differentiating mouse embryonic stem cells into a networked neuron population

Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population

Differentiation of ESCs into neurons starts during a routine cell passage. Dissociate the ESCs as before, but include an additional plate for neuronal differentiation. Transfer 350 microliters of the cell suspension into a 10-centimeter low-attachment dish containing 25 milliliters of differentiation medium.
Place the dish on an orbital shaker set to 30 to 45 RPM inside a tissue culture incubator at 37 degrees Celsius and 5% carbon dioxide, and incubate for 48 hours. After 48 hours, use a 25-milliliter pipette to transfer the differentiating cell aggregates to a 50-milliliter conical tube.
Immediately after, add 25 milliliters of fresh differentiation medium to the Petri dish. Allow the aggregates to settle for two to five minutes, producing a visible pellet that is one to two millimeters deep. Carefully aspirate the medium, ignoring any single cells or small aggregates still in suspension, and use a P1000 pipette to transfer the cell pellet back to the Petri dish. Return the dish to the rotary shaker in the incubator. After a further 48 hours, repeat the media change procedure. This time, add 30 milliliters of differentiation medium supplemented with six micromolar all-trans retinoic acid.
The pellet formed will now be two to four millimeters deep. After incubating the cells as before, repeat the media change procedure with retinoic acid supplementation for the final time. The pellet will be four to eight millimeters deep at this point.
The following day, prepare the plating surfaces as outlined in the written portion of the protocol. On the afternoon of the day of plating, thaw 5 milliliters each of pre-aliquoted NPC trypsinization medium and 0.1% soybean trypsin inhibitor at 37 degrees Celsius. Then, use a 25-milliliter pipette to transfer differentiating aggregates from the culture plate to a 50-milliliter conical tube.
Allow the aggregates to settle for three to five minutes, and then carefully aspirate the medium without disturbing the pellet. Next, wash the pellet with 5 to 10 milliliters of PBS, and after aggregates have settled, aspirate the PBS and repeat the wash. After the second PBS wash, add 5 milliliters of NPC trypsinization medium to the pellet and incubate at 37 degrees Celsius for five minutes, gently flicking the tube two to three times during the incubation.
Next, add 5 milliliters of 0.1% soybean trypsin inhibitor to the cells and quickly mix by inverting. Then, gently triturate the cells 10 to 15 times with a 10-milliliter pipette until a relatively homogeneous cell suspension is produced. Slowly filter the cell suspension through a 40 or 70-micron cell strainer placed on top of a 50-milliliter conical tube.
Then, once all of the suspension has been filtered, add 1 milliliter of N2 medium to the filter to wash the remaining cells through the strainer. Transfer the cell suspension to a 15-milliliter conical tube, and centrifuge for six minutes at 200 times g.
After aspirating the medium, without disturbing the pellet, triturate in 2 milliliters of N2 medium, then add N2 medium to a total volume of 10 milliliters. Centrifuge for five minutes at 200 times g. Aspirate the media and repeat the pellet washing process, again, resuspending the pellet in 10 milliliters of N2 medium.
Remove an aliquot of the cells, and count with a hemocytometer, and then repeat the centrifugation. Resuspend the cells in N2 medium to a final concentration of 1x times 107 cells per milliliter, and plate at a density of 150,000 to 200,000 cells per centimeter squared in dishes prepared at DIV minus 1, and place in the tissue culture incubator at 37 degrees Celsius. Maintain ESNs according to the instructions in the written protocol.

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1. Neuronal Differentiation
NOTE: Conduct steps 1.1 - 1.5 before noon and step 1.7 after noon. An overview of the differentiation procedures is presented in&#160;Figure 1A.
<ol>
	<li>Transfer 350 &#181;l (3.5 x 106) of ESCs to a 10 cm low-attachment dish containing 25 ml differentiation medium. Place on an orbital shaker set to 30 - 45 rpm inside a tissue culture incubator at 37 &#176;C with 5% CO2. This is the first day of differentiation, termed day&#160;in vitro&#160;(DIV) -8. 
	NOTE: The use of an ultra-low attachment dish increases the cost of the method but produces slightly larger yields than bacterial Petri dishes since aggregates can occasionally adhere to Petri dishes. If different dish sizes are preferred, medium volumes and cell numbers can be scaled accordingly.</li>
	<li>After 48 hr (DIV -6), use a 25 ml pipet to transfer the differentiating cell aggregates to a 50 ml conical tube. Immediately add a fresh 25 ml of differentiation medium to the Petri dish.</li>
	<li>Allow aggregates to settle over 2 - 5 min, producing a visible pellet that is 1 - 2 mm deep. Ignore single cells or small aggregates remaining in suspension. Carefully aspirate the medium and transfer the cell pellet back to the petri dish using a P1000. Place on rotary shaker in a tissue culture incubator.</li>
	<li>At DIV -4, repeat step 1.2. The pellet will be 2 - 4 mm deep. Replace 30 ml differentiation medium supplemented with 6 &#181;M all-trans retinoic acid (RA) to the petri dish. Return to the rotary shaker in the tissue culture incubator for an additional 48 hr.</li>
	<li>At DIV -2, repeat step 1.3. The pellet will be 4 - 8 mm deep at this point.</li>
	<li>At DIV -1, prepare plating surfaces as in Section 2.</li>
	<li>At DIV 0, thaw 5 ml of pre-aliquoted and frozen NPC trypsinization medium at 37 &#176;C for 5 - 10 min and place in a tissue culture hood. Using a 25 ml pipet, transfer differentiating aggregates to a 50 ml conical tube. Allow aggregates to settle and carefully aspirate the medium. Wash the pellet twice with 10 ml PBS, allowing the aggregates to settle between washes.</li>
	<li>After the second PBS wash, add 5 ml of NPC trypsinization medium to the pellet and incubate at 37 &#176;C for 5 min. Gently flick the tube after 2.5 min.</li>
	<li>Add 5 ml of 0.1% soybean trypsin inhibitor (STI) to inactivate trypsin and mix by inverting. Gently triturate 10 - 15 times with a 10 ml serological pipet until a relatively homogenous cell suspension is produced.</li>
	<li>Slowly transfer the cell suspension to a 40 &#181;m or 70 &#181;m cell strainer placed on the top of a 50 ml conical tube. Once all the suspension has been filtered, add 1 ml of N2 medium to wash the remaining cells through the filter and pellet the dissociated cell suspension for 6 min at 200 x g.</li>
	<li>Aspirate medium without disturbing the pellet. Wash cells twice with 10 ml N2 medium, pelleting cells for 5 min at 200 x g and triturating between washes with a P1000. Prior to the second wash, count cells using a hemocytometer.</li>
	<li>Resuspend cells in N2 medium at 1 x 107 cells per ml and plate ESNs at a cell density of 150,000 - 200,000 cells/cm2.</li>
	<li>Transfer newly plated ESNs to a humidified tissue culture incubator at 37 &#176;C and 5% CO2 and maintain as in Section 3.</li>
</ol>
2. Preparing Culture Surfaces for Plating Neural Precursors at DIV 0
<ol>
	<li>Prepare tissue culture-treated dishes at least 1 day prior to plating. Add sufficient polyethyleneimine (PEI; 25 &#181;g/ml in sterile H2O) or poly-D-lysine (PDL; 100 &#181;g/ml in sterile H2O) to cover tissue-culture-treated plastic dishes and incubate O/N at 37 &#176;C.</li>
	<li>On the morning of plating, wash dishes twice with double-distilled H2O and once with PBS. After the final wash, add sufficient N2 medium to cover the dish (e.g.,&#160;1 ml per well of a 12-well dish or 4 ml per 6 cm dish).</li>
	<li>Prepare 18 mm glass coverslips at least one day prior to neuron plating. Clean the coverslips by plasma-cleaning for 4 min.</li>
	<li>Immediately transfer cleaned coverslips to an ethanol-washed parafilm in the bottom of a large sterile dish and add 400 &#181;l of PEI or PDL solution, prepared as in step 2.1. Incubate O/N at 37 &#176;C in a tissue culture incubator.</li>
	<li>In the morning, wash coverslips three times with water and add 5 &#181;g/ml laminin in PBS for 1 - 3 hr at 37 &#176;C. Prior to dissociating neurons, aspirate the laminin and immediately transfer the coverslip to a well of a 12-well dish containing 1 ml of NPC medium, being sure to keep the treated side facing up.</li>
	<li>Store dishes and coverslips at 37 &#176;C until NPCs are ready to plate.</li>
</ol>
3. Maintenance of Neurons
<ol>
	<li>At DIV 1, aspirate medium and replace with N2 culture medium.</li>
	<li>At DIV 2 and 4, aspirate medium and replace with B27 culture medium.</li>
	<li>At DIV 8, aspirate medium and replace with B27 culture medium containing mitotic inhibitors to eliminate contaminating non-neuronal cells.</li>
	<li>At DIV 12, replace with B27 culture medium.</li>
	<li>Do not remove DIV 12+&#160;ESNs from 5% CO2&#160;until ready to use.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Knockout DMEM</td>
			<td>Life Technologies</td>
			<td>10829-018</td>
			<td>Media: ESC culture medium Formulation and notes: 500 mL</td>
		</tr>
		<tr>
			<td>100x MEM NEAA</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td>Media: store at 4 &#176;C for up to 1 month Formulation and notes: 6 mL</td>
		</tr>
		<tr>
			<td>200 mM L-Alanyl-L-Glutamine</td>
			<td>ATCC</td>
			<td>30-2115</td>
			<td>6 mL</td>
		</tr>
		<tr>
			<td>ES qualified FBS</td>
			<td>Applied Stem Cell</td>
			<td>ASM-5007</td>
			<td>90 mL</td>
		</tr>
		<tr>
			<td>100x antibiotics</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td>55 mM 2-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
			<td>1.1 mL</td>
		</tr>
		<tr>
			<td>107&#160;Units/mL LIF</td>
			<td>Millipore</td>
			<td>ESG1107</td>
			<td>60 &#956;L</td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Life Technologies</td>
			<td>10829-018</td>
			<td>Media: ESC differentiation medium Formulation and notes: 436.6 mL</td>
		</tr>
		<tr>
			<td>100x MEM NEAA</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td>Media: store at 4 &#176;C for up to 1 month Formulation and notes: 5 mL</td>
		</tr>
		<tr>
			<td>200 mM L-Alanyl-L-Glutamine</td>
			<td>ATCC</td>
			<td>30-2115</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>ESC-qualified serum</td>
			<td>Applied Stem Cell</td>
			<td>ASM-5007</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>100x antibiotics</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>2.5 mL</td>
		</tr>
		<tr>
			<td>55 mM 2-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
			<td>0.9 mL</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Media: NPC trypsinization medium Formulation and notes: 100 mL</td>
		</tr>
		<tr>
			<td>0.5 M EDTA (18.3%)</td>
			<td>Sigma-Aldrich</td>
			<td>3690</td>
			<td>Media: freeze in 5 mL aliquot Formulation and notes: 0.266 mL</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T8802</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>Polyethyleneimine (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>P3143</td>
			<td>Media: Surface coating solutions Formulation and notes: 2.5 &#181;g/mL in H20</td>
		</tr>
		<tr>
			<td>Poly-D-lysine (PDL)</td>
			<td>Sigma-Aldrich</td>
			<td>P7280</td>
			<td>Media: use within 1 wk Formulation and notes: 100 &#181;g/mL in H20</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>5 &#181;g/mL in H20</td>
		</tr>
		<tr>
			<td>DMEM/F12 + GlutaMAX</td>
			<td>Life Technologies</td>
			<td>10565-018</td>
			<td>Media: NPC culture medium Formulation and notes: 492.5 mL</td>
		</tr>
		<tr>
			<td>100x N2 vitamins</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>Media: store at 4 &#176;C for up to 1 month Formulation and notes: 5 mL</td>
		</tr>
		<tr>
			<td>100x antibiotics</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>2.5 mL</td>
		</tr>
		<tr>
			<td>Neurobasal A</td>
			<td>Life Technologies</td>
			<td>10888-022</td>
			<td>Media: ESN culture medium Formulation and notes: 482.5 mL</td>
		</tr>
		<tr>
			<td>50x B27 vitamins</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>Media: store at 4 &#176;C for up 1 month Formulation and notes: 10 mL</td>
		</tr>
		<tr>
			<td>200 mM L-Alanyl-L-Glutamine</td>
			<td>ATCC</td>
			<td>30-2115</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>100x antibiotics</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>2.5 mL</td>
		</tr>
		<tr>
			<td>5-fluoro-2&#39;-deoxyuridine (5FDU)</td>
			<td>Sigma-Aldrich</td>
			<td>F0503</td>
			<td>Media: 2000x Mitotic inhibitors aliquot and store at -20 &#176;C Formulation and notes: dd 150 mg 5FDU and 350 mg uridine to 10 mL Neurobasal A. Sterile filter, aliquot and freeze. Use 2000x in ESN culture medium.</td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Trypsin</td>
			<td>Life Technologies</td>
			<td>12605-010</td>
			<td>Media: Miscellaneous Formulation and notes: store at RT</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>soybean trypsin inhibitor (STI)</td>
			<td>Sigma-Aldrich</td>
			<td>T6414</td>
			<td>store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>store at RT</td>
		</tr>
		<tr>
			<td>ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td>Prepare 100 mM stock by dissolving 100 mg ascorbic acid in 5.7 mL of 50:50 DMSO/ethanol mix.</td>
		</tr>
		<tr>
			<td>retinoic acid (RA)</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>Resuspend 15 mg RA in 9 mL 50:50 DMSO/ethanol. Supplement with 1 mL of 100 mM ascorbic acid stock. Aliquot and stored at -80 &#176;C. Stable for 6 mos.</td>
		</tr>
		<tr>
			<td>plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX51 inverted microscope</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm glass coverslips</td>
			<td>Fisher</td>
			<td>12-545-84</td>
			<td></td>
		</tr>
		<tr>
			<td>cell strainer (40 &#181;m)</td>
			<td>Fisher</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Stovall Belly Dancer Shaker</td>
			<td>Fisher</td>
			<td>15-453-211</td>
			<td></td>
		</tr>
		<tr>
			<td>low adhesion dishes</td>
			<td>Fisher</td>
			<td>05-539-101</td>
			<td>Corning 3262</td>
		</tr>
		<tr>
			<td>bacterial dishes</td>
			<td>VWR</td>
			<td>25384-302</td>
			<td>100 x 15 mm</td>
		</tr>
	</tbody>
</table>

Source: Hubbard, K. et al., <a href="https://app.jove.com/v/52361">Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a technique for embryonic stem cell differentiation into a networked population of neurons with functioning synapses. It outlines the steps involved in embryonic stem cell differentiation into neurons, induction of neuronal network formation, and enrichment of the culture with networked neurons.

<img alt="Figure 1" src="/files/ftp_upload/22600/22600_Figure1.jpg" /> 
Figure 1. Suspension-adapted ESNs remain mitotically stable and express markers of pluripotency.&#160;(A) Schematic of ESC maintenance and differentiation. The presence or absence of retinoic acid (RA) or leukemia inhibitory factor (LIF) is marked by a + or &#8211;. A comparison between days&#160;in vitro&#160;(DIV) and classical developmental stages (DS) for primary neu...

1. Neuronal Differentiation
NOTE: Conduct steps 1.1 - 1.5 before noon and step 1.7 after noon. An overview of the differentiation procedures is presented ...

Seed mouse embryonic stem cells in a low-attachment dish containing differentiation media.&#160;
Incubate under rotation to facilitate cellular aggregation and differentiation into a neuronal lineage.
Transfer the aggregates to a tube and allow them to settle by gravity. Discard the media.
Resuspend and transfer the aggregates to the original dish containing differentiation media supplemented with retinoic acid.
Retinoic acid induces neuronal gene expression, triggering differentiation into neural progenitor cells.
Transfer the aggregates to a tube. Remove the media and add trypsin to dissociate the aggregates.
Add an inhibitor to neutralize the trypsin.
Pipette repeatedly to obtain a homogeneous cell suspension. Filter the suspension to remove aggregates.
Centrifuge and remove the supernatant. Resuspend the cells in media.
Seed the cells on a polymer-coated dish to facilitate cell adhesion.
Remove the media. Add fresh media containing growth factors to promote neuronal maturation and form a networked neuron population.

14 조회수. 마우스 배아 줄기세포를 네트워크화된 뉴런 집단으로 분화(Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population)

비디오: 마우스 배아 줄기세포를 네트워크화된 뉴런 집단으로 분화(Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population) - 실험

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.

마이크로 전극 배열에 네트워크 활동을 측정하기위한 유도 다 능성 줄기 세포의 빠른 신경 세포의 분화

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

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JoVE Journal

발생학

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

인간 배아 줄기 세포의 단세포 배양을 이용한 효율적인 신경 분화

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

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.

시험관 내 마우스 배아 줄기 세포로부터의 신경 분화

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

Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population

내레이션 대본

Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population