eoe sub articles

EoE Sub Articles

1. Experimental preparation 
<ol>
	<li>Prepare basement membrane matrix (BMM)-coated plates. 
	NOTE: BMM solidifies more quickly at warmer temperatures. Plates must be prepared rapidly and immediately placed at 4 &#176;C for storage.
	<ol>
		<li>Thaw the BMM (see Table of Materials) on ice, at 4 &#176;C for at least 2 h, or overnight.</li>
		<li>Mix Dulbecco&#39;s Modified Eagle Medium (DMEM)/F12 and BMM to a final concentration of 80 &#181;g/mL. Distribute the BMM solution in tissue culture dishes (1 mL for 35 mm and 2 mL for 60 mm dishes) and incubate at 37 &#176;C for at least 1 h or overnight before plating the cells. 
		&#8203;NOTE: The unused dishes can be stored at 4 &#176;C for 2 weeks.</li>
	</ol>
	</li>
	<li>Prepare poly-L-ornithine/laminin (PLO/laminin)-coated plates.
	<ol>
		<li>Prepare 20 &#181;g/mL PLO (see Table of Materials) in sterile Dulbecco&#39;s Phosphate Buffered Saline (DPBS+/+) and add 1 mL to each well of a 6-well plate. Incubate overnight at 37 &#176;C in the incubator.</li>
		<li>The following day, aspirate the PLO with a vacuum aspirator and wash two times with DPBS+/+. Air dry in the hood. 
		&#8203;NOTE: Air-dried plates can be stored at 4 &#176;C for up to 2 weeks, wrapped in aluminum foil, for future use.</li>
		<li>Prepare 10 &#181;g/mL laminin (see Table of Materials) in DPBS+/+ and add 1 mL to each well of a 6-well plate. Incubate at least for 3 h or overnight at 37 &#176;C in the incubator.</li>
		<li>Aspirate the laminin with a vacuum aspirator and add either 1 mL of medium or sterile DPBS+/+. 
		&#8203;NOTE: The laminin coating must not dry out. PBS or an appropriate culture medium should be added immediately to prevent this. Coated plates can be stored at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Prepare PSC passaging solution.
	<ol>
		<li>Dissolve 11.49 g of potassium chloride (KCl) and 0.147 g of sodium citrate dihydrate (HOC(COONa) (CH2COONa)2*2H2O) in 400 mL of sterile cell culture grade water (see Table of Materials).</li>
		<li>Measure the volume of the solution and record it as the initial volume (Vi).</li>
		<li>Measure the osmolarity and adjust it to 570 mOsm by adding sterile cell culture grade water using the formula Vi &#215; Oi = Vf &#215; 570 mOsm.</li>
		<li>Filter-sterilize the solution with a 0.20 &#181;m filter and make 10 mL aliquots. Store the aliquots at room temperature (RT) for up to 6 months.</li>
	</ol>
	</li>
	<li>Prepare pluripotent stem cell (PSC) medium. 
	&#8203;NOTE: iPSCs are maintained in a medium containing heat-stable Fibroblast growth factor 2 (FGF2) (see Table of Materials), providing a weekend-free feeding schedule. Other commercially available PSC media have not been tried for this protocol.
	<ol>
		<li>Thaw the PSC medium supplement overnight at 4 &#176;C.</li>
		<li>Add 10 mL of PSC medium supplement to 500 mL of basal PSC medium. Make 25 mL aliquots and store at &#8722;20 &#176;C. 
		&#8203;NOTE: Frozen aliquots can be stored at &#8722;20 &#176;C for 6 months. Thawed aliquots must be stored at 4 &#176;C and used within 2 weeks. Cells can be frozen for long-term storage in a PSC medium with 10% (v/v) dimethyl sulfoxide (DMSO).</li>
	</ol>
	</li>
	<li>Prepare PSC thawing medium.
	<ol>
		<li>Supplement PSC medium with 50 nM chroman, 1.5 &#181;M emricasan, 1x polyamine supplement, and 0.7 &#181;M trans-ISRIB (CEPT cocktail) (see Table of Materials).</li>
		<li>Filter-sterilize with a 0.20 &#181;m filter and store at 4 &#176;C for up to 4 weeks.</li>
	</ol>
	</li>
	<li>Prepare pulled glass pipettes.
	<ol>
		<li>Pull 22.9 cm (9 in) glass Pasteur pipettes into two pieces above a Bunsen burner, ~2 cm below the neck, creating two pipettes, with the thinner side being ~4 cm shorter than the other.</li>
		<li>With the help of the flame, bend the tips of the pulled side to create a smooth &#34;r&#34;.</li>
		<li>Place the pulled pipettes in autoclave sleeves and autoclave for sterilization.</li>
	</ol>
	</li>
	<li>Prepare cerebellar differentiation medium (CDM).
	<ol>
		<li>Mix Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM) and Ham&#39;s F12 nutrient mix in a 1:1 ratio. Supplement the mix with 1x L-alanine-L-glutamine supplement, 1% (v/v) chemically defined lipid concentrate, 0.45 mM 1-thio-glycerol, 15 &#181;L/ mL apo-transferrin, 5 mg/mL bovine serum albumin (BSA), and 7 &#181;g/mL insulin (see Table of Materials).</li>
		<li>Filter-sterilize with a 0.20 &#181;m filter, store at 4 &#176;C, and use within 1 month.</li>
	</ol>
	</li>
	<li>Prepare cerebellar maturation medium (CMM).
	<ol>
		<li>Supplement neurobasal medium with 1x L-alanine-L-glutamine supplement and 1x N-2 supplement (see Table of Materials).</li>
		<li>Filter-sterilize with a 0.20 &#181;m filter, store at 4 &#176;C, and use within 2 weeks.</li>
	</ol>
	</li>
</ol>
2. Feeder-free iPSC culture
<ol>
	<li>Thaw the cells following the steps below.
	<ol>
		<li>Place a BMM plate (step 1.1) into the 37 &#176;C incubator to gel for at least 1 h or overnight before thawing the cells.</li>
		<li>Pre-warm 10 mL of PSC thawing medium (step 1.5) at 37 &#176;C.</li>
		<li>Transfer the cryovial with cells from liquid nitrogen to a water bath at 37 &#176;C. 
		&#8203;NOTE: To avoid generating a source of contamination in the cell culture space, using a standard water bath is not recommended. Instead, fresh water can be heated in a small beaker to 37 &#176;C to generate a one-time-use water bath.</li>
		<li>When there is a small piece of ice left in the tube, remove it from the water bath, dry the tube, and spray with 70% ethanol. Transfer the tube to the hood and use a 2 mL or 5 mL serological pipette (see Table of Materials) to gently transfer the cells to a 15 mL conical tube.</li>
		<li>Add 8 mL of PSC thawing medium to the cells dropwise while swirling the conical tube. Centrifuge at 200 x g for 5 min at RT.</li>
		<li>Carefully aspirate the supernatant with a vacuum aspirator without disturbing the cell pellet. Resuspend the cells in 2 mL of PSC thawing medium.</li>
		<li>Aspirate the BMM from the plate and add the resuspended cells dropwise all over the plate for even distribution.</li>
		<li>Place the plate in the incubator at 37 &#176;C, 5% CO2. Refresh the medium the next day with PSC medium. After that, change the medium daily.</li>
	</ol>
	</li>
	<li>Maintain the cells following the steps below.
	<ol>
		<li>Pre-warm the necessary volume of PSC medium at 37 &#176;C (e.g., 1 mL of medium per 35 mm plate).</li>
		<li>Investigate the plates for differentiated cells or colonies and remove the differentiated cells with a pulled glass pipette (step 1.6). 
		NOTE: iPSC colonies have smooth edges with morphologically identical cells.</li>
		<li>Change the spent medium daily and passage every 3-4 days or when 70% confluency is reached.</li>
	</ol>
	</li>
	<li>Passage the cells.
	<ol>
		<li>Place the necessary amount of BMM plates into the incubator for at least 1 h or overnight before passaging. Pre-warm the necessary amount of PSC medium (step 1.3) at 37 &#176;C.</li>
		<li>Using a pulled glass pipette, clean off any differentiated cells or colonies.</li>
		<li>Aspirate the spent medium with a vacuum aspirator and add PSC dissociation medium, 1 mL per 35 mm dish. Incubate at 37 &#176;C for 1-3 min. 
		&#8203;NOTE: If colonies are lifting off in the PSC passaging solution before adding PSC medium, the incubation time can be reduced.</li>
		<li>Aspirate the PSC passaging solution and add pre-warmed PSC medium.</li>
		<li>Using a sterile 200 &#181;L pipette tip, scratch lines parallel to each other in one direction, turn the plate 90&#176;, and make a second set of scratch lines perpendicular to the previous ones (making a cross-hatched pattern across the plate).</li>
		<li>Aspirate the BMM solution from the set plates. Collect the colonies using a serological pipette and distribute them to new BMM plates.</li>
		<li>Incubate at 37 &#176;C, 5% CO2. Change the medium daily.</li>
	</ol>
	</li>
	<li>Freeze the cells.
	<ol>
		<li>Prepare cryovials by labeling the content and sterilize under ultraviolet (UV) light in the hood (see Table of Materials) for 30 min.</li>
		<li>Prepare PSC freezing medium by supplementing PSC medium with 10% (v/v) DMSO. Follow steps 2.3.2-2.3.5.</li>
		<li>Transfer the cells to a 15 mL conical tube with a 5 mL serological pipette and centrifuge at 200 x g for 5 min at RT.</li>
		<li>Carefully aspirate the medium and resuspend the cell pellet in PSC freezing medium.</li>
		<li>Distribute into cryovials. Place the vials into a freezing container and place the container in a &#8722;80 &#176;C freezer.</li>
		<li>The following day, transfer the cryovials to liquid nitrogen for long-term storage.</li>
	</ol>
	</li>
</ol>
3. Cerebellar differentiation
NOTE: Before starting the differentiation, iPSCs are passaged to six 35 mm dishes and are ready for the differentiation when they are at 70% confluency. Each 35 mm plate will be transferred to one well of the 6-well plate.
<ol>
	<li>On day 0, lift the healthy iPSC colonies for EB formation.
	<ol>
		<li>Add 1 mL of CDM (step 1.7) supplemented with 10 &#181;M Y-27632 and 10 &#181;M SB431542 (see Table of Materials) to each well of a 6-well ultra-low attachment (ULA) plate and place in the incubator until the lifted colonies are ready to be added to the wells.</li>
		<li>Clean the differentiated cells using a pulled glass pipette. Aspirate the medium and add 1 mL of PSC passaging solution for each 35 mm dish. Incubate for 3 min at 37 &#176;C, aspirate, and add 2 mL of CDM supplemented with 10 &#181;M Y-27632 and 10 &#181;M SB431542. 
		NOTE: If colonies are lifting off in the PSC passaging solution before adding CDM, the incubation time can be reduced.</li>
		<li>Under a transmitted-light inverted microscope, gently lift the colonies using the bent edge of the pulled glass pipette using 4x magnification. Once every colony is lifted, gently transfer all to one well of the 6-well ULA plate using a 10 mL serological pipette. Repeat this process for each iPSC plate. Incubate the cells at 37 &#176;C with 5% CO2. 
		NOTE: If the colonies are too large or are merged, they can be sliced with the tip of the pulled glass pipette to create smaller EBs.</li>
	</ol>
	</li>
	<li>On day 2, add FGF2 to each well to a final concentration of 50 ng/mL. 
	NOTE: The FGF2 used for cerebellar differentiation is not heat-stable. This protocol has not been tested with heat-stable FGF2.</li>
	<li>On day 7, do a 1/3 medium change. For 3 mL of total medium in a well, with a 1,000 &#181;L pipettor, gently aspirate 1 mL of spent medium and replace it with 1 mL of fresh CDM. Incubate the EBs for 7 days.</li>
	<li>On day 14, gently aspirate nearly all the spent medium using a 1,000 &#181;L pipettor. To ensure minimal damage to the EBs, swirl the plate to gather all the EBs in the center of the plate, and then tilt the plate and aspirate slowly from the edge.
	<ol>
		<li>As the medium amount decreases, slowly lay the plate flat and continue to aspirate. Leave enough medium to avoid drying the EBs out. Afterward, add 3 mL of fresh CDM supplemented with 10 &#181;M Y-27632. Transfer the EBs using a 10 mL serological pipette to a PLO/laminin-coated dish (step 1.2). 
		NOTE: Depending on the downstream application, the EBs can be transferred to a 6-well PLO/laminin plate or single EBs can be transferred to a single well of a PLO/laminin-coated 24-well plate or coverslip.</li>
	</ol>
	</li>
	<li>On day 15, aspirate the medium and replace it with fresh CDM. 
	NOTE: It is important to add enough medium to the wells to ensure enough medium between feedings as, over time, there will be evaporation (e.g., for a well in a 6-well plate, add 3 mL of medium). If the medium has started to acidify (turn clear and yellow), refresh the medium even if it is not on the feeding schedule until the end of the protocol.</li>
	<li>On day 21, aspirate the spent medium and replace it with CMM. On day 28, change the medium with fresh CMM. On day 35, harvest the cells for further applications.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-26D</td>
			<td></td>
		</tr>
		<tr>
			<td>1-thio-glycerol</td>
			<td>Sigma</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-26B</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Filter Unit, 0.2 &#181;m aPES, 50 mm Dia</td>
			<td>Fisher Scientific</td>
			<td>FB12566502</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Easy Grip Tissue Cluture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-25D</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Easy Grip Tissue Culture Dish</td>
			<td>Falcon</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well ultra-low attachment plates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>9&#34; Disposable Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture grade water</td>
			<td>Cytiva</td>
			<td>SH30529.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically defined lipid concentrate</td>
			<td>Gibco</td>
			<td>11905031</td>
			<td></td>
		</tr>
		<tr>
			<td>Chroman 1</td>
			<td>Cayman</td>
			<td>34681</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II, Type A2, Biological safety Cabinet</td>
			<td>NuAire, Inc.</td>
			<td>NU-540-600</td>
			<td>Hood, UV light</td>
		</tr>
		<tr>
			<td>Costar 24-well plate, TC treated</td>
			<td>Corning</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6-well plate, TC treated</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO (Dimethly sulfoxide)</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS+/+</td>
			<td>Gibco</td>
			<td>14040133</td>
			<td></td>
		</tr>
		<tr>
			<td>Emricasan</td>
			<td>Cayman</td>
			<td>22204</td>
			<td></td>
		</tr>
		<tr>
			<td>Epi5 episomal iPSC reprogramming kit</td>
			<td>Life Technologies</td>
			<td>A15960</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential 8-Flex</td>
			<td>Gibco</td>
			<td>A2858501</td>
			<td>PSC medium with heat-stable FGF2</td>
		</tr>
		<tr>
			<td>Fetal bovine serum - Premium Select</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>L-alanine-L-glutamine supplement</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12 Nutrient Mix</td>
			<td>Gibco</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>HERAcell VIOS 160i CO2 incubator</td>
			<td>Thermo Scientific</td>
			<td>50144906</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440053</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Gibco</td>
			<td>12585</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein</td>
			<td>Gibco</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>354234</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>MEM-NEAA</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Centrifuge</td>
			<td>Labnet International</td>
			<td>C1310</td>
			<td>Benchtop mini centrifuge</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyamine supplement</td>
			<td>Sigma</td>
			<td>P8483</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine (PLO)</td>
			<td>Sigma</td>
			<td>3655</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>746436</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Sigma</td>
			<td>54317</td>
			<td></td>
		</tr>
		<tr>
			<td>See through self-sealable pouches</td>
			<td>Steriking</td>
			<td>SS-T2 (90x250)</td>
			<td>Autoclave pouches</td>
		</tr>
		<tr>
			<td>Sodium citrate dihydrate</td>
			<td>Fisher Scientific</td>
			<td>S279-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filters, sterile, PES 0.22 &#181;m, 30 mm Dia</td>
			<td>Research Products International</td>
			<td>256131</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-ISRIB</td>
			<td>Cayman</td>
			<td>16258</td>
			<td></td>
		</tr>
		<tr>
			<td>Vapor pressure osmometer</td>
			<td>Wescor, Inc.</td>
			<td>Model 5520</td>
			<td>Osmometer</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Biogems</td>
			<td>1293823</td>
			<td></td>
		</tr>
	</tbody>
</table>

a technique to generate a 2d monolayer of cerebellar cells from induced pluripotent stem cells

Source: Madencioglu, D. A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/64462">Modeling Human Cerebellar Development In Vitro in 2D Structure.</a>&#160;J. Vis. Exp. (2022).
This video demonstrates a protocol for differentiating human induced pluripotent stem cells into cerebellar progenitors. It involves transferring stem cell colonies to an ultra-low attachment plate to form embryoid bodies, promoting differentiation with growth factors, and then transferring the embryoid bodies onto a matrix-coated plate for adhesion. Subsequent steps involve inducing cell migration and adding a maturation medium to guide the development of glutamatergic and GABAergic cerebellar neuronal precursors and Purkinje cell progenitors.

A Technique to Generate a 2D Monolayer of Cerebellar Cells from Induced Pluripotent Stem Cells

1. Experimental preparation 

	Prepare basement membrane matrix (BMM)-coated plates.
	NOTE: BMM solidifies more quickly at warmer temperatures. Plates ...

Begin with human induced pluripotent stem cell colonies cultured on an adherent basement membrane matrix.
Add a cerebellar differentiation medium containing a specific hormone, growth factor, and small molecule inhibitors.
Lift the colonies using a glass pipette, and transfer them to an ultra-low attachment plate.
The low-attachment surface maintains the cells in suspension, facilitating their aggregation into three-dimensional embryoid bodies, or EBs.
The hormone, growth factor, and inhibitors bind to their respective target sites, promoting stem cell differentiation into neuronal progenitors.
Transfer the EBs to culture plate wells coated with a synthetic polymer and an adhesion protein to facilitate EB attachment.
Replace the medium with an inhibitor-free differentiation medium.
The cells begin to migrate outward from the EBs, forming a 2D monolayer.
Remove the medium and add a cerebellar maturation medium.
Growth factors in the medium induce the maturation of neuronal progenitors into distinct cerebellar neuronal precursors.

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Advanced Neuronal Models

35 Views. Source: Madencioglu, D. A., et al. Modeling Human Cerebellar Development In Vitro in 2D Structure. J. Vis. Exp. (2022). This video demonstrates a protocol for differentiating human induced pluripotent stem cells into cerebellar progenitors. It involves transferring stem cell colonies to an ultra-low attachment plate to form embryoid bodies, promoting differentiation with growth factors, and then transferring the embryoid bodies onto a matrix-coated plate for adhesion. Subsequent steps invo...

Video: A Technique to Generate a 2D Monolayer of Cerebellar Cells from Induced Pluripotent Stem Cells - Concept

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

The objective of this report is to describe the protocols to derive the retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells using different sizes of embryoid bodies.

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

JoVE Journal

Developmental Biology

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

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

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

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

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.

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

A Technique to Generate a 2D Monolayer of Cerebellar Cells from Induced Pluripotent Stem Cells

Transcript

A Technique to Generate a 2D Monolayer of Cerebellar Cells from Induced Pluripotent Stem Cells

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

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