eoe sub articles

EoE Sub Articles

1. Generation of Induced Pluripotent Stem Cells
<ol>
	<li>Culture human fibroblasts in DMEM supplemented with 10% fetal bovine serum (FBS) in 6-well plates.</li>
	<li>Prior to commencing reprogramming of fibroblasts, coat 6-wells with Matrigel and incubate for at least an hour at room temperature. At 80% confluence, remove media from fibroblast cultures and wash once with 1x phosphate-buffered saline (PBS).</li>
	<li>Add sufficient 0.25% trypsin-EDTA to cover entire plate and incubate at 37 &#176;C for 10 min.</li>
	<li>Once cells have dislodged from wells, add twice the amount of DMEM with 10% FBS as that of trypsin to quench trypsin digestion. Gently pipette cell suspension up and down to break up cells. Count the number of single cells with a hemocytometer.</li>
	<li>Transfer 7 x 105 cells into a 15 ml centrifuge tube and centrifuge the cell suspension at 200 x g for 5 min.</li>
	<li>Remove supernatant and resuspend cell pellet in electroporation solution. Electroporate the fibroblasts according to manufacturer&#39;s instructions. Resuspend cells in 100 &#181;l of electroporation buffer and add 1 &#181;g of each episomal vector. Electroporate cells with settings of 1,650 V, 10 ms and 3 time pulses.</li>
	<li>Transfer cell suspension into Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% FBS and plate 5 x 104 cells per well of a 6-well plate. Incubate plates at 37 &#176;C and 5% CO2.</li>
	<li>After 48 hr, remove media from transfected fibroblast cultures and replace with mTeSR medium. Replace culture media daily until induced pluripotent stem cell (iPSC) colonies are ready to be isolated. iPSC colonies can be observed after 28-35 days.</li>
	<li>When ready for isolation, mechanically cut an iPSC colony and reseed in mTeSR medium with 10 &#181;M Rho-associated protein kinase (ROCK) inhibitor (Y-27632) onto a Matrigel coated 6-well plate. Replace culture medium daily.</li>
</ol>
2. Neural Induction and Maintenance of Human NPCs
<ol>
	<li>When iPSC cultures are around 20% confluence, remove mTeSR medium and replace with neural induction medium. Replenish medium every second day.</li>
	<li>After 7 days, remove neural induction medium and replace with human NPC maintenance medium. Replenish medium every second day up to 7 days before passaging cultures.</li>
	<li>Prior to passaging cultures, coat plates/flasks with Matrigel at room temperature for at least an hr.</li>
	<li>Remove media from confluent human NPC cultures and wash once with 1x PBS then aspirate PBS off.</li>
	<li>Add enough cell detachment solution to cover all wells then incubate for 5 min at 37 &#176;C.</li>
	<li>Ensure that all cells have detached. Add twice the amount of DMEM/F-12 (Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12) as that of cell detachment solution to the wells/flasks.</li>
	<li>Transfer DMEM/F-12 containing cells into a 15 ml centrifuge tube and centrifuge at 200 x g for 5 min.</li>
	<li>Remove supernatant and resuspend cells in 5 ml human NPC maintenance medium. Gently pipette cell suspension up and down to break up cells.</li>
	<li>Plate a third of total cells into Matrigel coated culture wells/flasks and top up sufficient medium supplemented with 10 &#181;M Y-27632. Incubate culture vessels at 37 &#176;C and 5% CO2.
	<ol>
		<li>Split cultures 1:3 every 3 to 4 days and replenish medium every other day. Maintain human NPC cultures at high density to prevent differentiation.</li>
	</ol>
	</li>
</ol>
3. Differentiation of Human NPCs into Neurons in Co-cultures
<ol>
	<li>Add the lentiviral vector Synapsin1-tdTomato to human NPC culture before initiating differentiation into neurons. Prepare astrocyte/cortical neuron co-cultures a day in advance before differentiating human NPCs.</li>
	<li>Wash human NPC cultures once with 1x PBS and add enough cell detachment solution to cover all wells/flasks then incubate for 5 min at 37 &#176;C.</li>
	<li>Ensure that all cells have detached. Add twice the amount of DMEM/F-12 as that of cell detachment solution to the wells/flasks. Transfer into a 15 ml centrifuge tube. Centrifuge at 200 x g for 5 min.</li>
	<li>Remove supernatant and resuspend cells in 5 ml neuronal differentiation medium. Gently pipette cell suspension up and down to break up cell pellet into single cells. Count the number of single cells with a hemocytometer.</li>
	<li>Carefully aspirate medium from astrocyte/cortical neuron cultures. Plate human NPCs at 5 x 103 cells/cm2 in 500 &#181;l of neuronal differentiation medium supplemented with 10 &#181;M Y-27632 for each 24-well. Gently add medium containing human NPCs into each well to avoid disturbing the astrocytes and cortical neurons.</li>
	<li>Incubate plates at 37 &#176;C and 5% CO2. Replace half of the medium with fresh medium every two to three days for at least 28 days.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Epi5 Episomal iPSC Reprogramming Kit</td>
			<td>Invitrogen</td>
			<td>A15960</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Lonza</td>
			<td>12719F</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo Scientific</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>BD Biosciences&#160;</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube</td>
			<td>BD Biosciences</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>Corning</td>
			<td>9761146</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>720083</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 flasks</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System&#160;</td>
			<td>Invitrogen</td>
			<td>MPK5000</td>
			<td>For electroporation of human fibroblasts</td>
		</tr>
		<tr>
			<td>Normal Dermal Human Fibroblasts</td>
			<td>PromoCell</td>
			<td>C12300</td>
			<td></td>
		</tr>
		<tr>
			<td>pLenti-Synapsin-hChR2(H134R)-EYFP-WPRE</td>
			<td>Addgene</td>
			<td>20945</td>
			<td></td>
		</tr>
	</tbody>
</table>

a co-culture technique for differentiating human neural progenitor cells into neurons

Source: Su, C. T. E., et al. <a href="https://app.jove.com/v/52408">An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System.</a> J. Vis. Exp. (2015).
This video demonstrates a method to differentiate human neural progenitor cells (NPCs) into neurons. The NPCs are introduced onto an established mouse astrocyte-rat cortical neuron co-culture, promoting NPC differentiation into neurons. The differentiation is confirmed by visualizing the cells under a confocal microscope.

A Co-culture Technique for Differentiating Human Neural Progenitor Cells into Neurons

1. Generation of Induced Pluripotent Stem Cells

	Culture human fibroblasts in DMEM supplemented with 10% fetal bovine serum (FBS) in 6-well plates.
	...

Take a culture of human neural progenitor cells, or NPCs, supported on an extracellular matrix.
The NPCs are engineered to express a fluorescent protein.
Discard the medium and wash the cells to remove any residual medium.
Incubate the culture with enzymes that disrupt the matrix, dissociating the cells.
Transfer the detached cells into a tube and add a medium to neutralize the enzymatic action.
Centrifuge to pellet the cells, discard the supernatant, and resuspend the NPCs in a neuronal differentiation medium.
Transfer the NPCs onto an established co-culture of mouse astrocytes and rat cortical neurons.
Growth factors released by the established co-culture, along with the cell-cell contacts of the NPCs with the cortical neurons and astrocytes, promote differentiation of the NPCs into mature neurons.
The mature neurons exhibit extended cellular processes and form synaptic connections.
Under a confocal microscope, use the signal from the fluorescent proteins to visualize the human NPC-derived neurons.

a-co-culture-technique-for-differentiating-human-neural-progenitor

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

65 Views. Fonte: Su, C. T. E., et al. Un approccio optogenetico per valutare la formazione di connessioni neuronali in un sistema di co-coltura. J. Vis. Exp. (2015). Questo video dimostra un metodo per differenziare le cellule progenitrici neurali umane (NPC) in neuroni. Le NPC vengono introdotte in una co-coltura di neuroni corticali di topo e ratto, promuovendo la differenziazione delle NPC nei neuroni. La differenziazione è confermata dalla visualizzazione delle cellule al microscopio confocale. 

Video: Una tecnica di co-coltura per differenziare le cellule progenitrici neurali umane in neuroni - 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.

Co-coltura di cellule simili a staminali di glioblastoma su neuroni modellati per studiare la migrazione e le interazioni cellulari

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

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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.

Co-coltura di neuroni glutamatergici e cellule di glioma pediatrico di alto grado in dispositivi microfluidici per valutare le interazioni elettriche

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

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

A Co-culture Technique for Differentiating Human Neural Progenitor Cells into Neurons

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A Co-culture Technique for Differentiating Human Neural Progenitor Cells into Neurons