eoe sub articles

EoE Sub Articles

1. Cloning of 2K7 Lentivector
<ol>
	<li>Before cloning the gene of interest into the 2K7 lentiviral vector, subclone the gene into the pENTR vector (3637 bp, Kanamycin resistant) using standard molecular techniques. Use the EcoRI and SacII restriction sites for the subcloning.</li>
	<li>Amplification of the 2K7 Lentivector
	<ol>
		<li>Transform 2K7 lentivector DNA by gently mixing 100 ng of plasmid DNA with competent cells, and incubate on ice for 30 min.</li>
		<li>Heat shock DNA/ competent cells mix at 42 &#176;C for 90 sec and plate them on LB-Agar plates containing both ampicillin (100 &#181;g/ml) and chloramphenicol (15 &#181;g/ml) at 37 &#176;C for 16-18 hr. 
		&#8203;NOTE: Chloramphenicol is used to prevent recombination between the long terminal repeats.</li>
		<li>The next day, grow selected bacterial clones in Luria-Bertani (LB) media containing both ampicillin (100 &#181;g/ml) and chloramphenicol (15 &#181;g/ml) at 37 &#176;C for 16-18 hr. Extract the DNA using a commercial plasmid DNA Maxiprep kit as per the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Sub-cloning from pENTR vector to 2K7 Lentivector (Figure 1).
	<ol>
		<li>Add the following to a 1.5 ml tube and mix gently: L4-R1 pENTR-pDNOR-CMV (promoter) (60 bng)1-2.5 &#181;l L1-L2 pENTR4IRES2GFP (with gene of interest) (80 bng)1-2.5 &#181;l K7 lentivector (80 ng/&#181;l)1 &#181;l TE-buffer, pH 82-5 &#181;l.Total 8 &#181;l.</li>
		<li>Remove the clonase enzyme mix from -80 &#176;C and thaw on ice for 2 min. Ensure that this enzyme is a fresh aliquot from -80 &#176;C as the enzyme has substantially reduced cloning efficiency with repeated freeze-thaw cycles</li>
		<li>Add 2 &#181;l of enzyme per reaction and mix well via vortex, and incubate the clonase reaction mixture at 23-25 &#176;C for 6-24 hr.</li>
		<li>Add 1 &#181;l of proteinase K solution (supplied with enzyme kit) to the clonase reaction mixture and incubate for 10 min at 37 &#176;C.</li>
		<li>Transform the clonase reaction mixture into competent cells and grow the select colonies in LB media containing ampicillin (100 &#181;g/ml) at 37 &#176;C for 16-18 hr.</li>
		<li>Extract and purify DNA using a commercial plasmid DNA Miniprep kit as per the manufacturer&#39;s instructions.</li>
		<li>Confirm the DNA via digestion using restriction enzymes Spe I and Sac II and an appropriate buffer as per the manufacturer&#39;s instruction to remove the fragment containing the promoter and gene of interest. 
		&#8203;NOTE: This step is critical to check if the subcloned DNA has the correct insert gene of interest with the right vector backbone. The size of the vector itself without the inserted fragment is 6.7 kb. The size of the released inserted fragment includes both the promoter and the insert gene of interest. Calculate the expected fragment sizes from the digest for the specific gene via a DNA sequence editing program, e.g., APE-A Plasmid Editor.</li>
		<li>Check the sizes of both the DNA vector backbone and the released fragment by running a 1% agarose gel in 1x TAE buffer (see Table 1) at 100 V.</li>
		<li>Amplify the confirmed DNA by growing bacteria in 500 ml of LB media containing ampicillin (100 &#181;g/ml) at 37 &#176;C for 16-18 hr.</li>
		<li>Extract and purify DNA using a commercial plasmid DNA Maxiprep kit as per the manufacturer&#39;s instructions. 
		NOTE: Maxiprep typically generates a sufficient amount of DNA required for viral production.</li>
	</ol>
	</li>
	<li>Repeat steps 1.3.9-1.3.10 to amplify the following DNAs for lentiviral preparations: Envelope vector (PMD2.G, 6.1 kb), Package vector (pBR8.91, 12.5 kb), and an empty lentiviral vector as a control (GFP-CMV-2K7, 8.7 kb).</li>
</ol>
2. 2K7 Virus Production 
NOTE: Day 1:
<ol>
	<li>On the day of transfection, plate 32 million HEK293 T cells in a T175 flask containing 25 ml Human Embryonic Kidney (HEK) 293 T cell media (see Table 1). Alternatively, plate 16 million cells the day before the transfection if time runs tight on the day of transfection. 
	NOTE: Transfection can be equally successful by plating cells on the day of transfection or prior to the day. Using either of the two alternatives, the critical point here is to make sure cells are stuck down on the culture dish surface prior to transfection. 
	NOTE: Day 2:</li>
	<li>Transfection
	<ol>
		<li>Prior to transfection, dilute DNA to 1 &#181;g/&#181;l in Tris-ethylenediaminetetraacetic acid (TE) Buffer that contains 10 mM Tris pH 8, 1 mM EDTA pH 8 in deionized water.</li>
		<li>In a 50 ml tube, prepare a master mix (Table 2) for transfection in the T175 flask. Add DNA to pre-warmed Dulbecco&#39;s Modified Eagle Medium (DMEM) and mix well by vortex, then add sterile polyethyleneimine (PEI) (see Table 1 to avoid premature precipitation.</li>
		<li>Mix well by inverting the tube 3-4x, and incubate for 15 min at room temperature (RT).</li>
		<li>Perform a full media change on HEK293T cells. Aspirate off the culture media from cells completely and feed with pre-warmed HEK293T cell culture media (25 ml per T175 flask).</li>
		<li>Add the transfection mixture (DNA/PEI mixture) dropwise to the monolayer cells. Move gently to mix well and incubate transfected cells at 37 &#176;C, 5% CO2, overnight. 
		&#8203;NOTE: Day 3:</li>
		<li>To ensure the transfection is successful, check green fluorescence protein (GFP) expression 24 hours post-transfection via fluorescent microscopy. 
		NOTE: Over 50% of cells expressing GFP typically indicate a good transfection. 
		NOTE: Day 4:</li>
	</ol>
	</li>
	<li>At 48 hr post-transfection, collect the viral supernatant and replace it with fresh HEK293 T media (25 ml per T175 flask).
	<ol>
		<li>Centrifuge the viral supernatant at 1,140 x g for 10 min at 4 &#176;C to clear up cell debris from the supernatant. Transfer the cleared supernatant to a 50 ml tube and store it at 4 &#176;C. 
		NOTE: Day 5:</li>
	</ol>
	</li>
	<li>At 72 hours post-transfection, collect the second batch of viral supernatant. Repeat step 2.3.1 and pool 48 and 72 hr cleared supernatants.</li>
	<li>To concentrate the virus, centrifuge viral supernatant at 170,000 x g for 90 min at 4 &#176;C using 30 ml ultracentrifuge tubes.</li>
	<li>Discard the supernatant and repeat step 2.6 until all the cleared supernatant has been centrifuged leaving an (invisible) pellet of virus plus precipitated PEI in the base of the tube.</li>
	<li>To resuspend the virus, add 500 &#181;l SATO media (see Table 1) to the ultracentrifuge tubes. Vortex for 30 sec and scrape the base of the tube with a pipette tip to mechanically loosen the virus. Repeat this step 6x in order to lose the viral pellet.</li>
	<li>Pool the resuspended virus into microcentrifuge tubes and spin very briefly to remove insoluble PEI. Filter the supernatant through a 0.45 &#181;m filter to remove proteins.</li>
	<li>Aliquot the virus into 20 &#181;l, 50 &#181;l, and 100 &#181;l aliquots and store at -80 &#176;C.</li>
</ol>
3. Transducing OPCs 
<ol>
	<li>Culture primary OPCs in SATO media (10ml/10cm plate) with Ciliary neurotrophic factor (CNTF, 10 ng/ml), Platelet-derived growth factor (PDGF, 10 ng/ml), Neurotrophin 3 (NT3, 1 ng/ml), and forskolin (4.2 &#181;g/ml) at 37 &#176;C, 8% CO2 for 24 hr after dissection.</li>
	<li>Completely aspirate off the OPC culture media, and feed cells with freshly made SATO media (10 ml) with growth factors (see above in step 3.1).</li>
	<li>Add virus to the OPCs to the optimal concentration, culture OPCs for a further 48 hr.</li>
</ol>
4. OPC Seeding for Myelinating Co-cultures (Figure 2)
<ol>
	<li>To remove OPCs off the surface, first rinse OPC plates with 8 ml Earle&#39;s balanced salt solution (EBSS) twice, then incubate cells with 5 ml of warm 0.05% Trypsin-EDTA diluted 1:10 with EBSS at 37 &#176;C for 2 min.</li>
	<li>Neutralize the trypsin with 30% FBS in EBSS (5 ml) and remove cells from the plate by pipetting. Transfer cell suspension to a 15 ml tube, and centrifuge at 180 x g for 15 min at RT.</li>
	<li>Aspirate off the supernatant and resuspend the cell pellet in 1 ml pre-warmed SATO media followed by cell count.</li>
	<li>Prior to the OPC seeding, completely aspirate media from the DRG culture plate. Gently seed 200,000 OPCs dropwise onto the DRG neurons as previously described. 
	NOTE: The total OPC seeding volume must be less than 200 &#181;l per 22 mm coverslip.</li>
	<li>Leave cells to settle without moving the plate for 10 min in the tissue culture hood, then gently top up with 1 ml pre-warmed SATO media per well.</li>
	<li>Replate the remaining sister OPCs with SATO media with growth factors (see step 6.1). Use these sister OPCs to verify the expression of the protein of interest.</li>
	<li>After 24 hr, replace the SATO media with the co-culture media (2 ml/well) containing SATO media (no factors) and neurobasal (v/v) with 1% B2Maintain the co-cultures for 14 days with media change every 2-3 days.</li>
	<li>Assess the co-cultures for myelin protein expression by western blot analysis and visualize the formation of myelinated axonal segments by double-immunostaining with antibodies against myelin basic protein markers and neuronal markers.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2K7 lentivector</td>
			<td></td>
			<td></td>
			<td>Kind gift from Dr Suter</td>
		</tr>
		<tr>
			<td>5-Fluoro-2&#8242;-deoxyuridine</td>
			<td>Sigma-Aldrich</td>
			<td>F0503-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma-Aldrich</td>
			<td>A9518-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 - NeuroCul SM1 Neuronal Supplement</td>
			<td>Stem Cell Technologies</td>
			<td>5711</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF (Human)</td>
			<td>Peprotech</td>
			<td>PT450021000</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin (d-Biotin)</td>
			<td>Sigma-Aldrich</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A4161</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>CNTF</td>
			<td>Peprotech</td>
			<td>450-13020</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO fluoresence mounting media</td>
			<td>DAKO</td>
			<td>S302380-2</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate, no glutamine</td>
			<td>Life Technologies</td>
			<td>10313039</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025-375KU</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, calcium, magnesium</td>
			<td>Life Technologies</td>
			<td>14040182</td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS</td>
			<td>Life Technologies</td>
			<td>14155063</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI-HF</td>
			<td>NEB</td>
			<td>R3101</td>
			<td></td>
		</tr>
		<tr>
			<td>Entry vectors for promoter and gene of interest</td>
			<td></td>
			<td></td>
			<td>Generate as per protocols 1-2</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>12003C</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma-Aldrich</td>
			<td>F6886-50MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (D-glucose)</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Chem Supply</td>
			<td>GL010-500M</td>
			<td>See stock solutions</td>
		</tr>
	</tbody>
</table>

an in vitro transduction-induced myelination assay for oligodendrocyte precursor cells

Source: Peckham, H. M., et al. <a href="https://app.jove.com/v/52179">Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays.</a> J. Vis. Exp. (2015).
The video demonstrates a protocol for differentiating oligodendrocyte precursor cells (OPCs) into oligodendrocytes and inducing myelination in a co-culture with dorsal root ganglion (DRG) neurons. The OPCs are transduced with lentiviral vectors encoding a signaling protein that regulates myelination. The transduced OPCs are co-cultured with DRG neurons to assess the differentiation into oligodendrocytes and the signaling protein-induced myelination.

Table 1. Table of stock solutions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Ingredients</td>
			<td>Notes</td>
		</tr>
		<tr>
			<td>TE Buffer pH 8</td>
			<td>10 mM Tris pH 8 
			1 mM EDTA pH 8</td>
			<td>Make up in deionized water.</td>
		</tr>
		<tr>
			<td>Polyethylenimine (PEI)</td>
			<td>1 g/L</td>
			<td>Make up in deionized water, filter-sterilize...

An In Vitro Transduction-Induced Myelination Assay for Oligodendrocyte Precursor Cells

1. Cloning of 2K7 Lentivector

	Before cloning the gene of interest into the 2K7 lentiviral vector, subclone the gene into the pENTR vector (3637 bp, ...

Take adherent cultures of primary oligodendrocyte precursor cells, or OPCs, in a growth medium.
Add control lentiviral vectors to the control well and test vectors, encoding signaling proteins that regulate myelination, to the test well, then incubate.
The cells internalize the vectors, and the viral RNA is reverse-transcribed into DNA and incorporated into the host genome, expressing the signaling proteins in the test cells.
Introduce enzymes to detach the OPCs from the substrate.
Neutralize the enzymes and centrifuge to remove the enzymes.
Add the growth medium and transfer the OPCs onto coverslips inside a microplate with adhered dorsal root ganglion or DRG neurons. Allow the OPCs to settle.
Fill the wells with the growth medium and incubate. Interaction with the DRG neurons triggers OPC maturation into oligodendrocytes.
The expressed signaling proteins induce oligodendrocytes to form myelin sheaths around the DRG neurons.
Assess the signaling protein-induced myelin sheath formation in the test cells compared to the control vector-transduced cells.

an-vitro-transduction-induced-myelination-assay-for-oligodendrocyte

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

36 Aufrufe. Quelle: Peckham, H. M., et al. Produktion und Verwendung von Lentiviren zur selektiven Transduktion von primären Oligodendrozyten-Vorläuferzellen für In-vitro-Myelinisierungsassays. J. Vis. Exp. (2015). Das Video demonstriert ein Protokoll zur Differenzierung von Oligodendrozyten-Vorläuferzellen (OPCs) in Oligodendrozyten und zur Induktion der Myelinisierung in einer Co-Kultur mit Neuronen des dorsalen Wurzelganglion (DRG). Die OPCs werden mit lentiviralen Vektoren transduziert, die für e...

Serie: Ein in vitro Transduktions-induzierter Myelinisierungsassay für Oligodendrozyten-Vorläuferzellen - Konzept

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

Generierung humaner Neuronen und Oligodendrozyten aus pluripotenten Stammzellen zur Modellierung von Neuron-Oligodendrozyten-Interaktionen

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Forschung

JoVE Journal

Neurowissenschaften

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation.
In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity.
Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs.
This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

We describe the production of mixed cultures of astrocytes and oligodendrocyte precursor cells derived from fetal or adult neural stem cells differentiating into mature oligodendrocytes, and in vitro modeling of noxious stimuli. The coupling with a cell-based high-content screening technique builds a reliable and robust drug screening system.

High-Content-Screening-Differenzierungs- und Reifungsanalyse von fetalen und adulten neuralen Stammzellen-abgeleiteten Oligodendrozyten-Vorläuferzellkulturen

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance ...

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

In vitro Myelinisierung peripherer Axone in einer Kokultur von Dorsalwurzelganglienexplantaten der Ratte und Schwann-Zellen

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

An In Vitro Transduction-Induced Myelination Assay for Oligodendrocyte Precursor Cells

Transkript

An In Vitro Transduction-Induced Myelination Assay for Oligodendrocyte Precursor Cells