generating neural progenitors from embryonic stem cells in serum-free monolayer cultures

Generating Neural Progenitors from Embryonic Stem Cells in Serum-Free Monolayer Cultures

Mix the gelatin with 500 milliliters of warm PBS and cover the bottom of the cell culture container with just enough of the solution to coat the surface. While the gelatin is coating, rinse the subconfluent culture of mouse embryonic stem cells two times in PBS. Then, add 1 milliliter of dissociation reagent to the culture.
After two to five minutes, tap the vessel to dislodge the monolayer into a single-cell suspension and collect the culture in a total of 10 milliliters of medium and cell dissociation reagent.
Transfer the cells into a 15-milliliter conical tube and spin them down in a centrifuge. Then, after careful aspiration of the supernatant, resuspend the pellet in 10 milliliters of pre-warmed N2B27 pipetting against the side of the tube to avoid creating bubbles.
Now, count the cells recording the concentration and resuspend them at the appropriate number of cells per unit of volume per well in pre-warmed N2B27 according to the table. Then, aspirate the gelatin from the culture vessel and plate the cells without swirling the container.
Place the cultures in a humid incubator at 37 degrees Celsius and 5% carbon dioxide, replacing the medium every one to two days with fresh N2B27.

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1. Media Preparation
NOTE: The protocol relies on using a mix of two separate media: DMEM/F12 supplemented with modified N2 supplement and Neurobasal supplemented with B27 supplement, typically in a 1:1 ratio.
<ol>
	<li>Prepare the modified N2 supplement by mixing the components in a 15 ml tube. Do not vortex or filter this; mix by inverting the tube until the solution is clear.
	<ol>
		<li>Start by pipetting 7.2 ml of DMEM/F12, then add 1 ml of 25 mg/ml insulin (made up in 0.01 M HCl) and mix well by inverting the tube. It takes a couple of minutes until the solution is clear.</li>
		<li>Add 1 ml of 100 mg/ml apo-transferrin (made up in water), 33 &#181;l of 0.6 mg/ml progesternone (made up in ethanol), 100 &#181;l of 160 mg/ml (1 M) putrescine (made up in water), 10 &#181;l of 3 mM sodium selenite (made up in water) and 666 &#181;l of 7.5% bovine serum albumin and mix the tube well. Aliquot into 2.5 ml and store at -20 &#176;C for up to 2 months.</li>
	</ol>
	</li>
	<li>Prepare the media.
	<ol>
		<li>To 250 ml of DMEM/F12 add 2.5 ml of N2. Mix well but do not shake or filter.</li>
		<li>To 250 ml of Neurobasal media, add 5 ml of B27 supplement. Mix well but do not shake or filter.</li>
		<li>Mix the media from steps 1.2.1 and 1.2.2 in a 1:1 ratio. In some cases a 1:3 mix may be required (supplemented as the 1:1 mix in step 1.2.4).</li>
		<li>To the mix add 0.5 ml of L-glutamine (final concentration 0.2 mM) and 3.5 &#181;l of 2-mercaptoethanol (final concentration 0.1 mM) and mix well without shaking. Store the media at 4 &#176;C for up to 3 weeks and avoid exposing to light. This final mix is called N2B27.</li>
	</ol>
	</li>
	<li>Prepare the gelatin solution.
	<ol>
		<li>Prepare a 1% gelatin solution in ultrapure water and autoclave at 121 &#176;C, 15 psi, for 15 min. It is important that the bottle used for this is completely clean from detergents or disinfectants, so it is recommended that a new bottle is used for this and is only ever rinsed in ultrapure water between uses. Aliquot the 1% solution into 50 ml aliquots and keep at 4 &#176;C for months.</li>
		<li>Warm an aliquot of 1% gelatin in a 37 &#176;C water bath until dissolved and add to 500 ml of warm phosphate-buffered saline (PBS). Mix well and pipet enough to cover the bottom of each well or plate. Allow the gelatin to coat the surface for at least 30 min.</li>
	</ol>
	</li>
</ol>
2. Plating the Cells
NOTE: This protocol applies equally to mouse ESC grown in 10% serum with leukemia inhibitory factor (LIF), serum replacement with LIF or serum-free media with LIF and bone morphogenetic protein 4 (BMP4) or MEK and GSK3 inhibitors (2i media) with or without LIF. However, the timing and efficiency of the differentiation may vary depending on the media and cells. For the experiments shown here we used the 46C mouse ESC line (with an EGFP reporter knocked into one of the endogenous Sox1 alleles), grown in GMEM with 10% serum and LIF. For optimal results, it is important that cells are dissociated and replated in the N2B27 media; simply changing of media from GMEM/serum/LIF to N2B27 always results in a reduced differentiation efficiency compared to replating the cells.
<ol>
	<li>Grow the cells in GMEM with 10% serum, 1 mM sodium pyruvate, 1x non-essential amino acids, 0.1 M 2-mercaptoethanol, and 100 units/ml LIF13. To get a subconfluent culture of mouse ESC, plate 1 x 106 cells into a T25 culture flask which will take 2 - 3 days.</li>
	<li>Observe cells under a bright-field microscope. When the cells are subconfluent and ready to passage, rinse them twice in PBS. Add 1 ml of cell dissociation reagent and return to the incubator for 2 - 5 min. Although trypsin and other enzymes can also be used for cell dissociation, they can negatively impact cell attachment, so the plating densities will need to be adjusted.</li>
	<li>Once the cells begin to lift from the plate, tap the vessel to dislodge them into a single cell suspension. Collect the cells into 10 ml of media and cell dissociation reagent and pipet into a 15 ml centrifuge tube.</li>
	<li>Spin the cells at 300 x g for 3 min at RT.</li>
	<li>Carefully aspirate the supernatant and resuspend the pellet in 10 ml of pre-warmed N2B27 by pipetting up and down 3 - 5 times. When pipetting the suspension down, pipet against the side of the tube to avoid creating bubbles. Count the cells with an automated cell counter or a hemocytometer and record the concentration.</li>
	<li>Prepare a cell suspension containing the desired number of cells per unit of volume to be plated per well in pre-warmed N2B27. A density of 10,500 cells per cm2 in a 6-well dish (i.e., 1 x 105 cells per well of a 6-well dish) is optimal. See Table 1 for the suggested number of cells per cm2 and plating media volumes for various cell culture vessels.</li>
	<li>Aspirate the gelatin from the culture vessel and plate the cell suspension according to the suggested density and volume in Table 1. Do not swirl the plates, as this will concentrate the cells to the center. Place the cultures in a humid incubator at 37 &#176;C, 5% CO2.</li>
	<li>Change media every 1 - 2 days by replacing all the media with fresh N2B27. Pipette gently as flushing the cells might affect the density and lessen differentiation efficiency in the following days. Where initial viability after plating is poor, plate the cells in a 1:3 mix of the N2 and B27-supplemented media and change this to N2B27 after the first two days. Cells will begin to differentiate and should show Sox1 expression (visible by green fluorescence of the reporter in the 46C cell line used here) within 4 - 6 days.</li>
</ol>
Table 1. Suggested plating densities and media volumes for different vessel sizes. The plating densities in this table were determined using the 46C cell line. For optimal results, the plating density of each individual line may have to be adjusted.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Platform</td>
			<td>Range of effective density (cells/cm2)</td>
			<td>Range of cell number to plate</td>
			<td>Suggested initial media volume (&#181;l)</td>
			<td>Suggested media volume after day 1 (&#181;l)</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>10,500 - 36,500</td>
			<td>99,750 - 346,750</td>
			<td>1,000</td>
			<td>2,000</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>15,600 - 46,800</td>
			<td>29,640 - 88,920</td>
			<td>500</td>
			<td>1,000</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>103,500 - 260,000</td>
			<td>33,120 - 83,200</td>
			<td>100</td>
			<td>200</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11320-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement, serum free</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td>Reconstitute with sterile 0.01M HCl</td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>Reconstitute with ethanol</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V</td>
			<td>Life Technologies</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td>Make sure it is completely dissolved before use as glutamine is usually sedimented</td>
		</tr>
		<tr>
			<td>Gelatine</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>GMEM</td>
			<td>Life Technologies</td>
			<td>11710-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>25cm2 tissue culture flask</td>
			<td>Thermo Scientific</td>
			<td>156367</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Wongpaiboonwattana, W., et al. <a href="https://app.jove.com/v/52823">Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture.</a> J. Vis. Exp.(2015)
The video demonstrates a protocol that uses serum-free media to generate neural progenitors from mouse embryonic stem cells. The desired concentration of embryonic stem cells, suspended in serum-free media, is plated on gelatin-coated coverslips. At an optimal cell density, cells produce autocrine signaling factors, which, along with the neural differentiation-inducing factors in the serum-free media, trigger the differentiation of embryonic stem cells into neural progenitors.

1. Media Preparation
NOTE: The protocol relies on using a mix of two separate media: DMEM/F12 supplemented with modified N2 supplement and Neurobasal ...

Begin with a culture of mouse embryonic stem cells.
Wash the cells with a buffer to remove media and cell debris.
Add an enzyme cocktail to detach cells from the flask surface.
Add media to stop the enzymatic activity and collect the cells in a tube. Centrifuge and discard the supernatant containing enzymes.
Resuspend the cells in serum-free neuronal culture media.&#160;
Seed the cells onto gelatin-coated multi-well plate wells.
Incubate. The embryonic stem cells adhere to the gelatin-coated surface. Media constituents, including nutrients, growth factors, and hormones, facilitate cell proliferation and form a monolayer.
At this cell density, the embryonic stem cells produce and release optimal levels of growth factors and cytokines.
These molecules trigger intracellular signaling pathways, facilitating the differentiation of embryonic stem cells into neural progenitors.

10 ビュー. 無血清単層培養における胚性幹細胞からの神経前駆細胞の生成

ビデオ: 無血清単層培養における胚性幹細胞からの神経前駆細胞の生成 - 実験

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

マイクロ電極アレイ上のネットワーク・アクティビティを測定するための人工多能性幹細胞の急速な神経分化

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

JoVE Journal

発生生物学

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

ニューロン回路の再学習：速い神経突起伸長と機能的ニューロン結合の新しい方法

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

神経科学

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.

インビトロにおけるマウス胚性幹細胞からの神経細胞分化

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 Embryonic Stem Cells in Serum-Free Monolayer Cultures

記録

Generating Neural Progenitors from Embryonic Stem Cells in Serum-Free Monolayer Cultures