generating neural progenitors from mouse embryonic stem cells by the hanging drop method

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Begin by setting up the hanging drop cell culture. For a 10-centimeter cell culture plate, adjust the concentration to 500 cells per 20 microliters of differentiation medium. Prepare enough cell suspension for 50 20-microliter droplets. Use a micropipette or a repeater pipette to place 20-microliter droplets of cell suspension onto the lid of a tissue culture plate, making sure that the droplets aren&#39;t too close together to prevent them from merging.
Fill the plate with 5 to 10 milliliters of PBS, and carefully put the lid back on the plate. Incubate the culture in the 37 degrees Celsius incubator. After two days, collect the droplets from the lid, and place them in a 10-centimeter cell culture suspension plate, filled with 10 milliliters of differentiation medium. Then, place the plate on an orbital shaker set to low speed in the incubator. After another two days, centrifuge the cells at 200 times g for three minutes, and remove the supernatant.
To induce embryoid body differentiation into neural progenitor cells, resuspend the cells in differentiation medium with 5-micromolar retinoic acid. Incubate the plate for two days. Then, replace the least half of the medium with fresh medium by tilting the plate and pipetting it out.

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Nanyang Technological University

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

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1. mESC culture
<ol>
	<li>Coat a 10 cm tissue-culture-treated plate with 0.1% gelatin and allow the gelatin to set for at least 15&#8211;30 min before aspirating it out.</li>
	<li>Seed &#947;-irradiated mouse embryonic fibroblasts (MEFs) one day before culturing the mESCs in the pre-warmed mESC medium (Dulbecco&#39;s modified Eagle medium (DMEM) with 15% fetal bovine serum (FBS), non-essential amino acids, &#946;-mercaptoethanol, L-glutamine, penicillin/streptomycin, sodium pyruvate, Leukemia inhibitory factor (LIF), PD0325901 (PD), and CHIR99021 (CH)).</li>
	<li>Allow the &#947;-irradiated MEFs to settle and attach to the plate surface before culturing E14 cells.</li>
	<li>Thaw E14 ESCs in 37 &#176;C water bath and quickly transfer the cells in a 15 cm conical tube with warm mESC medium. Pellet the cells at 200 x&#160;g&#160;for 3 min and remove the supernatant.</li>
	<li>Resuspend the cells in 10 mL of mESC medium and plate the cells on the culture plate containing the &#947;-irradiated MEFs seeded earlier. Incubate the cell culture in a 37 &#176;C incubator under 5% CO2.</li>
	<li>For culture passaging, aspirate the medium and wash the plate with sterile 1x Phosphate-buffered saline (PBS). Add enough 0.05% trypsin to cover the plate surface and incubate at 37 &#176;C for 3 min.</li>
	<li>Neutralize the trypsin with mESC medium and pipette to generate single-cell suspension. Centrifuge the cells at 200 x&#160;g&#160;for 3 min and remove the supernatant.</li>
	<li>Count the cells with a hemocytometer or cell counter and seed about 5.0 x 105&#160;cells in a 10 cm culture plate.</li>
	<li>Resuspend the cells in 10 mL of mESC medium, plate the cells on the gelatin-coated tissue culture plate and incubate cultures as described earlier. 
	NOTE: It is recommended that mESCs be passaged every 2 days to prevent the cells from differentiating in their colonies. Phenol red in the medium functions solely as a pH indicator and depending on the cellular density, it can turn yellowish (more acidic), sooner than 2 days. Hence, it may be necessary to change the medium every day. The &#947;-irradiated MEFs will eventually die off after a couple of passages.</li>
</ol>
2. EB, NPC, and neuron differentiation
<ol>
	<li>Perform culture passaging protocol and count the cells.</li>
	<li>Hanging drop method (day 0)
	<ol>
		<li>For a 10 cm cell culture plate, count roughly 2.5 x 104&#160;cells where 5.0 x 102&#160;cells will be suspended in 20 &#181;L differentiation medium (DMEM with 15% FBS, non-essential amino acids, &#946;-mercaptoethanol, L-glutamine, penicillin/streptomycin, and sodium pyruvate). Roughly fifty 20 &#181;L droplets containing the cells can be plated on one 10 cm plate.</li>
		<li>Aliquot the appropriate number of cells, and then centrifuge the cells at 200 x&#160;g&#160;for 3 min and remove the supernatant.</li>
		<li>Resuspend the cells in the appropriate volume of differentiation medium for a cell density of 5.0 x 102&#160;cells per 20 &#181;L (e.g., 2.5 x 104&#160;cells in 1 mL of differentiation medium).</li>
		<li>Using a micropipette or a repeater pipette, place 20 &#181;L droplets of the cell suspension onto the lid of the tissue culture plate. Make sure that the droplets are not too close to one another to prevent them from merging. 
		NOTE: The droplets can be plated on either a tissue-culture-treated attachment plate or suspension plate as they will be placed on the lid and not the plate itself. For a more feasible and sterile approach, plate the droplets on an attachment tissue-culture plate and transfer them to a suspension plate as described below.</li>
		<li>Fill up the plate with 5&#8211;10 mL of 1x PBS and carefully put the lid back on the plate. Incubate the culture in the 37 &#176;C incubator. 
		NOTE: PBS is added to the culture plate to prevent the droplets from drying up.</li>
	</ol>
	</li>
	<li>On&#160;day 2, use a micropipette to collect the droplets from the lid and place them in a 10 cm cell culture suspension plate filled with 10 mL of differentiation medium. Incubate the culture on an orbital shaker shaking at low speed in the incubator.</li>
	<li>On&#160;day 4, to harvest the EBs, collect the cells, centrifuge at 200 x&#160;g&#160;for 3 min, and remove the supernatant. 
	NOTE: The EBs can also be washed with 1x PBS as per the requirements of subsequent experiments.</li>
	<li>To continue the procedure and induce the EB differentiation into neural progenitor cells (NPCs), prepare the differentiation medium with 5 &#181;M retinoic acid (RA).</li>
	<li>Remove the old medium by pelleting the EBs at 100 x&#160;g&#160;for 3 min or allow the EBs to settle before aspirating out the old medium. Add 10 mL of the differentiation medium containing 5 &#181;M RA to the culture plate.</li>
	<li>On day 6, replace at least half of the medium with fresh medium containing 5 &#181;M RA by tilting the plate and pipetting out the medium as described above. 
	NOTE: It is recommended that at least half of the medium be replaced with fresh RA-containing medium on days 5 and 7. Take note of the phenol red indicator; if it turns yellowish, it is best to replace all of the medium.</li>
	<li>On&#160;day 8, harvest the NPCs by collecting the cells, centrifuging at 200 x&#160;g&#160;for 3 min, and removing the supernatant. 
	NOTE: The NPCs can also be washed with 1x PBS according to the needs of subsequent experiments. If needed, NPCs can be frozen down and thawed again for later culture and analysis. If the NPCs are to be cultured, accutase can also be used as an alternative to trypsin.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin + 0.53mM EDTA 1X</td>
			<td>Corning</td>
			<td>25-052-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>0.1% Gelatin</td>
			<td>Sigma</td>
			<td>G1890-100G</td>
			<td>Prepared in de-ionized water</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Cayman Chemicals</td>
			<td>13122</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-017-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>EB buffer</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>111000200</td>
			<td>Pharmco</td>
			<td>Diluted in de-ionized water</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S10250</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>LIF</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Collected from MEF supernatant</td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids</td>
			<td>Corning</td>
			<td>25-025-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>PD0325901</td>
			<td>Cayman Chemicals</td>
			<td>13034</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Prepared in de-ionized water</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>P217-500G</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic anhydrous</td>
			<td>0781-500G</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>BP358-10</td>
			<td>Fisher Bioreagents</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate, dibasic, heptahydrate</td>
			<td>SX0715-1</td>
			<td>Milipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Prepared in DMSO</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Corning</td>
			<td>25-000-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Fisher BioReagents</td>
			<td>BP176-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Hanafiah, A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61446">Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells.</a> J. Vis. Exp. (2020).
The video demonstrates the differentiation of mouse embryonic stem cells (mESCs) to embryoid bodies (EBs) by the hanging drop method. Upon suspending the mESCs in hanging drops and incubating, the cells aggregate to form EBs, which are further differentiated into neuronal progenitor cells (NPCs) using a differentiation medium supplemented with retinoic acid (RA).

1. mESC culture

	Coat a 10 cm tissue-culture-treated plate with 0.1% gelatin and allow the gelatin to set for at least 15&#8211;30 min before aspirating ...

Take a mouse embryonic stem cell suspension.
Centrifuge, remove the supernatant, and resuspend the cells in a neural differentiation medium.
Place droplets of the suspension on the inner side of a culture plate lid.
Add a buffer to the bottom plate to prevent the droplets from drying. Place the lid on the bottom plate, forming a hanging drop culture, and incubate.
Due to gravity, the cells cluster and form three-dimensional aggregates termed embryoid bodies or EBs.
Place the droplets into a plate containing the differentiation medium and incubate under agitation for the growth of EBs.
Centrifuge and remove the medium; resuspend the EBs in the differentiation medium supplemented with retinoic acid, transfer them to a culture plate, and incubate.
Retinoic acid enters the cells and triggers the formation of a transcription complex. The complex induces gene expression, triggering the differentiation into neural progenitor cells.
The differentiated EBs are ready for downstream applications.

13 Views. Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Video: Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method - Experiment

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

JoVE Journal

Developmental Biology

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.

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

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method

Transcript

Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method