generating induced pluripotent stem cell-derived cerebral organoids

Generating Induced Pluripotent Stem Cell-Derived Cerebral Organoids

Aspirate the medium with a pipette, and wash the cells once with DPBS. Then, add 500 microliters of cell detachment solution, or 0.5 millimolar EDTA, to dissociate the colonies, and incubate at 37 degrees Celsius for 3 to 5 minutes. Next, add 1 milliliter of fresh E8 media to each well, and pipette gently to detach all cells.
Transfer the entire cell suspension into a 15-millimeter centrifuge tube, and add another 1 milliliter of fresh E8 media. Then, spin down the cells at 290 g for 3 minutes at room temperature.
After discarding the supernatant, resuspend the cell pellet in 1 milliliter of E6 media, supplemented with 50 micromolar ROCK inhibitor, and count the cells using a hemocytometer.
Prepare a cell suspension of the desired seating density in E6 media, supplemented with the ROCK inhibitor. Then, add 150 microliters of the cell suspension into each well of a 96-well ultra-low attachment plate.
Next, centrifuge the plate at 290 g for 3 minutes at room temperature to force-aggregate the cells. Then, incubate the plate at 37 degrees Celsius in a humidified atmosphere with 5% carbon dioxide for embryoid body formation.
After 24 hours of incubation, transfer the plate to the hood, and carefully aspirate 120 microliters of the media using a pipette, taking care not to aspirate the embryoid body by lowering the pipette tip too far into the well.
Next, add 150 microliters of fresh E6 media, supplemented with 2 micromolar XAV939 and the SMAD inhibitors to each well. Transfer the plate back to the incubator, and replace the medium daily with freshly prepared E6, supplemented with the inhibitors.
At day seven of incubation, check whether all the embryoid bodies have reached a diameter of 550 to 600 micrometers and display a smooth, clear edge. At this stage, they are ready to be embedded in an extracellular matrix.
To embed the organoids, prepare dimpled embedding sheets by placing a 4-inch long thermoplastic sealing film sheet on an empty P200 box. Then use a 15-milliliter conical tube, or a 500-microliter microcentrifuge tube, and gently press down the film sheet into the holes to make 12, or the desired number of dimples.
Next, spray 70% ethanol on the film sheet, and let it dry inside the hood with the UV light on for at least 30 minutes. Meanwhile, thaw a sufficient quantity of basement membrane matrix on ice. Then, using a wide bore P200 tip, transfer one embryoid body from the 96-well plate to each dimple. Remove as much media as possible with a normal pipette tip, but do not let the embryoid body dry.
Next, with a regular pre-chilled P200 tip, add approximately 30 microliters of undiluted membrane matrix to each organoid, ensuring that the embryoid body is as close as possible to the center of the droplet.
Once all embryoid bodies are embedded in the matrix, place the film sheet in a sterile Petri dish. Then incubate the Petri dish at 37 degrees Celsius for 10 minutes in a humidified atmosphere with 5% carbon dioxide to allow the membrane matrix to solidify.
For organoid differentiation, add 5 milliliters of the prepared differentiation media with B27 without vitamin A to one well of an ultra-low attachment 6-well plate. Pre-warm the plate to 37 degrees Celsius in an incubator. Once the membrane matrix has solidified, transfer the embedded embryoid bodies to the 6-well plate by pushing out the dimples from the back of the film sheet. If necessary, use 1 milliliter of media from the well, and pipette it onto the sheet to detach the droplets from the film sheet.

generating-induced-pluripotent-stem-cell-derived-cerebral-organoids

Universidad de Cartagena UDEC

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Figure 1 illustrates a schematic overview of the workflow of this protocol.
1. iPSC reprogramming and maintenance
<ol>
	<li>Collect 8 mL of the subject&#39;s blood (aged or disease patient; ages 65 and older) into cell preparation tubes (CPT) with sodium citrate or into ethylenediaminetetraacetic acid (EDTA) or heparinized tubes.</li>
	<li>Centrifuge at 1,800 x g for 30 min at room temperature (RT) to collect pellet containing only peripheral blood and no serum.</li>
	<li>Use specialized reprogramming vectors according to the manufacturer&#39;s protocol (see Table of Materials) to obtain iPSCs.</li>
	<li>Culture iPSCs in feeder-free, lactose dehydrogenase elevating virus (LDEV)-free reduced growth factor basement membrane matrix coated 6-well culture plates in Essential 8 (E8) media (see Table of Materials) at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
	<li>Maintain iPSCs in E8 media for 3-4 days in order to avoid overcrowding or spontaneous differentiation.</li>
	<li>Validate the pluripotency of the iPSC lines using immunofluorescent markers (step 2) and test for mycoplasma (step 3).</li>
</ol>
2. Pluripotency staining
<ol>
	<li>To conserve solutions and antibodies, seed the cells on coated (step 1.4) 24-well culture plates 3-4 days prior to analysis. Aspirate the medium with a pipette and fix the cells with 4% formaldehyde in phosphate-buffered saline (1x PBS) for 15-20 minutes at RT.</li>
	<li>Wash the cells 3x with 1x PBS and permeabilize with 0.1% Triton-X 100 in 1% bovine serum albumin (BSA) in PBS for at least 15 min, but no more than 1 h at RT.</li>
	<li>Wash 3x with 1x PBS and block with 5% BSA in PBS for 30 min at RT.</li>
	<li>Wash again 3x with 1x PBS and add the antibodies Sox2 and Oct3/4 (1:100 dilution; see Table of Materials), and the nucleic stain DAPI in 1% BSA (1:4,000 dilution) overnight at 4 &#176;C . Wrap in aluminum foil to protect from light.</li>
	<li>Wash 3x with 1x PBS and leave the cells in PBS. Image with a fluorescence microscope at a 10-20x magnification. Ensure that the pluripotency markers sex determining region Y-box 2 (Sox2) and octamer binding transcription factor 3/4 (Oct3/4) are localized in the nuclei of the cells.</li>
</ol>
3. Mycoplasma testing
NOTE: Refer to the detection kit&#39;s protocol (see Table of Materials) for detailed assay execution and analysis steps. The detection kit provides the reagent, the substrate, and the assay buffer for mycoplasma testing.
<ol>
	<li>Prior to passaging cells or refreshing medium, collect 2-3 mL of cell culture medium in a centrifuge tube and pellet any cells or debris at 200 x g for 5 min at RT. Store the supernatant at 4 &#176;C for &#8804;5 days. Incubate the cells with the medium for at least 24 h to ensure a detectable signal.</li>
	<li>Add 100 &#181;L of cell supernatant to a fresh tube or well of a white-walled 96-well plate (opaque bottom recommended, see Table of Materials). Reconstitute the reagent and substrate in the assay buffer and equilibrate for 15 min at RT.</li>
	<li>Add 100 &#181;L of the reagent to the sample and incubate for 5 min at RT. Measure the luminescence with a luminometer (measurement #1).</li>
	<li>Add 100 &#181;L of the substrate to the sample and incubate for 10 min at RT. Measure the luminescence (measurement #2).</li>
	<li>Determine the mycoplasma contamination by the ratio of measurement #2 to #1. Refer to the kit&#39;s manual for interpretation of the results.</li>
</ol>
4. Microfilament preparation
<ol>
	<li>Begin the preparation of the poly (lactic-co-glycolic acid) (PLGA, see Table of Materials) microfilaments by fraying the suture strand with the blunt end of a scalpel. Spay the frayed fiber lightly with 70% ethanol.</li>
	<li>Under the microscope and using a ruler, begin cutting the PLGA fiber into fragments of 500 &#181;m to 1 mm long strands. Cut about 25 mm of the fiber in total. Keep the filaments in a 15 mL tube with a 1 mL antibiotic-antimycotic solution.
	<ol>
		<li>In the hood, dilute the fiber solution with 10 mL of DMEM/F-12 (Table 1). Vortex well to mix the solution. 
		&#8203;NOTE: Work in a cell culture flow hood in a sterile environment.</li>
	</ol>
	</li>
	<li>Add 20 &#181;L of the fiber solution to three embryoid body (EB) forming wells of a 96-well plate. Under brightfield microscopy, count and average the fibers per well. Dilute or concentrate to average 5-10 PLGA microfilaments per well. Prepare each well in this manner.</li>
	<li>The wells are now ready to be seeded with cells. Store the plate at room temperature until needed or at 4 &#730;C for the following day.</li>
</ol>
5. Embryoid Body (EB) formation
NOTE: All media and solutions must be warmed to RT.
<ol>
	<li>Once the iPSCs have reached 70%-80% confluency (Figure 2A), they are ready to be passaged and used for EB formation. Check the cells with a microscope at 10x-20x magnification. Ensure that the colonies display minimal (&#60;10%) areas of spontaneous differentiation.</li>
	<li>Aspirate the medium with a pipette and wash the cells once with Dulbecco&#39;s Phosphate-Buffered Saline (DPBS). Dissociate the colonies by adding 500 &#181;L of cell detachment solution (see Table of Materials) or 0.5 mM of EDTA and incubate for 3-5 min at 37 &#176;C. 
	NOTE: Work in a cell culture flow hood in a sterile environment.</li>
	<li>Collect the released cells by adding 1 mL of fresh E8 media to each well and pipette gently until all cells are detached.</li>
	<li>Transfer 1.5 mL of cell suspension into a 15 mL tube, and add another 1 mL of fresh E8 media to reach a total volume of 2.5 mL.</li>
	<li>Centrifuge at 290 x g for 3 min at RT.</li>
	<li>Aspirate the supernatant with a pipette, resuspend the cell pellet in 1 mL of Essential 6 (E6, see Table of Materials) media supplemented with 50 &#181;M Rho-associated protein kinase (ROCK) inhibitor, and count the cells using a hemocytometer.</li>
	<li>Prepare a cell suspension of 60,000-90,000 cells/mL, depending on the desired seeding density, in E6 media supplemented with 50 &#181;M of ROCK inhibitor (see Table of Materials).</li>
	<li>Add 150 &#181;L of the cell suspension into each well of a 96-well ultra-low attachment (ULA) plate (or a prepared concave plate). Seed 9,000-11,000 cells per well.</li>
	<li>Centrifuge the plate to force-aggregate the cells at 290 x g for 1 min at RT. Place the plate in the incubator at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
</ol>
6. Neuroepithelial induction
<ol>
	<li>After 24 h, carefully aspirate 120 &#181;L of the media with a pipette. Ensure not to aspirate the EB by lowering the pipette tip too far into the well.</li>
	<li>Add 150 &#181;L of E6 media (at RT) supplemented with 2 &#181;M of XAV939 and the Suppressor of Mothers against Decapentaplegic (SMAD) inhibitors: 10 &#181;M of SB431542 and 500 nM of LDN 193189 per well (see Table of Materials).</li>
	<li>Change the medium daily with freshly prepared E6 media supplemented with 2 &#181;M of XAV939, 10 &#181;M of SB431542, and 500 nM of LDN 193189. 
	NOTE: By day 6 (DIV6), the EBs should have a diameter of 550-600 &#181;m and be ready for further differentiation.</li>
</ol>
7. Organoid differentiation and maturation
NOTE: All media needs to be warmed to RT.
<ol>
	<li>At approximately DIV7, check whether all the EBs have reached a diameter of 550-600 &#181;m and display a smooth and clear edge (Figure 2B); at this stage, they are ready to be embedded in an extracellular matrix (ECM). 
	NOTE: Work in a sterile environment.</li>
	<li>Prepare dimpled embedding sheets from the thermoplastic sealing film (see Table of Materials) by placing a film sheet (about 4 in long) on an empty P200 box. Using a 15 mL conical tube or a 500 &#181;L microcentrifuge tube, gently press down the film sheet into the holes to make 12 dimples. Spray the film sheet with 70% ethanol and let it dry inside the flow hood with the UV light switched on for at least 30 min.</li>
	<li>Thaw a sufficient quantity of basement membrane matrix (Matrigel, see Table of Materials) on ice and place it inside the flow hood. 
	NOTE: Per EB, approximately 30 &#181;L of undiluted membrane matrix is needed. Always keep the membrane matrix below 4 &#176;C to prevent it from gelling. Pre-chilled pipette tips are also recommended as they decelerate the matrix from polymerizing in the tip during pipetting, thereby reducing material loss.</li>
	<li>Using a wide-bore P200 tip, transfer one EB to each dimple, and remove as much media as possible with a normal pipette tip. Be mindful not to let the EBs dry. Using a regular P200 tip, add ~30 &#181;L of undiluted membrane matrix to each organoid, ensuring that the EB is at the center of the droplet.</li>
	<li>Once all EBs are embedded in the matrix, place the film sheet containing the EBs in a sterile Petri dish. 
	NOTE: Optionally, a small Petri dish filled with sterile water can be placed in the larger Petri dish next to the film sheet to prevent evaporation.
	<ol>
		<li>Transfer the dish to an incubator and incubate at 37 &#730;C in a humidified atmosphere with 5% CO2 for approximately 10 min to let the membrane matrix solidify.</li>
	</ol>
	</li>
	<li>For each set of 12 embedded organoids, prepare 5 mL of differentiation media with B27 without vitamin A (Table 1) in one well of a ULA 6-well plate. Pre-warm the plate to 37 &#176;C in an incubator.</li>
	<li>Once the incubation time is finished, transfer the embedded EBs to the ULA 6-well plate by taking the film sheet and pushing out the dimples from the back of the sheet. If necessary, take 1 mL from the well and pipette it onto the sheet to help the droplets detach from the film. 
	NOTE: Important morphological changes can be seen 1 day after embedding; EBs go from having smooth edges to bulging protrusions forming buds (Figure 2C).</li>
	<li>After 2 days (DIV9), perform a half-media change. Be careful not to aspirate or damage the membrane matrix droplets in the process.</li>
	<li>After 2 more days (DIV11), perform a full media change, supplementing the media with 3 &#181;M of CHIR99021 (see Table of Materials).</li>
	<li>At DIV14, change the media to differentiation media with B27 with vitamin A (Table 1) for a gradual increase in organoid size.</li>
	<li>At DIV16, place the well-plate on an orbital shaker at 90 rpm inside an incubator. Change the media every 2 days.</li>
	<li>Every 40 DIV, dilute 500 &#181;L of the membrane matrix for every 50 mL of media for additional nutrients in the media.</li>
</ol>
Table 1: Composition of the differentiation media used in the present study.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Final Concentration</td>
			<td>Volume (50 mL total)</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>50%</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>50%</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>N2 Supplement (100x)</td>
			<td>1x</td>
			<td>0.25 mL</td>
		</tr>
		<tr>
			<td>B27 Supplement -/+ Vitamin A (50x)</td>
			<td>0.5x</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>0.25%</td>
			<td>12.5 &#181;L</td>
		</tr>
		<tr>
			<td>GlutaMAX (100x)</td>
			<td>1x</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>MEM-NEAA (100x)</td>
			<td>0.5x</td>
			<td>0.25 mL</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>10 mM</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>Antibiotic/Antimycotic (100x)</td>
			<td>1x</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td>2-&#946;-mercaptoethanol</td>
			<td>50 &#181;M</td>
			<td>17.5 &#181;L</td>
		</tr>
		<tr>
			<td colspan="3">NOTE: some DMEM-F12 already contains GlutaMAX, no need to add additional.</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-&#946;-Mercaptoethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>31350010</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindoledihydrochloride (DAPI)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>67:40:00</td>
		</tr>
		<tr>
			<td>6-well Clear Flat Bottom CELLSTAR Cell Culture Multiwell Plate</td>
			<td>Greiner Bio-One</td>
			<td>657185</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Clear Flat Bottom Ultra-Low Attachment Well Plate</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Clear Round Bottom Ultra-Low Attachment Microplate</td>
			<td>Corning</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate</td>
			<td>Thermo Fisher Scientific</td>
			<td>136101</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma-Aldrich</td>
			<td>46964</td>
			<td>cell detachment solution</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Suppement (with Vitamin A) (50x)</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement (minus Vitamin A) (50x)</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer&#8482; Glass Mononuclear Cell Preparation (CPT) Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-685-125</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td>With plate holders</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Selleck Chemicals</td>
			<td>S2924</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoTune Sendai Reprogramming Vector</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1378001</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR primers | human | MAPT</td>
			<td>Bio-Rad</td>
			<td>dHsaCPE192234</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR primers | human | RBFOX3 (NeuN)</td>
			<td>Bio-Rad</td>
			<td>dHsaCPE5052108</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320074</td>
			<td></td>
		</tr>
		<tr>
			<td>Doublecortin (DCX)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-8066</td>
			<td>09:20</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td>no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Eppendorf cups, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030 125.215</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential 6</td>
			<td>Gibco</td>
			<td>A1516401</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential 8</td>
			<td>Gibco</td>
			<td>A1517001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes, 15 mL, conical</td>
			<td>Greiner Bio-One</td>
			<td>188271-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>37% Stock solution, diluted to 4% in PBS</td>
		</tr>
		<tr>
			<td>Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1413202</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax (100x)</td>
			<td>Gibco</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer cell counter</td>
			<td>Hausser scientific</td>
			<td>1490</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>15-630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN 193189</td>
			<td>StemCell Technologies</td>
			<td>72147</td>
			<td></td>
		</tr>
		<tr>
			<td>MAP2</td>
			<td>Abcam</td>
			<td>ab32454</td>
			<td>04:20</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free</td>
			<td>Corning</td>
			<td>356230</td>
			<td>basement membrane matrix</td>
		</tr>
		<tr>
			<td>MEM-Non Essential Amino Acid Solution (MEM-NEAA; 100x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Multilabel Counter Victor 3 Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>1420</td>
			<td>luminometer</td>
		</tr>
		<tr>
			<td>MycoAlert Mycoplasma Detection Kit</td>
			<td>Lonza</td>
			<td>LT07-318</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>NeuN</td>
			<td>Millipore</td>
			<td>MAB377</td>
			<td>09:20</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Oct-3/4 Antibody (C-10) Alexa Fluor 647</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-5279 AF647</td>
			<td>02:40</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td>thermoplastic film sheet</td>
		</tr>
		<tr>
			<td>PAX6</td>
			<td>Thermo Fisher Scientific</td>
			<td>42-6600</td>
			<td>04:20</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(lactic-co-glycolic acid) (PLGA) microfilaments</td>
			<td>Ethicon</td>
			<td>J463</td>
			<td></td>
		</tr>
		<tr>
			<td>QX200 Droplet digital PCR system</td>
			<td>Bio-Rad</td>
			<td>1864001</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor (Y27632)</td>
			<td>Selleck Chemicals</td>
			<td>S1049</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>R&#38;D Systems</td>
			<td>1614/50</td>
			<td></td>
		</tr>
		<tr>
			<td>SOX2 Monoclonal Antibody (Btjce), Alexa Fluor 488, eBioscience</td>
			<td>Invitrogen</td>
			<td>53-9811-80</td>
			<td>02:40</td>
		</tr>
		<tr>
			<td>Synapsin I (SYN)</td>
			<td>Calbiochem</td>
			<td>574777</td>
			<td>04:20</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>TUJ1</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-80005</td>
			<td>Beta-3-tubulin; 1:500</td>
		</tr>
		<tr>
			<td>XAV939</td>
			<td>Tocris Bioscience</td>
			<td>3748</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Koch, L. S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/64586">Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research.</a> J. Vis. Exp. (2023).
This video demonstrates an assay to generate cerebral organoids from induced pluripotent stem cells (iPSCs). Human iPSCs are induced to form embryoid bodies (EBs). The EBs are cultured over a polymer scaffold using an induction medium, promoting differentiation into neural stem cells, progenitor cells, and immature neurons. Finally, the EBs are embedded in a matrix and transferred to a maintenance medium for cerebral organoid formation.

<img alt="Figure 1" src="/files/ftp_upload/22352/22352_Figure1.jpg" /> 
Figure 1: Schematic illustration of the workflow and timeline of this method.
<img alt="Figure 2" src="/files/ftp_upload/22352/22352_Figure2.jpg" /> 
Figure 2: Differentiation and maturation of cerebral organoids. (A) Representative image of an iPSC culture at 70%-80% confluency. (B</strong...

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Figure 1 illustrates a schematic ...

Take colonies of induced pluripotent stem cells, or iPSCs, cultured on a supporting matrix.
Add a solution containing enzymes to dissociate the colonies into single cells.
Transfer the cells into a tube, centrifuge them, and discard the supernatant.
Resuspend the iPSCs in a growth medium.
Transfer the cells into a microplate containing biocompatible polymer fibers.
Centrifuge to force-aggregate iPSCs, forming embryoid bodies, or EBs, on the polymer scaffold.
Transition to a neural induction medium that promotes iPSC proliferation and differentiation into neural stem cells, progenitor cells, and immature neurons.
Upon incubation, the cells undergo self-organization.
Place the embryoid bodies onto a dimpled sheet and add drops containing extracellular matrix proteins.
Incubate to solidify the matrix, embedding the EBs.
Transfer the EBs into a microplate containing a neural maintenance medium. Incubate.
The EBs undergo cellular proliferation to form protrusions and buddings and differentiate into mature neurons, forming cerebral organoids.

56 Views. Generazione di organoidi cerebrali derivati da cellule staminali pluripotenti indotte

Video: Generazione di organoidi cerebrali derivati da cellule staminali pluripotenti indotte - Experiment

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

Generazione di organoidi cardiaci umani auto-assemblanti derivati da cellule staminali pluripotenti

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

Ricerca

JoVE Journal

Biochimica

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

Generazione di un chip di barriera emato-encefalica basata sull'iPSC umano

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Biologia dello sviluppo

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental defects whose underlying mechanisms remain to be elucidated. A major roadblock is the lack of effective models recapitulating the early-onset neuronal impairment seen in the patients. Advances in the technology of induced pluripotent stem cells (iPSCs) enable the generation of three-dimensional (3D) brain organoids that can be used to investigate the impact of diseases on the development and organization of the nervous system. Researchers, including these authors, have recently introduced human brain organoids to model mitochondrial disorders. This paper reports a detailed protocol for the robust generation of human iPSC-derived brain organoids and their use in mitochondrial bioenergetic profiling and imaging analyses. These experiments will allow the use of brain organoids to investigate metabolic and developmental dysfunctions and may provide crucial information to dissect the neuronal pathology of mitochondrial diseases.

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Generazione di organoidi del cervello umano per la modellazione della malattia mitocondriale

Mitochondrial diseases represent the largest class of inborn errors of metabolism and are currently incurable. These diseases cause neurodevelopmental ...

Generating Induced Pluripotent Stem Cell-Derived Cerebral Organoids

Trascrizione

Generating Induced Pluripotent Stem Cell-Derived Cerebral Organoids