a lentiviral transcriptional reporter system to generate a glioblastoma stem cell differentiation reporter line

A Lentiviral Transcriptional Reporter System to Generate a Glioblastoma Stem Cell Differentiation Reporter Line

To begin seed 1 million glioblastoma stem cells or GSCs in 2 milliliters of complete neural stem cell growth medium in a 6-well plate. Next, transduce the cells with the lentivirus reporter at a multiplicity of infection equal to five.
To the cell and virus mixture, add 2 microliters of polybrene such that the final concentration is 8 micrograms per milliliter, and incubate the cells at 37 degrees Celsius and 5% CO2 overnight. After incubation, replace the growth medium to remove unbound virus, and centrifuge cells at 360 times g for 5 minutes. Then, carefully aspirate medium and plate cells back in a 6-well plate, and continue to incubate the cells for 24 hours.
Harvest 0.5 milliliters of the total culture volume. Pellet cells by centrifugation at 360 times g for 5 minutes at room temperature. Resuspend cells in 0.5 milliliters fresh neural stem cell growth medium.
Place the plate back in the incubator. To allow for expansion for at least 72 hours. Add 200 microliters of dissociation reagent, and incubate for five minutes in a water bath set to 37 degrees Celsius. Dissociate cells by pipetting up and down gently. To the dissociated cells, add 800 microliters of HBSS to ensure minimal cell attachment or aggregation.
Next, transfer 200 microliters of each cell suspension into wells of a 96 multi-well plate. Determine the percentage of cells expressing the green fluorescent protein by flow cytometry. Record at least 10,000 viable cells in each acquisition.

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1. Lentiviral Transcriptional Reporter System
NOTE: In all flow cytometric analyses, use parental, non-transduced, cells or vector-transduced (non-fluorescent) cells to establish baseline fluorescence. Also, note that in all steps where mechanical trituration is called for, be gentle. Harsh trituration can kill a significant number of the fragile GSCs and influence flow cytometry results.
<ol>
	<li>Plate 1x106&#160;cells in 2 mL of complete neural stem cell growth medium in a 6-multiwell plate.</li>
	<li>Transduce cells by adding lentivirus reporter at a multiplicity of infection (moi) equal to 5.</li>
	<li>Add 2 &#181;L of polybrene for a final concentration of 8 &#181;g/mL.</li>
	<li>Incubate the cells at 37 &#176;C and 5% CO2&#160;overnight.</li>
	<li>The next day, replace growth medium to remove unbound virus. Place the plate back in the incubator at 37 &#176;C and 5% CO2&#160;and incubate for 24 h.</li>
	<li>Harvest 0.5 mL of the total volume; spin down cells at 360 x g for 5 min at room temperature in a 15- mL conical tube and remove supernatant by aspiration. Add 0.5 mL of fresh neural stem cell growth medium to the remaining cells and return the plate to the incubator for continued expansion.</li>
	<li>Add 200 &#181;L of dissociation reagent and incubate for 5 min at 37 &#176;C.</li>
	<li>Dissociate cells by gently pipetting up and down. 
	NOTE: Harsh trituration may result in significant killing of GSCs; complete dissociation is usually achieved after 20 - 30 times.</li>
	<li>To minimize cell attachment or aggregation, add 800 &#181;L of Hank&#39;s Balanced Salt Solution (HBSS) for a final volume of 1 mL.</li>
	<li>Transfer 200 &#181;L of each cell suspension to a well of a 96-multiwell plate.</li>
	<li>For this procedure, use a benchtop flow cytometer with 96-well plate capability equipped with a blue laser for excitation and with the capability of detecting green fluorescent protein. Perform flow cytometric analysis with at least 10,000 viable cells for each acquisition.</li>
	<li>Determine the percentage of GFP-positive cells by flow cytometry.</li>
</ol>
2. Determination of GFAP:GFP Expression by Flow Cytometry
<ol>
	<li>Harvest an aliquot of cells (0.5 mL) from each reporter clone and non-transduced controls to determine the exact percentage of GFP-positive cells. Considering reporter clones that contain ~1 - 5% GFP-positive cells.</li>
	<li>Spin cells at 360 x g for 5 min at room temperature and remove supernatant by aspiration.</li>
	<li>To each pellet, add 200 &#181;L of cell dissociation reagent and incubate the tubes in a water bath set to 37 &#176;C.</li>
	<li>Triturate cells gently to achieve a single-cell suspension (usually between 20 to 30 times).</li>
	<li>To minimize cell attachment or aggregation, add 800 &#181;L HBSS to a final volume of 1 mL.</li>
	<li>Transfer 200 &#181;L per well of a 96-multiwell plate from each cell suspension.</li>
	<li>For this procedure, use a benchtop flow cytometer with 96-well plate capability. Perform flow cytometric analysis with at least 10,000 viable cells for each acquisition.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ESGRO Complete Accutase</td>
			<td>EMD Millipore</td>
			<td>SF006</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (Hank&#39;s Balanced Salt Solution)</td>
			<td>Sigma Aldrich</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GFAP Differentiation Reporter (pGreenZeo, Virus)</td>
			<td>SBI (System Biosciences)</td>
			<td>SR10015VA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml sterile disposable reagent reservoirs</td>
			<td>Corning</td>
			<td>4870</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Centrifuge tubes</td>
			<td>Celltreat</td>
			<td>229411</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml Microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Guava easyCyte 5HT Benchtop Flow Cytometer</td>
			<td>EMD Millipore</td>
			<td>0500-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>For the preparation of neural stem cell media (500 mL)</td>
			<td></td>
			<td></td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>GoldBio.com</td>
			<td>A-421-250</td>
			<td>0.20%</td>
		</tr>
		<tr>
			<td>DMEM/F12 10X</td>
			<td>Corning</td>
			<td>90-091-PB</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>H3149</td>
			<td>0.00%</td>
		</tr>
		<tr>
			<td>HEPES 1M</td>
			<td>Sigma Aldrich</td>
			<td>H4036</td>
			<td>5.4 mM</td>
		</tr>
		<tr>
			<td>Insulin-Transferrin- Selenium (ITS -G) (100X)</td>
			<td>Life Technologies</td>
			<td>41400-045</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S-5761</td>
			<td>14.5 mM</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL) 100X</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma Aldrich</td>
			<td>P8783</td>
			<td>16 nM</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma Aldrich</td>
			<td>P5780</td>
			<td>4.8 &#181;M</td>
		</tr>
		<tr>
			<td>Basic FGF (FGF2), Human</td>
			<td>GoldBio</td>
			<td>1140-02-50</td>
			<td>10 ng/ml</td>
		</tr>
		<tr>
			<td>EGF, Human</td>
			<td>GoldBio</td>
			<td>1150-04-100</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td>Bottle-Top Filter, 150ml, 33mm, 0.22um, Pes, S, Ind</td>
			<td>Corning</td>
			<td>431160</td>
			<td>Use to filter sterlize media</td>
		</tr>
	</tbody>
</table>

Source: Spina, R. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56176">Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells.</a>&#160;J. Vis. Exp. (2018).
This video demonstrates a technique to generate a glioblastoma stem cell reporter line to identify the differentiation of glioblastoma stem cells (GSCs) into astroglia. It outlines the steps involved in seeding GSCs, transducing them with a lentivirus encoding GFP, and determining GFP reporter expression through flow cytometry.

1. Lentiviral Transcriptional Reporter System
NOTE: In all flow cytometric analyses, use parental, non-transduced, cells or vector-transduced ...

Begin with glioblastoma stem cells, or GSCs.&#160;
Add the lentivirus reporter encoding a green fluorescent protein or GFP driven by the GFAP promoter.
Introduce a cationic polymer.
The polymer neutralizes the negative charges on the virus and GSC surface, enabling viral-host cell membrane fusion and RNA release.
The viral RNA is reverse-transcribed into DNA and integrated into the host genome.
Collect the cells, centrifuge, and discard the supernatant.
Add fresh media and re-plate the cells. Incubate.
The GSCs that differentiate into astroglia express transcription factors that activate the GFAP promoter, inducing GFP expression.
Undifferentiated GSCs fail to express GFP.
The GSCs expand and aggregate to form neurospheres. Collect the neurospheres, centrifuge, and discard the supernatant.
Add enzymes to dissociate the cells. Pipette repeatedly to obtain a single-cell suspension.
Add buffer and transfer the cells to a multi-well plate.
Using flow cytometry, detect the GFP-expressing cells.

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Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

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Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

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An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

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A Lentiviral Transcriptional Reporter System to Generate a Glioblastoma Stem Cell Differentiation Reporter Line

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A Lentiviral Transcriptional Reporter System to Generate a Glioblastoma Stem Cell Differentiation Reporter Line