a technique for generating neural progenitor cells from mouse embryonic stem cells

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

Add 1.5 million mouse embryonic stem cells into a non-adhesive bacterial dish in 10 milliliters of basal differentiation medium, and incubate the plate at 37 degrees Celsius and 5% carbon dioxide to allow for embryoid body formation.
After two days, transfer the cell aggregates into 15-milliliter centrifuge tubes, and let them settle by gravity. Remove the supernatant and add 10 milliliters of fresh basal differentiation medium to resuspend the embryoid bodies. Then, replant them into a new non-adhesive dish and incubate them for another two days.
Collect and seed about 50 embryoid bodies in 2 milliliters of basal differentiation medium per well onto a gelatin-coated six-well plate. For retinoic acid induction, add 2 microliters of RHA stock into each well to a final concentration of 1 micromolar. Return the plate to the incubator and differentiate the cells for another four days.

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1. Mouse embryonic stem cell culture
<ol>
	<li>Prepare 0.1% gelatin coated cell culture dishes or plates.
	<ol>
		<li>Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in water) to 60 mm cell culture dishes. Rock gently to ensure even coating of the cell culture dishes.</li>
		<li>Put the dishes into a 5% CO2 incubator at 37 &#176;C and allow coating for 1 h.</li>
		<li>Remove the 0.1% gelatin solution before seeding the cells. 
		NOTE: After removing the gelatin, there is no need to dry or wash the coated dishes.</li>
	</ol>
	</li>
	<li>Mouse embryonic stem cells (A2lox and 129) culture
	<ol>
		<li>Incubate mESCs (A2lox and 129) cells in the 0.1% gelatin coated 60 mm cell culture dishes in mESC growth medium at 37 &#176;C in a 5% CO2 incubator, respectively. The mESC growth medium consists of 85% knock-out Dulbecco&#39;s modified Eagle medium/nutrient mixture F-12 (DMEM/F12), 15% Knock-out serum replacement (KSR), 0.1 mM &#946;-mercaptoethanol (2ME), 2 mM GlutaMAX, 1% non-essential amino acid (NEAA), 1% penicillin/streptomycin (P/S), 1000 U/mL leukemia inhibitory factor (LIF), 10 nM CHIR-99021 (GSK-3 inhibitor) and 0.33 nM PD0325901 (MEK inhibitor). 
		CAUTION: &#946;-mercaptoethanol is flammable and has inhalation toxicity. Keep away from fire sources and wear a mask to avoid inhalation when use.</li>
		<li>Change the mESC growth medium daily for better growth of A2lox and 129.</li>
		<li>When the cells reach 80% confluence, remove the medium and add 1 mL of 0.1% trypsin to the dish. Gently rock for 30 s to ensure even cover of trypsin on all cells.</li>
		<li>Leave the cells for about 1 min to trypsinize and then remove the trypsin using a 1 mL pipette.</li>
		<li>Add 2 mL of mESC growth medium to the dish, pipette up and down several times to make a single cell suspension.</li>
		<li>Count the density of the cells in the suspension as accurately as possible using a hemocytometer.</li>
		<li>Divide the cells into 7 groups and induce differentiation using different protocols shown in Table 1.</li>
	</ol>
	</li>
</ol>
2. Differentiation from mESCs to NPCs
<ol>
	<li>Prepare 0.1% gelatin coated cell culture plates or coverslips.
	<ol>
		<li>Before use, prepare 0.1% gelatin coated 6-well plates or coverslips as in step 1.1.</li>
	</ol>
	</li>
	<li>Differentiation (Table 1)
	<ol>
		<li>Add 1.5 x 106 mESCs into a non-adhesive bacterial dish in 10 mL of basal differentiation medium I to allow for embryoid body formation at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>After 2 days, transfer cell aggregates into 15 mL centrifuge tubes and let them settle by gravity.</li>
		<li>Remove the supernatant and add 10 mL of fresh basal differentiation medium I to resuspend the embryoid bodies. Replant them into a new non-adhesive bacterial dish and allow differentiation for another 2 days.</li>
		<li>Check the formation of embryoid bodies under the microscope (Figure 1A).</li>
		<li>Collect embryoid bodies as described in steps 2.2.2-2.2.3. Seed about 50 embryoid bodies in 2 mL of basal differentiation medium I per well onto 0.1% gelatin-coated 6-well plates.</li>
		<li>Prepare 1 mM all-trans retinoic acid (RA) stock (in DMSO) and store away from light in a -80 &#176;C freezer after sub-packaging. 
		NOTE: RA is unstable, and attention should be paid to keeping it away from light and reducing air contact during preparation of RA stock.</li>
		<li>For RA induction, add 2 &#181;L of RA stock into each well to make a final concentration of 1 &#181;M.</li>
		<li>Place the plate into the 5% CO2 incubator at 37 &#176;C and differentiate for another 4 days.</li>
		<li>Change the entire 2 mL of basal differentiation medium I (with 1 &#181;M RA) every 2 days.</li>
	</ol>
	</li>
</ol>
Table 1: Details of the protocol used in differentiation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protocol</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>4-day Embryoid Bodies formation + 4-day RA induction</td>
			<td>Basal differentiation medium: 
			DMEM/F12 +15%FBS + 1%NEAA+0.1mM 2ME+ 1%P/S</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>stored at -20 &#176;C, and protect from light</td>
		</tr>
		<tr>
			<td>CHIR-99021 (CT99021)</td>
			<td>Selleck</td>
			<td>S1263</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>NEST</td>
			<td>801007</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Gibco</td>
			<td>CM0635B</td>
			<td>stored at room temperature</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12</td>
			<td>Gibco</td>
			<td>12660012</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Gibco</td>
			<td>11140076</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1X)</td>
			<td>HyClone</td>
			<td>SH30256.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin/ Streptomycin Solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>PD0325901(Mirdametinib)</td>
			<td>Selleck</td>
			<td>S1036</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>stored at -80 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>Strain 129 Mouse Embryonic Stem Cells</td>
			<td>Cyagen</td>
			<td>MUAES-01001</td>
			<td>Maintained in feeder-free culture system</td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1X) Solution</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>stored at 4 &#176;C and protect from light</td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>NUNC</td>
			<td>339650</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Mao, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
This video demonstrates a method for the in vitro generation of neural progenitor cells (NPCs) from mouse embryonic stem cells (mESCs). The mESCs are cultured in suspension to form embryoid bodies (EBs), which are then guided to differentiate into NPCs via retinoic acid treatment in an adherent culture.

<img alt="Figure 1" src="/files/ftp_upload/22409/22409_Figure1.jpg" /> 
Figure 1: The morphology of the embryoid bodies. (A) Embryoid bodies cultured for 4 days. (B) Embryoid bodies cultured for 2 days.

1. Mouse embryonic stem cell culture

	Prepare 0.1% gelatin coated cell culture dishes or plates.
	
		Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in ...

Take mouse embryonic stem cells, or mESCs, suspended in a differentiation medium, and incubate.
In the suspension culture, mESCs aggregate to form a structure termed embryoid bodies or EBs.
EBs are three-dimensional aggregates that mimic a developing embryo and can be differentiated into neural cells.
Transfer the EBs into a tube and allow them to settle by gravity.
Remove the nutrient-depleted medium; resuspend the EBs in a fresh medium, and transfer them to a culture dish.
Incubate the suspension culture to facilitate the growth of EBs.
Transfer the EBs suspended in the medium onto a multiwell plate. The wells are coated with an adhesion protein, allowing the EBs to attach.
Introduce retinoic acid, which enters the cells and triggers the formation of a transcription complex.
The complex induces gene expression, promoting the differentiation of the cells into neural progenitor cells, or NPCs, which exhibit extended cellular processes.

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Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended effects that extrinsic patterning factors might introduce into human pluripotent stem cell (hPSC) models of brain development or pathophysiology, such as masking disease phenotype. Here, we present two cerebellar differentiation protocols for hPSCs, designed with simpler startup method, fewer patterning factors, and less material requirements than previous protocols. Recently, we developed culture procedures, which generate free-floating 3-dimensional (3D) products consistent with other brain &#34;organoid&#34; protocols, including morphologies relevant to modeling brain development such as sub/ventricular zone- and rhombic lip-like structures. The second uses an adherent, 2D monolayer procedure to complete differentiation, which is shown capable of generating functional cerebellar neurons, as products are positive for cerebellar-associated markers, and exhibit neuron-like calcium influxes. Together, these protocols offer scientists a choice of options suited to different research purposes, as well as a basic model for testing other types of streamlined neural differentiations.

We describe a simplified 3D differentiation protocol for hPSCs, using defined medium and reduced growth factors, capable of generating cell aggregates with early neuroepithelial structures and positive for cerebellar-associated markers, as well as an optional 2D modification for differentiating cells as a monolayer to generate functional neurons.

İsteğe bağlı 2D değişiklik ile akıcı 3D serebellar farklılaşma Protokolü

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended ...

JoVE Journal

Gelişim Biyolojisi

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

Nöral Progenitörlerin ve Nöronların Fare Embriyonik Kök Hücrelerinden Farklılaşması ve Karakterizasyonu

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

Fare embriyonik kök hücre farklılaşma içine kortikal Interneuron Kara filmin tarih öncesi

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

TRANSKRIPT

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells