differentiating human induced pluripotent stem cells into neurons on micro-electrode arrays

Differentiating Human Induced Pluripotent Stem Cells into Neurons on Micro-Electrode Arrays

For the six-well MEAs, dilute the cells to obtain a cell suspension of 7.5 x 105 cells per milliliter. Aspirate the diluted laminin from the six-well MEAs. For the six-well MEAs, plate the cells by adding 100 microliters of cell suspension to the active electrode area in each well.
Next, place the six-well MEAs in a humidified 37 degrees Celsius incubator. After two hours, carefully add 500 microliters of the prepared E8 medium to each well of the six-well MEAs. Subsequently, place the six-well MEAs overnight in a humidified 37 degrees Celsius incubator.
On day three, warm 0.05% Trypsin-EDTA to room temperature. Warm DPBS in DMEM/F-12 with 1% penicillin-streptomycin to 37 degrees Celsius. Then, aspirate the spent medium of the rat astrocyte culture. Wash the culture by adding 5 milliliters of DPBS, and swish it around gently.
Afterward, aspirate DPBS and add 5 milliliters of 0.05% Trypsin-EDTA. Swish the Trypsin-EDTA around gently. Then, incubate the cells in a humidified 37 degrees Celsius incubator for 5 to 10 minutes. Subsequently, check under the microscope to examine whether the cells are detached.
Detach the last cells by hitting the flask a few times. Then, add 5 milliliters of DMEM/F-12 to the flask. Triturate the cells gently inside the flask with a 10-milliliter pipette. Afterward, collect the cell suspension in a 15-milliliter tube. Spin the tube at 200 times g for eight minutes.
After that, aspirate the supernatant and resuspend the cells in 1 milliliter of DMEM/F-12. Determine the number of cells per milliliter using a hemocytometer.
Next, add 7.5 x 104 astrocytes to each well of the six-well MEAs, and add 2 x 104 astrocytes to each well of the 24-well plate. Incubate the cells overnight in a humidified 37 degrees Celsius incubator.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Differentiation of rtTA/Ngn2-positive hiPSCs to Neurons on 6-well MEAs and Glass Coverslips
NOTE: In this protocol, the details are provided for differentiating rtTA/Ngn2-positive hiPSCs on two different substrates, i.e. 6-well MEAs (devices composed of 6 independent wells with 9 recording and 1 reference embedded microelectrodes per well) and glass coverslips in the wells of a 24-well plate. The protocols, however, can easily be adapted for larger substrates (e.g., for the wells of 12- or 6-well plates), by scaling up the mentioned values according to the surface area.
<ol>
	<li>Prepare the MEAs or glass coverslips (day 0 and day 1)
	<ol>
		<li>The day before the start of the differentiation, sterilize the MEAs according to the manufacturer&#39;s recommendation.</li>
		<li>Dilute the adhesion protein Poly-L-Ornithine (PLO) in sterile ultrapure water to a final concentration 50 &#181;g/mL. Coat the active electrode area of 6-well MEAs by placing a 100 &#181;L drop of the diluted PLO in each well. Place the coverslips in the wells of the 24-well plate using sterile tweezers. Add 800 &#181;L of the diluted PLO in each well. Prevent the coverslips from floating by pushing them down with the 1000 &#181;L pipette tip.</li>
		<li>Incubate the 6-well MEAs and 24-well plate O/N in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2. The next day, aspirate the diluted PLO. Wash the glass surfaces of the 6-well MEAs and the coverslips twice with sterile ultrapure water.</li>
		<li>Dilute laminin in cold DMEM/F12 to a final concentration of 20 &#181;g/mL (for the 6-well MEAs) and 10 &#181;g/mL (for the glass coverslips). Immediately coat the active electrode area of the 6-well MEAs by placing a 100 &#181;L drop in each well. Similarly, add 400 &#181;L of the diluted laminin in each well of the 24-well plate to coat the coverslips. Prevent the coverslips from floating by pushing them down with the 1,000 &#181;L pipette tip.</li>
		<li>Incubate the 6-well MEAs and 24 well plate in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2 for at least 2 h.</li>
	</ol>
	</li>
	<li>Plate the hiPSCs (day 1) 
	NOTE: The volumes that are mentioned in steps 3.2.1 - 3.2.4 assume that the rtTA/Ngn2-positive hiPSCs are cultured in a 6-well plate and that the cells of one well are harvested. The volumes that are required for plating the cells on the 6-well MEAs and/or the coverslips depends on the number of 6-well MEAs and/or the number of coverslips that are used in the experiment; the numbers specified in steps 3.2.6 - 3.2.8 allow scaling to different experiment sizes.
	<ol>
		<li>Warm DMEM/F12, CDS and E8 medium with 1% (v/v) penicillin/streptomycin to R/T. Add doxycycline to a final concentration of 4 &#181;g/mL and ROCK inhibitor to the E8 medium.</li>
		<li>Aspirate the spent medium of the rtTA/Ngn2-positive hiPSCs and add 1 mL CDS to the hiPSCs. Incubate 3 - 5 min in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2. Check under the microscope whether the cells are detaching from one another.</li>
		<li>Add 2 mL DMEM/F12 in the well, gently suspend the cells with a 1,000 &#181;L pipette and transfer the cells to a 15 mL tube. Add 7 mL DMEM/F12 to the cell suspension. Spin the cells at 200 x g for 5 min.</li>
		<li>Aspirate the supernatant and add 2 mL of the prepared E8 medium. Dissociate the hiPSCs by putting the tip of a 1,000 &#181;L pipette against the side of the 15 mL tube and resuspending the cells gently. Check under the microscope whether the cells are dissociated.</li>
		<li>Determine the number of cells (cells/mL) using a hemocytometer chamber. 
		NOTE: A 6-well plate well at 80 - 90% confluency will typically yield 3.0 - 4.0 x 106 cells in total.</li>
		<li>Aspirate the diluted laminin. For the 6-well MEAs, dilute the cells to obtain a cell suspension of 7.5 x 105 cells/mL. Plate the cells by adding a drop of 100 &#181;L of the cell suspension on the active electrode area in each well of the 6-well MEAs. For the coverslips, dilute the cells to obtain a cell suspension of 4.0 x 104 cells/mL. Plate the cells by adding 500 &#181;L of the cell suspension to the wells of the 24-well plate. 
		NOTE: The final cell density on the MEAs is higher than on the coverslips (Figure 1A and B). We found that this high cell density was required to properly record the network activity. In the protocol, the numbers are provided that were optimal for the assays.</li>
		<li>Place the 6-well MEAs and 24-well plate in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2 for 2 h (MEAs) or O/N (24-well plate).</li>
		<li>After 2 h, carefully add 500 &#181;L of the prepared E8 medium to each well of the 6-well MEAs. Place the 6-well MEAs O/N in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2.</li>
	</ol>
	</li>
	<li>Change the medium (day 2)
	<ol>
		<li>The next day, prepare DMEM/F12 with 1% (v/v) N-2 supplement, 1% (v/v) non-essential amino acids, and 1% (v/v) penicillin/streptomycin. Add human recombinant neurotrophin-3 (NT-3) to a final concentration of 10 ng/mL, human recombinant brain-derived neurotrophic factor (BDNF) to a final concentration of 10 ng/mL, and doxycycline to a final concentration of 4 &#181;g/mL. Warm the medium to 37 &#176;C.</li>
		<li>Add laminin to a final concentration of 0.2 &#181;g/mL to the medium. Filter the resulting medium. Aspirate the spent medium from the wells of the 6-well MEAs and the 24-well plate and replace it with the prepared medium. Incubate the 6-well MEAs and 24-well plate O/N in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2.</li>
	</ol>
	</li>
	<li>Add rat astrocytes (day 3) 
	NOTE: The volumes that are mentioned in this protocol assume that the rat astrocytes are cultured in T75 culture flasks. It is critical that the rat astrocytes that are added to the cultures are of good quality. We use two criteria to check if the rat astrocytes are of good quality. First, the rat astrocyte culture should be able to grow confluent within ten days after the isolation from the rat embryonic brains. Second, after splitting the rat astrocyte culture, the rat astrocytes should be able to form a confluent, tessellated monolayer (Figure 1C). If the rat astrocyte culture does not fulfill these two criteria, we advise not to use this culture for differentiation experiments.
	<ol>
		<li>Warm 0.05% trypsin-EDTA to RT. Warm the DPBS and DMEM/F12 with 1% (v/v) penicillin/streptomycin to 37 &#176;C.</li>
		<li>Aspirate the spent medium of the rat astrocyte culture. Wash the culture by adding 5 mL DPBS and swish it around gently.</li>
		<li>Aspirate the DPBS and add 5 mL 0.05% trypsin-EDTA. Swish the trypsin-EDTA around gently. Incubate in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2 for 5 - 10 min.</li>
		<li>Check under the microscope whether the cells are detached. Detach the last cells by hitting the flask a few times.</li>
		<li>Add 5 mL of DMEM/F12 to the flask. Triturate the cells gently inside the flask with a 10 mL pipette. Collect the cell suspension in a 15 mL tube. Spin the tube at 200 x g for 8 min.</li>
		<li>Aspirate the supernatant and resuspend the cells in 1 mL of DMEM/F12. Determine the number of cells (cells/mL) using a hemocytometer chamber.</li>
		<li>Add 7.5 x 104 astrocytes per well of the 6-well MEAs. Add 2.0 x 104 astrocytes per well of the 24-well plate. Incubate the MEAs and the 24-well plate O/N in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2.</li>
	</ol>
	</li>
	<li>Change the medium (day 4)
	<ol>
		<li>Prepare neurobasal medium with 2% (v/v) B-27 supplement, 1% (v/v) L-alanyl-L-glutamine and 1% (v/v) penicillin/streptomycin. Add NT-3 to a final concentration of 10 ng/mL, BDNF to a final concentration of 10 ng/mL, and doxycycline to a final concentration of 4 &#181;g/mL. In addition, add cytosine &#946;-D-arabinofuranoside to a concentration of 2 &#181;M. 
		&#8203;NOTE: Cytosine &#946;-D-arabinofuranoside is added to the medium to inhibit astrocyte proliferation and to kill the remaining hiPSCs that are not differentiating into neurons.</li>
		<li>Filter the medium and warm to 37 &#176;C. Aspirate the spent medium from the wells of the 6-well MEAs and the 24-well plate and replace it with the prepared medium. Maintain the 6-well MEAs and the 24-well plate in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2.</li>
	</ol>
	</li>
	<li>Refresh the medium (day 6 - 28) 
	NOTE: Starting from day 6, refresh half of the medium every two days. From day 10 onwards, the medium is supplemented with FBS to support the astrocyte viability.
	<ol>
		<li>Prepare neurobasal medium with 2% (v/v) B-27 supplement, 1% (v/v) L-alanyl-L-glutamine, and 1% (v/v) penicillin/streptomycin. Add NT-3 to a final concentration of 10 ng/mL, BDNF to a final concentration of 10 ng/mL, and doxycycline to a final concentration of 4 &#181;g/mL. From day 10 onwards, also supplement the medium with 2.5% (v/v) FBS. Filter the resulting medium and warm it to 37 &#176;C.</li>
		<li>Remove half of the spent medium from the wells of the 6-well MEAs and the 24-well plate using a 1000 &#181;L pipette and replace it with the prepared medium. Maintain the 6-well MEAs and the 24-well plate in a humidified 37 &#176;C incubator with an atmosphere of 5% CO2.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B-27 supplement</td>
			<td>Gibco</td>
			<td>0080085SA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ca2+/Mg2+-free HBSS</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05 % Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 % Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>High-glucose DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrix</td>
			<td>Gibco</td>
			<td>A1413201</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell detachment solution</td>
			<td>Sigma-Aldrich</td>
			<td>A6964</td>
			<td></td>
		</tr>
		<tr>
			<td>E8 medium</td>
			<td>Gibco</td>
			<td>A1517001</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor</td>
			<td>Gibco</td>
			<td>A2644501</td>
			<td>Alternatively, ROCK inhibitors like thiazovivin can be used.</td>
		</tr>
		<tr>
			<td>6-well MEAs</td>
			<td>Multi Channel Systems</td>
			<td>60-6wellMEA200/30iR-Ti-tcr</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>VWR</td>
			<td>631-0899</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>Sigma-Aldrich</td>
			<td>P3655-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycyclin</td>
			<td>Sigma-Aldrich</td>
			<td>D9891-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>NT-3, human recombinant</td>
			<td>Promokine</td>
			<td>C66425</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF, human recombinant</td>
			<td>Promokine</td>
			<td>C66212</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-arabinofuranoside</td>
			<td>Sigma-Aldrich</td>
			<td>C1768-100MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Frega, M., et al., <a href="https://app.jove.com/v/54900">Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.</a> J. Vis. Exp. (2017)
The video demonstrates a method for generating excitatory cortical neurons from human-induced pluripotent stem cells (hiPSCs). Upon doxycycline induction, the hiPSCs overexpress neurogenin-2, a neural-specific transcription factor. By using specific media and co-culturing with astrocytes, this process facilitates the hiPSCs&#39; differentiation into neurons with synaptic connections.

<img alt="Figure 1" src="/files/ftp_upload/22359/22359_Figure1.jpg" /> 
Figure 1: hiPSC Differentiation into Neurons. A. Three time points of hiPSCs differentiation into neurons on coverslips. B. Plating of hiPSCs on MEAs. C. Astrocytes at 100% confluency in T75 flask (note that the cells form a tessellated monolayer). Scale bars: 150 &#181;m.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Differentiation of ...

Take transduced human-induced pluripotent stem cells or hiPSCs, overexpressing neurogenin-2, driven by the antibiotic doxycycline-inducible system.&#160;
Transfer them to the adhesion protein-coated active electrode areas in the wells of a multi-well microelectrode array.
Allow hiPSCs to attach to the coated active electrode areas.
Add media supplemented with doxycycline, which induces neurogenin-2 overexpression, a neural-specific transcription factor.
Neurogenin-2 triggers molecular pathways, and leads to hiPSC differentiation into neural precursor cells, while media constituents aid cell survival.
Introduce astrocytes to the micro-electrode array wells, where they adhere to the electrode&#39;s active area.
Replace media with neurobasal media containing neurogenic growth factors, doxycycline, and a cell growth inhibitor.
The inhibitor prevents astrocyte proliferation and eliminates undifferentiated hiPSCs.
Over time, sustained neurogenin-2 expression and growth factors drive neural precursor cell differentiation into neurons.
Further, replace media with neurobasal media containing serum for astrocyte survival.
Astrocytes secrete growth factors promoting neuron maturation and synapse formation, forming a neuronal network.

19 ビュー. ヒト人工多能性幹細胞の微小電極アレイ上のニューロンへの分化

ビデオ: ヒト人工多能性幹細胞の微小電極アレイ上のニューロンへの分化 - 実験

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

共培養系における神経結合の形成を評価するための光遺伝学的アプローチ

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

JoVE Journal

発生生物学

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

ヒト人工多能性幹細胞由来の錐体ニューロンからの樹状突起棘の三次元定量

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

領域特異的ヒト多能性幹細胞由来アストロサイトおよびニューロンを用いたALSモデリングのための電気生理学的基盤の確立

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

Differentiating Human Induced Pluripotent Stem Cells into Neurons on Micro-Electrode Arrays

記録

Differentiating Human Induced Pluripotent Stem Cells into Neurons on Micro-Electrode Arrays