generating postganglionic sympathetic neurons from human pluripotent stem cells

Generating Postganglionic Sympathetic Neurons from Human Pluripotent Stem Cells

One day before differentiation induction, coat the appropriate number of six-well plates with two milliliters of basement membrane matrix per well, and store the plates at four degrees Celsius overnight. The next morning, warm the plates to room temperature, and wash the ready-to-split human pluripotent stem cell culture two times with fresh PBS per wash.
After the second wash, treat the cells with seven milliliters of 0.5 millimolar EDTA for 15 minutes in the cell culture incubator, before aspirating the floating human pluripotent stem cells and transferring the harvested cells into a 50-milliliter tube.
Add an equal volume of PBS to the tube to dilute the EDTA, and collect the cells by centrifugation. Resuspend the pellet in one milliliter of day 01 differentiation medium. Before adding an appropriate volume of medium for counting, dilute the cells to a 1.25 x 105 cells per square centimeter concentration in two milliliters of differentiation medium per well.
Aspirate all of the basement membrane matrix solution from each well of the previously prepared six-well plate. Then, seed 2 milliliters of cells into each well, and place the plate in the cell culture incubator. The next morning, feed the cells with 3 milliliters of day 01 differentiation medium per well.
On day two of differentiation, feed the cells with three milliliters of day two to ten differentiation medium per well. Then, feed the cells every other day until day 10. To aggregate the neural crest cells into spheroids, on day 10, wash the cells with PBS, and add two milliliters of dissociation medium to each well. After 20 minutes in the cell culture incubator, transfer the floating cells into a 50-milliliter conical tube, and bring the volume of suspension in the tube to 50 milliliters with fresh PBS.
Collect the cells by centrifugation, and resuspend the pellet in a sufficient volume of day 10 to 14 spheroid medium for counting. After counting, dilute the cells to a 5 x 105 cells per 500 microliters concentration using day 10 to 14 spheroid medium, and plate 500 microliters of cells into each well of an ultra-low attachment 24-well plate. Then return the cells to the cell culture incubator.
To induce a minimal spheroid culture, on day 11 of culture, feed the neural crest spheroids with an additional 500 microliters of day 10 to 14 spheroid medium per well. On day 12, tilt the plate to accumulate the spheroids on one side of the plate, and carefully aspirate as much medium as possible from each well without aspirating spheroids. Then, feed the spheroids with one milliliter of day 10 to 14 spheroid medium per well.
To induce an expanded spheroid culture on day 15, feed the spheroids with 1.5 milliliters of day 10 to 14 spheroid medium supplemented with 0.5 micromolar retinoic acid per well. Then, return the spheroids to the cell culture incubator.
For sympathetic neuron differentiation on day 14 or 28, tilt the plate to accumulate the spheroids on one side of the plate, and carefully aspirate as much medium as possible. Feed the cells with one milliliter of sympathetic neuron medium.
To break up big spheroid aggregates into smaller spheroids, add no more than one milliliter of medium per well, and pipette the spheroids five to 10 times before splitting.
And remove the excess laminin fibronectin from previously prepared 24-well plates. Split the plate one milliliter of spheroids from each well on the 24-well spheroid culture plate between four separate wells of the newly coated 24-well plate. Add 250 microliters of fresh sympathetic neuron medium to each well, and place the plate in the cell culture incubator. The next morning, replace the medium in each well with one milliliter of sympathetic neuron medium supplemented with 0.125 micromolar retinoic acid, and return the plate to the cell culture incubator. After day 35, feed the neurons by carefully replacing only half of the existing medium in each well with fresh medium.

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1. Set-up for dish coating, media preparation, and hPSC maintenance
<ol>
	<li>Dish coating
	<ol>
		<li>Vitronectin (VTN) coating
		<ol>
			<li>Place vials of VTN in a 37 &#176;C water bath until fully thawed, then mix thoroughly.</li>
			<li>For a 100 mm Petri dish, mix 7 mL of 1x phosphate buffered saline (PBS) with 0.5 mg/mL VTN, add VTN solution to the dish, and incubate at room temperature (RT) for 1 h.</li>
		</ol>
		</li>
		<li>Basement membrane matrix coating&#8203;
		<ol>
			<li>Thaw vials of basement membrane matrix (see Table of Materials) on ice at 4 &#176;C overnight.</li>
			<li>For one well of a 6 well plate, mix 2 mL of Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) with 20 &#181;L of 100x basement membrane matrix, add basement membrane matrix solution to the dish, wrap the dish with paraffin film, and store in a clean container at 4 &#176;C overnight. Work as quickly as possible. Coated dishes can be stored in 4 &#176;C for up to 2 weeks.</li>
		</ol>
		</li>
		<li>Polyornithine (PO)/laminin (LM)/fibronectin (FN) coating
		<ol>
			<li>On the first day, for one well of a 24 well plate, mix 15 &#181;g/mL of PO with 1 mL of 1x PBS, incubate at 37 &#176;C, 5% CO2 overnight. Thaw both LM and FN at -20 &#176;C overnight and store at 4 &#176;C until fully thawed.</li>
			<li>On the second day, aspirate PO solution, wash the wells 2x with 1x PBS, add 1 mL of 1x PBS containing 2 &#181;g/mL of LM and 2 &#181;g/mL of FN and incubate at 37 &#176;C in 5% CO2 overnight. At this point the dish with the LM/FN solution can be kept in the incubator for months as long as it does not dry out. Add more 1x PBS to prevent the dish from drying out.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Media preparation
	<ol>
		<li>Prepare the Essential 8 medium (E8) by thawing one bottle of E8 supplement at 4 &#176;C overnight. Mix the supplement with 500 mL of E8 medium and antibiotics if needed. 
		NOTE: Working E8 solution should be used up within 2 weeks.</li>
		<li>Prepare the hPSC freezing medium by mixing 90 mL of complete E8 medium with 10 mL of dimethyl sulfoxide (DMSO) for a total volume of 100 mL. Filter sterilize.</li>
		<li>Prepare the day 0 to day 1 differentiation medium by mixing 100 mL of essential 6 (E6) medium with 10 &#181;M SB431542, 1 ng/mL bone morphogenetic protein 4 (BMP4), 300 nM CHIR99021, and 10 &#181;M Y27632 for a total volume of 100 mL.</li>
		<li>Prepare the day 2 to day 10 differentiation medium by mixing 100 mL of E6 medium with 10 &#181;M SB and 0.75 &#181;M CHIR99021 for a total volume of 100 mL.</li>
		<li>Prepare the day 10 to day 14 spheroid medium by mixing neurobasal medium with 2 mL of B27 (50x), 1 mL of N2 (100x), 2 mM L-Glutamate, 3 &#181;M CHIR99021, and 10 ng/mL fibroblast growth factor-2 (FGF2) for a total volume of 100 mL.</li>
		<li>Prepare the day 14 to day 28 medium for spheroid long term maintenance by adding 0.5 &#181;M of fresh retinoic acid (RA) to the day 10 to day 14 spheroid medium for every feeding. 
		NOTE: Always keep RA at -80 &#176;C.</li>
		<li>Prepare the SymN maturation medium by mixing neurobasal medium with 2 mL of B27 (50x), 1 mL of N2 (100x), 2 mM L-glutamate, 25 ng/mL glial cell line-derived neurotrophic factor (GDNF), 25 ng/mL brain-derived neurotrophic factor (BDNF), 25 ng/mL nerve growth factor (NGF), 200 &#181;M ascorbic acid, and 0.2 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) for a total volume of 100 mL. The solution should be used within 2 weeks. Before each feeding, add 0.125 &#181;M fresh RA. This solution is used from day 14 (option 1) or day 28 (option 2).</li>
		<li>Prepare the fluorescence-activated cell sorting (FACS) buffer by mixing DMEM with 2% FBS, 2 mM L-glutamate and antibiotics if needed for a total volume of 100 mL.</li>
	</ol>
	</li>
	<li>hPSC maintenance
	<ol>
		<li>Thawing and keeping hPSCs
		<ol>
			<li>Prepare one VTN coated 100 mm dish.</li>
			<li>To thaw a vial of hPSCs directly from liquid nitrogen, put the vial into a 37 &#176;C water bath, carefully swinging the tube in the water until it thaws. Transfer the thawed hPSCs to a 15 mL tube containing 10 mL of 1x PBS, and centrifuge at 200 x g for 4 min.</li>
			<li>Discard the supernatant and add 1 mL of E8 medium to the tube. Pipette a few times to fully resuspend the pellet and then add another 9 mL of E8 medium to reach 10 mL total.</li>
			<li>Aspirate the VTN solution from the 100 mm dish.</li>
			<li>Transfer the hPSCs to a 100 mm dish, shake gently (up-down and left-right, not in circles) to make sure cells are distributed evenly in the dish</li>
			<li>Incubate at 37 &#176;C, in 5% CO2.</li>
			<li>On the following day, aspirate all medium and feed with 10 mL of E8.</li>
			<li>Feed this way every day for the next 3&#8211;4 days and then prepare to split.</li>
		</ol>
		</li>
		<li>Splitting hPSCs 
		NOTE: hPSCs at the point of splitting should be 80%&#8211;90% confluent. Big colonies with smooth and bright edges should be observed. However, contact between each colony should be avoided (Figure 1B, day 0 and Figure 4B).
		<ol>
			<li>Prepare VTN-coated 100 mm dishes as needed.</li>
			<li>Aspirate the E8 and wash the dish that needs to be split 1x with 1x PBS.</li>
			<li>Aspirate the 1x PBS and add 4 mL of 0.25 M EDTA. Incubate for 2 min at 37 &#176;C, 5% CO2. 
			NOTE: The hPSCs should be split/replated as small colonies. Do not treat with ethylenediaminetetraacetic acid (EDTA) longer than 2 min to prevent separation into single cells. The cells should be still attached to the dish surface after the 2 min treatment.</li>
			<li>Aspirate the EDTA, detach the colonies by strongly pipetting 10 mL of E8 medium onto the dish surface, and collect all the medium and cells in a 15 mL tube.</li>
			<li>With hPSCs at 80%&#8211;90% confluency, split colonies by 1:15-1:20. For example, to split hPSCs by 1:20 into one 100 mm dish, take 500 &#181;L of E8/hPSCs solution and mix with 9.5 mL of fresh E8 medium.</li>
			<li>Plate hPSCs in VTN-coated 100 mm dishes. 
			NOTE: It is advised to establish the ideal split ratio for each researcher and cell line independently.</li>
		</ol>
		</li>
		<li>Freezing hPSCs
		<ol>
			<li>For one 100 mm dish of hPSCs that is ready to be split, prepare three cryovials and 3.5 mL of freezing medium. 
			&#8203;NOTE: Media and vials should be kept in 4 &#176;C or on ice until usage.</li>
			<li>Aspirate E8 and wash the dish 2x with 1x PBS.</li>
			<li>Aspirate 1x PBS and add 4 mL of 0.25 M EDTA, incubate for 2 min at 37 &#176;C, in 5% CO2. 
			NOTE: hPSCs should be frozen as small colonies at the time that they would be split. Do not treat the cells with EDTA longer than 2 min to prevent separation into single cells. Cells should be still attached on the dish&#39;s surface after the 2 min treatment.</li>
			<li>Aspirate the EDTA, detach the colonies by strongly pipetting 10 mL of 1x PBS on the dish&#39;s surface, and collect all medium and cells in a 50 mL tube.</li>
			<li>Add 20 mL of 1x PBS and centrifuge at 200 x g for 4 min to wash out the remaining EDTA.</li>
			<li>Discard the supernatant and resuspend the pellet in 3 mL of freezing medium.</li>
			<li>Distribute hPSCs evenly into the three cryovials, 1 mL each.</li>
			<li>Store at -80 &#176;C overnight in a controlled freezing box or a styrofoam sandwich to ensure a slow temperature drop, and then transfer to a liquid nitrogen tank for long term storage.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Seeding hPSCs to start the differentiation (day 0)
NOTE: hPSCs should be ready for differentiation after being stabilized (i.e., being split 2&#8211;3x after thawing). Be sure that the colonies are healthy, with smooth, shiny edges, and minimal differentiation (Figure 2B).
<ol>
	<li>Prepare basement membrane matrix-coated dishes (24 well or 6 well dishes) one day before day 0 as needed. Bring the dishes to RT at the start of differentiation.</li>
	<li>Make the day 0&#8211;1 differentiation medium as needed.</li>
	<li>Aspirate the E8 from the confluent, ready to split hPCSs, and wash the dish 2x with 1x PBS.</li>
	<li>Add 7 mL of 0.25 M EDTA per 100 mm dish, incubate at 37 &#176;C, 5% CO2, for 15 min. 
	NOTE: At this point, EDTA treatment is prolonged to disperse into single cells.</li>
	<li>Pipet off all the hPSCs (they should be floating) and transfer to a 50 mL tube. Add the same amount or more of 1x PBS as EDTA solution to dilute out the EDTA.</li>
	<li>Centrifuge at 200 x g for 4 min.</li>
	<li>Discard the supernatant, add 1 mL of day 0&#8211;1 differentiation medium and pipet to homogenize the cells. Follow by adding more medium and then mix to dilute the cell solution to a concentration ideal to count the cells. 
	NOTE: Do not overdilute the cell solution. Cells from one full 100 mm dish should be diluted in 5 mL of medium to start.</li>
	<li>Count the cell number using an automated cell counter or hemocytometer.</li>
	<li>Dilute the cell solution as needed to reach 125,000 cells/cm2 in a low final volume (e.g., 2 mL per well for a 6 well dish or 500 &#181;L per well for a 24 well dish). 
	NOTE: A low volume helps the cells attach faster.</li>
	<li>Aspirate all of the basement membrane matrix solution from the coated dishes and plate the cell solution into the wells.</li>
	<li>Incubate at 37 &#176;C, in 5% CO2.</li>
</ol>
3. Neural crest cell induction (day 1 to day 10, Figure 1A)
<ol>
	<li>On day 1 feed the cells with day 0&#8211;1 differentiation medium (3 mL per well for 6 well dishes and 1 mL per well for 24 well dishes).</li>
	<li>On day 2 feed the cells with day 2&#8211;day 10 differentiation medium (3 mL per well for 6 well dishes and 1 mL per well for 24 well dishes).</li>
	<li>From now on, the cells should be fed every other day until day 10 (i.e., the next feeding day will be day 4). 
	NOTE: From day 6 on, NC ridges should be detected (Figure 1B). To check if differentiation is taking place, it is advised to carry a parallel differentiation culture in smaller wells (i.e., 24 wells), that can be stained for SOX10/AP2a and used for marker gene expression along the time of differentiation (Figure 1B,C).</li>
	<li>If sorting cells, proceed to section 4. Otherwise, proceed to section 5.</li>
</ol>
4. Fluorescence activated cell sorting (FACS) for neural crest marker CD49D and aggregating NC cells in spheroids
NOTE: For FACS sorting, the samples should be kept on ice and not be exposed to light after staining until sorting.
<ol>
	<li>Prepare FACS buffer if the cells are sorted.</li>
	<li>Prepare day 10&#8211;14 spheroid medium.</li>
	<li>On day 10, remove the medium, and wash 1x with 1x PBS.</li>
	<li>Add dissociation solution (see Table of Materials) at 2 mL per well for a 6 well dish or 1 mL per well for a 24 well dish, and incubate at 37 &#176;C, 5% CO2 for 20 min.</li>
	<li>Pipet off all the hPSCs and transfer to a 50 mL tube.</li>
	<li>Fill up the rest of the tube with FACS buffer and centrifuge at 200 x g for 4 min. 
	NOTE: Each 50 mL tube can accommodate up to 20 mL or less of cell solution. The volume of the FACS buffer should be high enough to neutralize the dissociation solution.</li>
	<li>Discard the supernatant, resuspend the cells with the appropriate amount of FACS buffer (~2 mL per well of a 6 well plate), and count to determine the cell number.</li>
	<li>Prepare the following samples.
	<ol>
		<li>Sample 1 (unstained control): 1 x 106 cells in 400 &#181;L of FACS buffer. Filter the cells through a 20 &#181;m strainer cap and keep the tube on ice.</li>
		<li>Sample 2 (DAPI only control): 1 x 106 cells in 400 &#181;L of FACS buffer containing 0.5 ug/mL 4&#8242;,6-diamidino-2-phenylindole (DAPI). Filter the cells through a FACS tube with a strainer cap and keep the tube on ice.</li>
		<li>Sample 3 (CD49D-labeled): Suspend the rest of the cells with FACS buffer containing phycoerythrin/cyanine7 (PE/Cy7)-conjugated CD49D antibody (5 &#181;L for 1 x 106 cells per 100 &#181;L of FACS buffer) in a 15 mL tube and incubate on ice for 20 min.</li>
	</ol>
	</li>
	<li>Fill up the tubes with FACS buffer and centrifuge at 200 x g for 4 min.</li>
	<li>Discard the supernatant and resuspend every 5&#8211;10 x 106 cells in 1 mL of FACS buffer containing 0.5 ug/mL DAPI according to the manufacturer&#39;s instructions.</li>
	<li>Filter the cells through the FACS tube with the strainer cap and keep the tube on ice.</li>
	<li>Prepare collection FACS tubes containing 2 mL of FACS buffer.</li>
	<li>Sort through the FACS machine with lasers that can detect DAPI and PE-Cy7 to isolate the CD49D+ population.</li>
	<li>After sorting, count the sorted cells.</li>
	<li>Centrifuge all sorted cells and resuspend in day 10&#8211;14 spheroid medium to a final concentration of 0.5 x 106 cells per 500 &#181;L of medium.</li>
	<li>Plate 0.5 x 106 cells per well into ultra-low attachment 24 well plates.</li>
	<li>Incubate the cells at 37 &#176;C, in 5% CO2.</li>
</ol>
5. Aggregating NC cells in spheroids
<ol>
	<li>If not using FACS to isolate the NC cells and instead aggregating them into spheroids directly, first prepare cells as described in steps 4.2&#8211;4.5.</li>
	<li>Fill up the rest of the tube with 1x PBS, and centrifuge at 200 x g for 4 min.</li>
	<li>Discard the supernatant, resuspend the cells with an appropriate amount of day 10&#8211;14 spheroid medium (e.g., ~2 mL of medium per well for a 6 well plate), and count to determine the cell number.</li>
	<li>Dilute the cells in day 10&#8211;14 spheroid medium to 0.5 x 106 cells per 500 &#181;L of medium.</li>
	<li>Plate 500 &#181;L of the cell suspension per well in ultra-low attachment 24 well plates.</li>
	<li>Incubate the cells at 37 &#176;C, in 5% CO2.</li>
</ol>
6. NC spheroid maintenance and sympathetic progenitor induction (day 10 to day 14, Figure 3A)
<ol>
	<li>Option 1: Minimal spheroid culture&#8203;
	<ol>
		<li>On day 11, add 500 &#181;L of day 10&#8211;14 spheroid medium to the NC spheroids without aspirating existing medium from day 10. Incubate at 37 &#176;C, in 5% CO2.</li>
		<li>On day 12, tilt the plate to accumulate the NC spheroids on one side of the wells. Carefully aspirate and discard as much medium as possible, and feed with 1 mL of day 10&#8211;14 spheroid medium.</li>
		<li>Keep feeding the cells every day until day 14.</li>
		<li>Optional: If the spheroids aggregate and generate a large clump, use a pipette to break the spheroid clumps up. This also ensures that individual spheroids do not get too large. 
		NOTE: The ideal spheroid size range should be around 100&#8211;500 &#956;m. Within that range, the size of individual spheroids is not critical. However, the morphology, such as a smooth and clear edges (Figure 2 and Figure 4) is important for further success. At day 14, each 24 well plate ideally contains about 50&#8211;60 spheroids of different sizes within the abovementioned size range.</li>
	</ol>
	</li>
	<li>Option 2: Expanded spheroid culture&#8203;
	<ol>
		<li>On day 15, to keep NC spheroids, feed with 1.5 mL of day 10&#8211;14 spheroid medium containing 0.5 &#181;M RA. Incubate at 37 &#176;C, 5% CO2. 
		NOTE: RA should be added fresh for every feeding and always be stored at -80 &#176;C.</li>
		<li>From now on, feed every other day up to day 28 and then continue with plating of the spheroids (section 7.1). 
		NOTE: The growing spheroids are split approximately 1x per week by pipetting them with a 1 mL pipette to break them up. They are split at an approximate ratio of 1:2&#8211;1:4. Within the 2 week expansion period, the cells should roughly quadruple in number.</li>
	</ol>
	</li>
</ol>
7. Sympathetic neuron (SymN) differentiation and maturation (Option 1: after day 14; Option 2: after day 28)
<ol>
	<li>Plating spheroids in regular dishes
	<ol>
		<li>Prepare PO/LM/FM-coated 24 well plates.</li>
		<li>Prepare symN medium containing 0.125 &#181;M RA (add fresh every feed) and 10 &#181;M Y27632 (day 14 only).</li>
		<li>On day 14, tilt the plate to accumulate NC spheroids on one side of the wells. Carefully aspirate and discard as much medium as possible, and feed with 1 mL of symN medium.</li>
		<li>Remove LM/FN from the coated plates.</li>
		<li>Split and plate each well from the 24 well plate into 4 separate wells of the new, coated 24 well plate. Each original well will have 1 mL, containing ~50&#8211;60 spheroids. This yields 250 &#181;L, containing approximately 10&#8211;15 spheroids for each well on the new plate.</li>
		<li>Add 250 &#181;L of additional medium per well. 
		NOTE: This is a split of 1:4; make sure that the spheroids are distributed properly within the solution so that the split is relatively even. The number of spheroids is not counted because the final number does not affect the success of generating symNs.</li>
		<li>Incubate at 37 &#176;C, 5% CO2.</li>
		<li>On day 15 (or day 29 for option 2), feed by replacing all medium with 1 mL of symN medium containing 0.125 &#181;M RA. From now on, the neurons should be fed every 2 days until day 20 (or day 35 for option 2).</li>
		<li>After day 20 (or day 35 for option 2), the neurons should be fed by carefully replacing only half of the existing medium (500 &#181;L). From now on, feed every week unless the medium quickly turns yellow.</li>
		<li>Keep feeding weekly until the desired time point. 
		NOTE: symNs tend to aggregate in ganglia-like structures and are prone to detach from the culture dishes. To prevent this, half-feedings and minimal handling is recommended.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm cell culture dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tissue culture tubes</td>
			<td>VWR/Corning</td>
			<td>89039-664</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plates</td>
			<td>Falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well ultra-low-attachment plates</td>
			<td>Corning</td>
			<td>07 200 601 and 07 200 602</td>
			<td></td>
		</tr>
		<tr>
			<td>5% CO2/20% O2 tissue culture incubator</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>Heracell VIOS 160i</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tissue culture tubes</td>
			<td>VWR/Corning</td>
			<td>89039-656</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovation Cell Technologies</td>
			<td>AT104500</td>
			<td>Cell dissociation solution</td>
		</tr>
		<tr>
			<td>Anti-AP2a antibody</td>
			<td>Abcam</td>
			<td>ab108311</td>
			<td>Host: Rabbit; 1:400 dilution</td>
		</tr>
		<tr>
			<td>Anti-Ascl1 antibody</td>
			<td>BD Pharmingen</td>
			<td>556604</td>
			<td>Host: Mouse IgG1; 1:200 dilution</td>
		</tr>
		<tr>
			<td>Anti-CD49D antibody</td>
			<td>BioLegend</td>
			<td>304313</td>
			<td>Host: Mouse IgG1; 5 &#956;l/million cells in 100 &#956;l volume</td>
		</tr>
		<tr>
			<td>Anti-CD49D antibody (isotype)</td>
			<td>BioLegend</td>
			<td>400125</td>
			<td>Host: Mouse IgG1; 5 &#956;l/million cells in 100 &#956;l volume</td>
		</tr>
		<tr>
			<td>Anti-DAPI antibody</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Anti-DBH antibody</td>
			<td>Immunostar</td>
			<td>22806</td>
			<td>Host: Rabbit; 1:500 dilution</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td>Host: Chicken; 1:1000 dilution</td>
		</tr>
		<tr>
			<td>Anti-HOXC9 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-365692</td>
			<td>Host: Mouse IgG1; 1:100 dilution</td>
		</tr>
		<tr>
			<td>Anti-NET1 antibody</td>
			<td>Mab</td>
			<td>NET17-1</td>
			<td>Host: Mouse; 1:1000 dilution</td>
		</tr>
		<tr>
			<td>Anti-PRPH antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-377093/H0112</td>
			<td>Host: Mouse IgG2a; 1:200 dilution</td>
		</tr>
		<tr>
			<td>Anti-SOX10 antibody</td>
			<td>Abcam</td>
			<td>ab50839</td>
			<td>Host: Mouse; 1:100 dilution</td>
		</tr>
		<tr>
			<td>Anti-TH antibody</td>
			<td>Pel-Freez</td>
			<td>P40101- 150</td>
			<td>Host: Rabbit; 1:500 dilution</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A8960-5G</td>
			<td>Stock concentration: 100 mM</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>12587-010</td>
			<td>Stock concentration: 50x</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>R&#38;D Systems</td>
			<td>248-BD</td>
			<td>Stock concentration: 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-BP</td>
			<td>Stock concentration: 6 mM</td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>Countess II</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting chamber slides</td>
			<td>Invitrogen</td>
			<td>C10312</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>57021&#38;5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>R&#38;D Systems</td>
			<td>4423</td>
			<td>Stock concentration: 6 mM</td>
		</tr>
		<tr>
			<td>Cryo-vial</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>375353</td>
			<td></td>
		</tr>
		<tr>
			<td>dbcAMP</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>Stock concentration: 100 mM</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>10829-018</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>11330-057</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>E6 medium</td>
			<td>gibco</td>
			<td>A15165-01</td>
			<td></td>
		</tr>
		<tr>
			<td>E8 medium</td>
			<td>gibco</td>
			<td>A15169-01</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>E8 supplement</td>
			<td>gibco</td>
			<td>A15171-01</td>
			<td>Stock concentration: 50x</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>ED2SS</td>
			<td>Stock concentration: 0.5 M</td>
		</tr>
		<tr>
			<td>FACS machine</td>
			<td>Beckman Coulter</td>
			<td>CytoFLEX (for FACS)</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS machine</td>
			<td>Beckman Coulter</td>
			<td>MoFlo Astrios EQ (for sorting)</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes (blue filter cap)</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes (white cap)</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>PeproTech</td>
			<td>450</td>
			<td>Stock concentration: 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Geltrex</td>
			<td>Invitrogen</td>
			<td>A1413202</td>
			<td>Basement membrane matrix; Stock concentration: 100x</td>
		</tr>
		<tr>
			<td>hPSCs</td>
			<td>Thomson et al., (1998)</td>
			<td>WA09</td>
			<td></td>
		</tr>
		<tr>
			<td>hPSCs</td>
			<td>Oh et al. (2016)</td>
			<td>H9-PHOX2B::eGFP</td>
			<td></td>
		</tr>
		<tr>
			<td>Human fibronectin (FN)</td>
			<td>VWR/Corning</td>
			<td>47743-654</td>
			<td>Stock concentration: 1 mg/mL</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher/Gibco</td>
			<td>25030-081</td>
			<td>Stock concentration: 200 mM</td>
		</tr>
		<tr>
			<td>LN tank</td>
			<td>Custom Biogenic Systems</td>
			<td>V-1500AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse laminin I (LM)</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td>Stock concentration: 1 mg/mL</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>17502-048</td>
			<td>Stock concentration: 100x</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>gibco</td>
			<td>21103-049</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>NGF</td>
			<td>PeproTech</td>
			<td>450-01</td>
			<td>Stock concentration: 25 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td>Stock concentration: 1x</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide (PO)</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td>Stock concentration: 15 mg/mL</td>
		</tr>
		<tr>
			<td>Primocin (antibiotics)</td>
			<td>InvivoGen</td>
			<td>ANTPM1</td>
			<td>Stock concentration: 50 mg/mL</td>
		</tr>
		<tr>
			<td>qPCR machine</td>
			<td>Bio-Rad Laboratories</td>
			<td>C1000 Touch</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR plates</td>
			<td>Bio-Rad Laboratories</td>
			<td>HSP9601</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant FGF2</td>
			<td>R&#38;D Systems</td>
			<td>233-FB/CF</td>
			<td>Stock concentration: 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Stock concentration: 1 mM</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris/R&#38;D Systems</td>
			<td>1614</td>
			<td>Stock concentration: 10 mM</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Corning</td>
			<td>MT-25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitronectin (VTN)</td>
			<td>Thermo Fisher/Life Technologies</td>
			<td>A14700</td>
			<td>Stock concentration: 0.5 mg/mL</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>VWR/Corning</td>
			<td>706308</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>R&#38;D Systems</td>
			<td>1254</td>
			<td>Stock concentration: 10 mM</td>
		</tr>
	</tbody>
</table>

Source: Wu, H. F., et al. <a href="https://app.jove.com/v/60843">Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions.</a> J. Vis. Exp. (2020).
This video demonstrates an in vitro method to generate postganglionic sympathetic neurons from human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural crest cells (NCCs) on a basement membrane matrix support using a differentiation medium. The NCCs are then dissociated and formed into spheroids using another differentiation medium. Finally, the spheroids are transformed into postganglionic sympathetic neurons using a sympathetic neuron medium.

<img alt="Figure 1" src="/files/ftp_upload/22346/22346_Figure1.jpg" /> 
Figure 1: NC induction. (A) Timeline and treatments for NC induction from day 0 to day 10. (B) The morphology and formation of NC ridges were monitored every 2 days by bright field microscopy. Cells at each time point after day 2 were co-stained for AP2A (red) and SOX10 (green). All immunofluorescence pictures were counterstained with DAPI. Red arrows indicate th...

1. Set-up for dish coating, media preparation, and hPSC maintenance

	Dish coating
	
		Vitronectin (VTN) coating
		
			Place vials of VTN in a 37 &#176;C ...

Take an extracellular matrix-coated microplate and add human pluripotent stem cells suspended in a differentiation medium.
The medium contains signaling molecules that influence cellular signaling pathways, directing differentiation into neural progenitor cells.
Introduce another differentiation medium containing signaling molecules that induce differentiation into neural crest cells, or NCCs, precursors to neuronal and non-neuronal cells.
Add enzymes to disrupt the matrix, thereby dissociating the cells.
Wash to neutralize the enzymes, then centrifuge and discard the supernatant. Resuspend the NCCs in a spheroid medium and transfer them to a microplate.
The medium&#39;s growth factors and small molecules induce spheroid formation.
Supplement with retinoic acid, which promotes differentiation into sympathetic progenitor neurons.
Transfer the spheroid onto an adhesion protein-coated microplate in a sympathetic neuron maturation medium, causing spheroid attachment.
Neural growth factors and retinoic acid in the medium promote maturation into postganglionic sympathetic neurons, which are part of the peripheral nervous system and regulate involuntary processes.

21 次观看. 从人多能干细胞产生节后交感神经神经元

视频: 从人多能干细胞产生节后交感神经神经元 - 实验

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, wound healing, root canal treatment, etc., forming a new research field named plasma medicine. Being a mixture of electrical, chemical, and biological reactive species, CAPs have shown their abilities to enhance nerve stem cells differentiation both in vitro and in vivo and are becoming a promising way for neurological disease treatment in the future. The much more exciting news is that using CAPs may realize one-step, and safely directed, differentiation of neural stem cells (NSCs) for tissue transportation. We demonstrate here the detailed experimental protocol of using a self-made CAP jet device to enhance NSC differentiation in C17.2-NSCs and primary rat neural stem cells, as well as observing the cell fate by inverted and fluorescence microscopy. With the help of immunofluorescence staining technology, we found both the NSCs showed an accelerated differential rate than the untreated group, and ~75% of the NSCs selectively differentiated into neurons, which are mainly mature, cholinergic, and motor neurons.

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

This protocol aims to provide detailed experimental steps of a cold atmospheric plasma treatment on neural stem cells and immunofluorescence detection for differentiation enhancement.

一步冷常压等离子体体外处理神经干细胞分化

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, ...

科研

JoVE Journal

工程学

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.

小鼠胚胎干细胞中神经祖细胞和神经元的分化与表征

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

Generating Postganglionic Sympathetic Neurons from Human Pluripotent Stem Cells

成績單

Generating Postganglionic Sympathetic Neurons from Human Pluripotent Stem Cells