, M., Garcia, J. M., Prough, D. S., Hellmich, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+and+analysis+of+amplified+antisense+RNA+from+distinct+cell+populations+of+the+young+and+aged+rat+brain:+effect+of+traumatic+brain+injury+on+hippocampal+gene+expression.">Laser capture microdissection and analysis of amplified antisense RNA from distinct cell populations of the young and aged rat brain: effect of traumatic brain injury on hippocampal gene expression.</a> Brain Res. Mol. Brain Res. 122, 47-61 (2004).</li><li>Pugazhenthi, S., Wang, M., Pham, S., Sze, C. I., Eckman, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+CREB+expression+in+Alzheimer's+brain+and+in+Abeta-treated+rat+hippocampal+neurons.">Downregulation of CREB expression in Alzheimer's brain and in Abeta-treated rat hippocampal neurons.</a> Mol. Neurodegener. 6, 60 (2011).</li><li>Dworkin, S., Mantamadiotis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+CREB+signalling+in+neurogenesis.">Targeting CREB signalling in neurogenesis.</a> Expert Opin. Ther. Targets. 14, 869-879 (2010).</li><li>Trujillo, C. 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=Novel+perspectives+of+neural+stem+cell+differentiation:+from+neurotransmitters+to+therapeutics.">Novel perspectives of neural stem cell differentiation: from neurotransmitters to therapeutics.</a> Cytometry A. 75, 38-53 (2009).</li><li>Pugazhenthi, 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=Varicella-zoster+virus+infection+of+differentiated+human+neural+stem+cells.">Varicella-zoster virus infection of differentiated human neural stem cells.</a> J. Virol. 85, 6678-6686 (2011).</li><li>Kamme, F., 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=Single-cell+microarray+analysis+in+hippocampus+CA1:+demonstration+and+validation+of+cellular+heterogeneity.">Single-cell microarray analysis in hippocampus CA1: demonstration and validation of cellular heterogeneity.</a> J. Neurosci. 23, 3607-3615 (2003).</li></ol>

No conflicts of interest to declare.

We describe in this study a neuron-rich cell culture model by differentiation of self-renewing human neuroprogenitor cells and a method to isolate a pure population of neurons by laser capture microdissection. We have used a combination of NGF, BDNF, DBC and retinoic acid for neuronal differentiation of NPCs. DBC is used to activate CREB, a transcription factor that enhances neurogenesis 12. Retinoic acid induces cell cycle exit and reduces the glial population 13. The method described in this study...

Life-long neurogenesis is known to occur in the subventricular zone of the lateral ventricles and in the subgranular layer of the dentate gyrus of the adult mammalian brain 1. Neuroprogenitor cells (NPCs) that originate from these regions are multipotent cells that can differentiate into neurons, astrocytes and oligodendrocytes 2. NPCs have generated interest because of their potential to be transplanted in patients with various neurodegenerative disorders including Parkinson&#8217;s disease, amyotrophic lateral sclerosis, stroke and Alzheimer&#8217;s disease (AD) 3. Studies with NPCs have generally focused on this transplantation angle but the potential of NPC-derived neurons as a cell culture model to determine the mechanism of neurodegeneration has not been fully exploited. Previous studies have generally used post-mitotic neurons isolated from rodent brain tissues which need to be isolated for each experiment as they are not self-renewing. Although human neuroblastoma cell lines including SH-SY5Y and SK-N-MC cells can be expanded, they do not have the characteristics of primary neurons. Human NPCs, on the other hand, offer both advantages because they can be expanded for multiple passages and can be differentiated to generate a cell population with the characteristics of primary neurons 4,5. In the current study, we describe a new differentiation protocol to obtain a consistent neuron-rich population from commercially available NPCs isolated from human fetal brain. Because these cultures do contain a small percent of glial cells, we need additional methods to isolate a pure population of neurons for molecular characterization. Laser capture microdissection (LCM) is a novel technique by which a homogenous population of cells from a tissue section can be selectively captured for gene expression analysis 6. The brain is a heterogeneous tissue consisting of neurons, glia and other cell types. LCM has been used to determine neuron-specific gene expression analyses 7-10. We have previously performed LCM of hippocampal neurons from AD (Tg2576) mouse brain sections to show decreased expression of cyclic AMP response element binding protein (CREB) and BDNF specifically in hippocampal neurons 11. In the present study, we describe procedures for the expansion of human NPCs, neuronal differentiation, immunofluorescent staining for neuronal markers and LCM for the isolation of neurons cultured on PEN membrane slides.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Neuroprogenitor cells (NPCs) </td>
			<td>Lonza</td>
			<td>PT-2599</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A human basal medium</td>
			<td>Stem cell technology</td>
			<td>5750</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A Proliferation supplement</td>
			<td>Stem cell technology</td>
			<td>5753</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Neurocult NS-A Differentiation supplement</td>
			<td>Stem cell technology</td>
			<td>5754</td>
			<td></td>
		 </tr>
		 <tr>
			<td>B-27 Supplement (50x) </td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Human epidermal growth factor</td>
			<td>Stem cell technology</td>
			<td>2633</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fibroblast growth factor </td>
			<td>Sigma</td>
			<td>F0291</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Brain Derived Neurotrophic Factor </td>
			<td>Cell signaling </td>
			<td>3897S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Nerve Growth Factor </td>
			<td>Invitrogen</td>
			<td>13257-019</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dibutyryl cyclic AMP</td>
			<td>Sigma</td>
			<td>D-0627</td>
			<td></td>
		 </tr>
		 <tr>
			<td>poly-L-lysine </td>
			<td>Sigma</td>
			<td>P-5899</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse laminin </td>
			<td>Sigma</td>
			<td>L-2020</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R-2625</td>
			<td></td>
		 </tr>
		 <tr>
			<td>STAT3 antibody</td>
			<td>Cell signaling</td>
			<td>9132</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GFAP antibody</td>
			<td>Cell signaling</td>
			<td>3670</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GAP43 antibody</td>
			<td>Transduction laboratories</td>
			<td>612262</td>
			<td></td>
		 </tr>
		 <tr>
			<td>BDNF antibody</td>
			<td>Millipore</td>
			<td>AB1534SP</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Acetyl cholinesterase antibody </td>
			<td>Santz cruz</td>
			<td>Sc-11409</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synaptophysin antibody</td>
			<td>Abcam</td>
			<td>ab18008-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>NeuN antibody</td>
			<td>Chemicon</td>
			<td>MAB377</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Synapsin antibody</td>
			<td>Novus</td>
			<td>NB300-104</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti mouse FITC</td>
			<td>Jackson Immuno </td>
			<td>115-095-146</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td>Research Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Anti Rabbit Cy3</td>
			<td>Jackson Immuno </td>
			<td>711-165-152</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td>Research Laboratories</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A1653</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Triton-X 100</td>
			<td>Acros</td>
			<td>21568-2500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Paraformaldehye </td>
			<td>Fisher</td>
			<td>4042</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Coverslip (Big circle cover slip)</td>
			<td>Fisherbrand</td>
			<td>12-545-102</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mounting medium (Prolong Gold)</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pen membrane</td>
			<td>Applied biosystems</td>
			<td>LCM0521</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Histogene LCM frozen section staining kit</td>
			<td>Applied biosystems</td>
			<td>KIT0401</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RiboAmp RNA Amplification kit</td>
			<td>Applied biosystems</td>
			<td>KIT0201</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Picopure RNA Isolation kit</td>
			<td>Applied biosystems</td>
			<td>KIT0202</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CapsureMacro LCM caps</td>
			<td>Applied biosystems</td>
			<td>LCM0211</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

laser capture microdissection of neurons from differentiated human neuroprogenitor cells in culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Expansion of NPCs (Figure 1)
When neurospheres are broken down to a single cell suspension by trituration, the pipette tip needs to touch the bottom of the tube so that there is some resistance when the suspension is pipetted up and down. The number of times of trituration will vary between individuals and needs to be decided by trial and error by examining the resulting cell suspension under a microscope. It is critical to provide sufficient density of cell suspension because th...

Watch this Scientific Journal Video about Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture at JoVE.com

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

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12.0K Views.  Denver VA Medical Center. Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.