eoe sub articles

EoE Sub Articles

1. Human neuron induction from human pluripotent stem cells
<ol>
	<li>Lentivirus preparation (~5 days)
	<ol>
		<li>Plate ~1 million Human embryonic kidney 293T (HEK293T) cells each T75 flask, to have them ~40% confluent when performing transfection. Transfect them with plasmids expressing tetracycline-inducible neurogenin 2 (Ngn2) and puromycin-resistant gene (PuroR; under the same Tet operator (TetO) promoter control), reverse tetracycline transactivator (rtTA) and the three helper plasmids pRSV-REV, pMDLg/pRRE, and VSV-G (12 &#181;g of lentiviral vector DNA and 6 &#181;g of each of the helper plasmid DNA). Prepare at least three flasks per lentivirus preparation. Use polyethylenimine (PEI) for transfection following the manufacturer&#39;s instruction. Change the media after 16 h and discard.</li>
		<li>Harvest released viral particles by collecting culture media every day and replace with fresh media for 3 days. Pool the collected media containing viral particles for purification. Filter the virus through a 0.22 &#181;m filter and centrifuge at 49,000 x g for 90 min. Resuspend the pellet in the appropriate volume of phosphate-buffered saline (PBS)-glucose (~150 &#181;L).</li>
	</ol>
	</li>
	<li>Neuron Induction (~5 days) 
	NOTE: This induction protocol (Figure 1A; flow diagram) is highly effective for both induced pluripotent stem (iPS) and embryonic stem (ES) cells of validated pluripotency (which can be assayed by immunohistochemistry staining of well-characterized pluripotency markers; Figure 1B).
	<ol>
		<li>Use commercially available H1 human ES cells at the passage of 52 (see Table of Materials). Culture the cells on extracellular matrix solution coated 6-well plates (~0.5 mg of matrix solution per 6-well plate; see Table of Materials) using ES cell maintenance medium (see Table of Materials) and incubate the plates at 37 &#176;C with 5% CO2.</li>
		<li>On Day -2, detach ES cells (80% confluent) with 1 mL of cell detachment solution (see Table of Materials) and incubate at room temperature for 10 min. Transfer the cells to a tube; wash the well with 2 mL of media and combine in the same tube. Centrifuge at 300 x g for 5 min, resuspend the pellet in media, and plate the cells onto matrix coated 6-well plates at the seeding density of 1 x 105 cells per well.</li>
		<li>On Day -1, add lentiviruses expressing Ngn2 plus PuroR and rtTA together with polybrene (8 &#181;g/ml) to the ES cells in fresh ES cell maintenance medium (see Table of Materials). The exact number of viruses should be determined by actual titers or the titration. We typically add 5 &#181;L each virus per well in a 6-well plate.</li>
		<li>On Day 0, add Doxycycline (2 &#181;g/mL, to activate Ngn2 expression) in Dulbecco&#39;s modified Eagle medium/nutrient mixture F-12 (DMEM-F12) medium with N2 supplement without morphogens.</li>
		<li>On Day 1, add Puromycin in fresh medium of DMEM-F12 plus N2 and doxycycline, to the final concentration of 1 &#181;g/mL medium. Select the transduced cells in Puromycin for at least 24 h. Higher Puromycin concentration (up to 5 &#181;g/mL) and longer selection period (up to 48 h) may be required to adequately remove the under-transduced cells if the virus titer is low.</li>
		<li>On Day 2, detach differentiating neurons with cell detachment solution (see Table of Materials), and re-plate them on 24-well plates (between 80,000&#8211;200,000 cells/well) coated with matrix solution (see Table of Materials), and maintain them in neurobasal-A (NBA)/B27 medium without doxycycline. The seeding density is critical.</li>
		<li>At this stage, detached neurons can be frozen in specialized commercial freezing medium (see Table of Materials) and stored in liquid nitrogen for up to 3 months. Pure neurons can be plated accounting for the typical ~15%&#8211;20% cell death post-thaw, cultured alone or co-cultured with other brain cell types (see step 3.2.3. for co-culturing with OPCs).</li>
		<li>Culture pure iNs on the plates coated with extracellular matrix-based solutions as instructed by the manufacturer (see Table of Materials). The characteristic pyramidal morphology should be apparent by Day 4 (and Day 6; Figure 1C). The synapse formation can be detected as early as Day 14 to 16 and is prominent at Day 24 by immunohistochemical staining with standard pre- and post-synaptic markers. (Figure 1D; labeled with the pre-synaptic marker Synapsin 1 and the dendritic marker Map2).</li>
	</ol>
	</li>
</ol>
2. Human oligodendrocyte precursor cell (OPCs) induction from pluripotent stem cells and oligodendrocyte maturation
<ol>
	<li>Neural Progenitor Cell (NPC) generation: monolayer protocol (~7 days). See Figure 2A for the flow diagram.
	<ol>
		<li>Culture H1 human ES cells as described earlier (see step 1.2.1.) and trans-differentiate them into neural progenitor cells (NPCs) by an established approach called dual SMADi, with small molecule inhibitors for multiple signaling pathways. Here we use a widely accepted commercial kit and follow the monolayer protocol provided by the manufacturer (see Table of Materials).</li>
		<li>On Day -1, plate 0.5&#8211;1 x 106 cells per well in a 6-well plate coated by a growth factor reduced matrix solution (see Table of Materials; ~0.5 mg of matrix solution per 6-well plate) with ES cell maintenance medium (see Table of Materials). This growth factor reduced matrix solution is used to coat all the plates that will be used in the following steps.</li>
		<li>On Day 0, treat cells for 24 h with ES cell maintenance medium (see Table of Materials) supplemented by 2% DMSO.</li>
		<li>On Day 1&#8211;6, change the full media with warm (37 &#176;C) neural induction medium containing the SMAD inhibitors from the commercial kit (see Table of Materials). If cells divide and reach confluence before Day 7, passage them to the seeding density of 0.5&#8211;1 x 106, as described earlier in step 2.1.2.</li>
		<li>On Day 7, passage NPCs using cell detachment solution (see Table of Materials) and plate at a seeding density of 1&#8211;2 x 105 cells/well of a 24-well plate.</li>
		<li>Assay the differentiation efficiency by immunohistochemical (IHC) staining for absence of pluripotency marker, Octamer-binding transcription factor 4 (OCT4) for example, and presence of NPC markers such as paired box 6 (PAX6), Nestin, and sex determining region Y-box transcription factor 1 (Sox1).</li>
		<li>At this stage, detached NPCs can be frozen in the specialized commercial NPC freezing media (see Table of Materials) and stored in liquid nitrogen for up to 3 months. After freeze-and-thaw for once, NPCs still retain the multipotency to give rise to neurons, astrocytes, and OPCs with reliable protocols.</li>
	</ol>
	</li>
	<li>Oligodendrocyte precursor cell (OPC) generation (~7 days). Please see Figure 2A for the flow diagram.
	<ol>
		<li>On Day 7, passage NPCs using cell detachment solution (see Table of Materials) and plate them at a seeding density of 1&#8211;2 x 105 cells per well in a 24-well plate in warm (37 &#176;C) neural induction medium plus SMAD inhibitors from the commercial kit (see Table of Materials).</li>
		<li>On Day 8, prepare a solution of 1% dimethyl sulfoxide (DMSO) in the OPC differentiation medium and treat the plated NPCs for 24 h. The OPC differentiation medium is composed of: DMEM/F12 medium, 1% N2 supplement, 1% B27 supplement, basic fibroblast growth factor (bFGF) at 20 ng/mL, smoothened agonist (SAG) at 1 &#181;M, platelet-derived growth factor-AA (PDGF-AA) at 10 ng/mL (see Table of Materials).</li>
		<li>On Day 9, replace media with fresh OPC differentiation medium without DMSO. Feed the cells every other day until Day 15. If the cells reach confluence before Day 15, passage them to the seeding density of 1&#8211;2 x 105 cells per well as described in step 2.2.1.</li>
		<li>On Day 14, plate OPCs in OPC differentiation medium at a density of 1&#8211;2 x 105 cells/well in a 24-well plate.</li>
		<li>At this stage (Day 15), test cells for the presence of OPC-specific markers by IHC staining or qPCR (e.g., O4, oligodendrocyte lineage transcription factor [Olig]-1/2, Chondroitin sulfate proteoglycan 4/neuron-glial antigen 2 [CSPG4/Ng2[, NK2 Homeobox 2 [NKX2.2], platelet-derived growth factor receptor alpha [PDGFRa]; Figure 2B) and for the absence of NPC markers (Pax6 or Nestin; Figure 2D). We typically detect the O4 immunoreactivity in more than 95% of the cells at Day 15. Of particular relevance to Alzheimer&#8217;s disease, the expression of APP (amyloid precursor protein), BACE1 (the processing protease &#946;-secreatase 1), and peptide amyloid-&#946; (A&#946;) is abundant in OPCs (Figure 2F).</li>
	</ol>
	</li>
</ol>
3. Co-culturing of human induced neurons (iNs) and oligodendrocyte precursor cells (iOPCs)
<ol>
	<li>iOPC plating (~3 days)
	<ol>
		<li>Plate iOPCs at Day 14 at a density of 1 x 105 cells per well in a 24-well plate (as described above in step 2.2.4.) in OPC differentiation medium (as described in step 2.2.2.).</li>
	</ol>
	</li>
	<li>iN-iOPC co-culture set up
	<ol>
		<li>On Day 15, detach the induced human neurons at the step of Day 2 after the Puromycin selection (as described in step 1.2.6.) with cell detachment solution (see Table of Materials).</li>
		<li>Add neurons onto the cultured OPCs, plating at the seeding density of 2 x 105 cells per well in the 24-well plate with growing OPCs (from step 3.1.1). Use the co-culture medium containing Neurobasal-A medium, 2% B27 supplement, and 100 ng/mL T3 triiodothyronine. Change the medium on the next day and then every other day afterwards. If OPCs proliferate too fast and reach confluency in less than 3 days, add cytosine arabinoside (Ara-C) at a concentration of 2&#8211;5 &#181;M. A representative image of the iNs and iOPCs grown in co-culture after 7 days neurons is shown in Figure 3A.</li>
		<li>Use frozen neurons prepared as described above in step 1.2.7 for co-culturing with OPCs. Plate freeze-and-thaw neurons at a higher density of 3 x 105 cells per well.</li>
		<li>After Day 14&#8211;16 in co-cultures, the synapse formation in iNs can be observed by IHC staining of pre- and post-synaptic markers, and by Day 21 the synaptic puncta should be abundant (Figure 3C) and neuronal activities can be reliably recorded.</li>
		<li>Starting at Day 21, test cells for OL specific markers (for example, myelin basic protein [MBP] and proteolipid protein 1 [PLP1]). By Day 28, we normally observe the phenomenon ensheathing of iN axons by iOL processes, labeled by IHC staining for specific markers (Figure 3B; neurofilament [NF] for iN axons and MBP for iOPC processes).</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>ThermoFisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>ThermoFisher</td>
			<td>PHG 0266</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>MilliporeSigma</td>
			<td>A9501</td>
			<td></td>
		</tr>
		<tr>
			<td>Clemastine</td>
			<td>MilliporeSigma</td>
			<td>SML0445</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>STEMCELL Technologies</td>
			<td>36254</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>ThermoFisher</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>MilliporeSigma</td>
			<td>D3072</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ScienCell</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>H1 human ES cells</td>
			<td>WiCell</td>
			<td>WA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR plus</td>
			<td>STEMCELL Technologies</td>
			<td>5825</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>ThermoFisher</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal A medium</td>
			<td>ThermoFisher</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>ThermoFisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-AA</td>
			<td>R&#38;D Systems</td>
			<td>221-AA-010</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>VWR</td>
			<td>71002-812</td>
			<td></td>
		</tr>
		<tr>
			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>MilliporeSigma</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-REV</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>ThermoFisher</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Tocris</td>
			<td>4366</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Neural Progenitor Freezing Media</td>
			<td>STEMCELL Technologies</td>
			<td>5838</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff SMADi Neural Induction Kit</td>
			<td>STEMCELL Technologies</td>
			<td>8581</td>
			<td></td>
		</tr>
		<tr>
			<td>T3 triiodothyronine</td>
			<td>MilliporeSigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Tempo-iOlogo: Human iPSC-derived OPCs</td>
			<td>Tempo BioScience</td>
			<td>SKU102</td>
			<td></td>
		</tr>
		<tr>
			<td>TetO-Ng2-Puro</td>
			<td>Addgene</td>
			<td>52047</td>
			<td></td>
		</tr>
		<tr>
			<td>VSV-G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
	</tbody>
</table>

a co-culture method to model neuron-oligodendrocyte interactions

Source: Assetta, B.,et al. <a href="https://app.jove.com/v/61778">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates the establishment of a co-culture of human induced neurons (iNs) and induced oligodendrocyte precursor cells (iOPCs). iNs are mixed with iOPCs in a co-culture and then incubated to support the maturation of both iNs into neurons and iOPCs into oligodendrocytes. Neurons form synaptic connections with other neurons and oligodendrocytes, while oligodendrocytes develop myelin sheaths around the neurons&#39; axons.

<img alt="Figure 1" src="/files/ftp_upload/22424/22424_Figure1.jpg" /> 
Figure 1: Direct Generation of human induced neurons (iNs) from hPSCs. (A) Flow diagram of iN generation. (B) Representative bright field and immunofluorescence images of the starting culture of human pluripotent stem cells (H1) to confirm the pluripotency. Oct4 is shown in red and Sox2 in green. (C) Representative bright field images of iNs at D...

A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

1. Human neuron induction from human pluripotent stem cells

	Lentivirus preparation (~5 days)
	
		Plate ~1 million Human embryonic kidney 293T (HEK293T) ...

Take human-induced neurons, or iNs, cultured on an extracellular matrix.
Remove the media and add digestive enzymes that disrupt the matrix, dissociating the cells.
Transfer the iNs into a tube. Centrifuge the suspension and discard the supernatant containing the enzymes.
Resuspend the iNs in a co-culture medium; transfer them onto a culture of induced oligodendrocyte precursor cells, or iOPCs.
iOPCs are precursors to oligodendrocytes that form myelin sheaths around the axons of neurons.
Upon incubation, the iOPCs form synaptic connections with the iNs. Trophic factors released by the iOPCs and growth factors in the medium promote iN maturation into neurons, exhibiting increased cellular processes that form synaptic connections with other neurons.
The electrical activity of the mature neurons and growth factors in the medium induces iOPC maturation into oligodendrocytes.
The oligodendrocytes extend cellular processes, forming myelin sheaths around the neuronal axons.
The established co-culture is ready for analysis.

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Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

183 Views. Source: Assetta, B.,et al. Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions. J. Vis. Exp. (2020). This video demonstrates the establishment of a co-culture of human induced neurons (iNs) and induced oligodendrocyte precursor cells (iOPCs). iNs are mixed with iOPCs in a co-culture and then incubated to support the maturation of both iNs into neurons and iOPCs into oligodendrocytes. Neurons form synaptic connections with...

Video: A Co-culture Method to Model Neuron-Oligodendrocyte Interactions - Concept

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

JoVE Journal

Cancer Research

Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

Recent works uncover the neuronal impact on high-grade pediatric glioma (pHGG) cells and their reciprocal interactions. The present work shows the development of an in vitro model co-culturing pHGG cells and glutamatergic neurons and recorded their electrophysiological interactions to mimic those interactivities.

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

Transcript

A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments