generating forebrain-type cerebral organoids from human-induced pluripotent stem cells

Generating Forebrain-Type Cerebral Organoids from Human-Induced Pluripotent Stem Cells

To generate pluripotent stem cell aggregates, when the stem cell cultures reach 70% to 90% confluency, add 500 microliters of cell dissociation reagent to one well of the six-well culture plate to detach the cells.
Induced pluripotent stem cells, which are adapted to monolayer culture conditions, are a good cell type to generate aggregates as they are less prone to stress-induced cell death during the dissociation and aggregation procedure.
After 5 to 10 minutes at 37 degrees Celsius, gently tap the plate, and use 2 milliliters of DMEM/F-12 to wash the cells from the bottom of the well. Transfer the resulting cell suspension into a 15-milliliter conical tube, and bring the volume of the cell solution up to 10 milliliters with more DMEM/F-12 medium.
After counting, transfer 4.5 times 10 to the third cells per pluripotent stem cell aggregate into a new 15-milliliter tube, and collect the cells by centrifugation. Resuspend the pellet in the appropriate volume of pluripotent stem cell medium, supplemented with 50 micromolar ROCK inhibitor to achieve a 4.5 x 103 cells per 150 microliters of medium concentration.
Next, add 150 microliters of cells to individual wells of a 96-well low-attachment U-bottom plate, and place the plate in a 37 degrees Celsius and 5% carbon dioxide incubator. For monitoring anterior neuroectoderm induction, use a tissue culture microscope to closely observe the morphologic changes of the pluripotent stem cell aggregates every day under low magnification.
On day 1, cell aggregates with clear borders should be observed. On day 2, carefully aspirate approximately 2/3 of the medium without disturbing the cell aggregates at the bottom of each well, and replace the discarded medium with 100 microliters of fresh pluripotent stem cell medium.
Between four and six days later, when the cell aggregates reach 350 to 450 micrometers in diameter and exhibit smooth edges, use a modified 100-microliter pipette tip to transfer up to 20 aggregates into a single 6-centimeter low-attachment culture plate containing 5 milliliters of cortical induction medium.
Replace the cortical induction medium with fresh medium once every three days, monitoring the morphology of the aggregates daily under the 4x objective. After four to five days in the cortical induction medium, the edges of the cell aggregates should begin to brighten at the surface, indicating neuroectodermal differentiation, and the radial organization of a pseudostratified epithelium should emerge.
To embed the neuroectodermal aggregates in a matrix scaffold, fill a basement membrane extract on ice for two to three hours. While the extract is thawing, use sterile scissors to cut plastic paraffin film into one 4-by-4 centimeter square per 16 organoids, and place each piece of film over an empty 100-microliter micropipette tip tray.
Press the paraffin film with a gloved fingertip so that small dimples appear, and clean the film with 70% ethanol. After UV irradiation in a closed, sterile biosafety cabinet for 30 minutes, use a modified 100-microliter pipette tip with a 1 and 1/2 to 2-millimeter opening to transfer each cell aggregate into one dimple in the film.
When all of the aggregates have been transferred, use an uncut 100-microliter pipette tip to carefully aspirate the medium from each dimple, and add 40 microliters of undiluted basement membrane extract to each cell aggregate.
Be careful not to damage the organoids by rough manipulation or aspiration into the pipette tip, as this will damage the developing neuroepithelium.
Use the pipette tip to position the aggregates into the middle of each drop, and use sterile forceps to carefully transfer the plastic paraffin film sheet into a 10-centimeter Petri dish. Place the dish in the incubator for 15 to 20 minutes.
While the extract is solidifying, add 5 milliliters of fresh cortical induction medium to one six-centimeter low attachment culture dish. At the end of the incubation, flip the paraffin film sheet over, and use sterile forceps to gently squeeze up to 16 polymerized droplets into each 6-centimeter dish. Then, return the aggregates to the cell culture incubator.
The next day, transfer the organoid culture dishes onto a rocking cell culture shaker tilted at a 5-degree angle at 14 RPM within a cell culture incubator with daily light microscopy monitoring until the aggregates reach the differentiation stage of interest.

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1. Generation of iPSC Aggregates
<ol>
	<li>Generation of single-cell monolayer cultures from iPSC colonies
	<ol>
		<li>Prepare a basement membrane extract (BME) coated 6-well plate. Thaw BME on the ice at 4 &#176;C for 2-3 h, dilute it with cold Dulbecco&#39;s Modified Eagle Medium F12 (DMEM-F12; 1:50 dilution), cover the plate with 1 mL/well of the diluted BME solution, and store the plate overnight at 4 &#176;C.</li>
		<li>Aspirate the medium and wash intact iPSC colonies of at least two wells of a 6-well plate with 0.5 mM EDTA in phosphate-buffered saline (PBS) twice before incubating colonies with 0.5 mM ethylenediaminetetraacetic acid (EDTA) in PBS for 4 min at room temperature (RT). Aspirate EDTA solution and gently detach the colonies by washing them off the bottom of the dish with 5 mL of DMEM-F12 medium. Collect them in a 15-mL tube and pellet them by centrifugation (4 min at 1,200 x g at RT). 
		NOTE: iPSCs reprogrammed from commercially available fibroblasts have been successfully used.</li>
		<li>Aspirate the supernatant and incubate the iPSC colonies with 500 &#181;L of cell-dissociation reagent for 6 min at 37 &#176;C.</li>
		<li>Pipet the cell suspension several times gently up and down with a 1 mL pipette to break the remaining cell clusters into single cells.</li>
		<li>Add 4 mL of DMEM-F12 to the cell suspension to dilute the cell-dissociation reagent.</li>
		<li>Spin the cells down at 1,200 x g for 4 min at RT.</li>
		<li>Resuspend the cells in 2 mL of iPSC medium supplemented with 5 &#181;M Y-27632 and seed cells into one well of a BME-coated 6-well plate. 
		NOTE: Use the iPSC medium specified in the Table of Materials for a single-cell monolayer iPSC cultures.</li>
		<li>On the next day, replace the medium with fresh iPSC medium lacking Y-27632. From this point, continue to culture the cells, changing the medium every day until iPSCs are 100% confluent. Depending on the cell line and the confluency of the starting wells, this will take between 2-4 days.</li>
		<li>Once confluent, passage the iPSCs. Aspirate medium and apply 500 &#181;L of cell-dissociation reagent on the cells.</li>
		<li>Incubate the cells for 5-10 min at 37 &#176;C. Gently rock the plate to detach the cells.</li>
		<li>Wash the cells from the well using 2 mL of DMEM-F12 and collect them in a 15-mL tube. Add DMEM-F12 for a total volume of 5 mL.</li>
		<li>Spin down the cells at 1,200 x g for 4 min at RT and aspirate the supernatant.</li>
		<li>Seed the cells in iPSC medium supplemented with 5 &#181;M Y-27632 in a 1:2 to 1:4 ratio on a BME coated 6-well plate (2 mL/well of medium).</li>
		<li>Continue to culture the cells for 2-5 days and change iPSC medium lacking Y-27632 every day. Once confluent, passage iPSCs (steps 1.1.4-1.1.8). 
		NOTE: Culture the iPSCs for at least 2 passages as a monolayer in the iPSC medium specified in the Table of Materials before using them for the generation of iPSC aggregates to allow the cells to adapt to the culture conditions.</li>
	</ol>
	</li>
	<li>Use monolayer iPSC cultures when they are 70-90% confluent for the generation of iPSC aggregates. 
	&#8203;NOTE: iPSC adapted to the culture conditions as single cells are less prone to stress that leads to cell death during the dissociation and aggregation procedure. Monolayer iPSC cultures need to display typical pluripotent morphology with no evidence of differentiation. Test the cultures on a regular basis for mycoplasma contamination. Work only with mycoplasma-free iPSC cultures.
	<ol>
		<li>Aspirate the culture medium from one well of a 6-well plate and apply 500 &#181;L of cell-dissociation reagent on the cells.</li>
		<li>Incubate the cells for 5-10 min at 37 &#176;C. Gently rock the plate to detach the cells.</li>
		<li>Wash cells from the well using 2 mL of DMEM-F12 and collect them in a 15-mL tube. Add DMEM-F12 for a total volume of 10 mL.</li>
		<li>For cell counting, take 25 &#181;L from the cell suspension and mix it with 25 &#181;L of trypan blue to mark dead cells. Count the living cells using a counting chamber.</li>
		<li>Collect enough cells (4,500 cells per iPSC aggregate) from the cell suspension in a 15 mL tube.</li>
		<li>Spin down the cells at 1,200 x g for 4 min at RT and aspirate the supernatant.</li>
		<li>Resuspend the cells in an appropriate volume of iPSC medium supplemented with 50 &#181;M Y-27632 to obtain 4,500 live cells per 150 &#181;L. 
		NOTE: Using a high concentration of Y-27632 (50 &#181;M) is critical for the survival of the iPSCs.</li>
		<li>Plate 150 &#181;L in each well of a low-attachment 96-well U-bottom plate and place it in the incubator at 37 &#176;C and 5% CO2. 
		NOTE: When generating iPSC aggregates, consider that at least 6 organoids are needed for quality control at day 20 (see Section 5).</li>
	</ol>
	</li>
</ol>
2. Induction of Anterior Neuroectoderm
<ol>
	<li>Closely monitor morphology changes of the iPSC aggregates every day under the tissue culture microscope using a 4X or 10X power lens. Observe at day 1, cell aggregates with clear borders. Continue to culture iPSC aggregates in the incubator at 37 &#176;C and 5% CO2. 
	NOTE: A certain number of dead cells/well is normal and will not affect organoid generation.</li>
	<li>Feed the iPSC aggregates starting from day 2 and continuing with every other day by gently aspirating approximately 2/3 of the medium without disturbing the cell aggregates at the bottom. Add an additional 100 &#181;L of iPSC medium lacking Y-27632. 
	NOTE: Within 4-6 days the cell aggregates will be 350-450 &#181;m in diameter and exhibit smooth edges.</li>
	<li>At this stage, pool the cell aggregates with a cut 100 &#181;L pipette tip into a low-attachment 6 cm dish (maximum of 20 aggregates/dish) in cortical induction medium containing DMEM-F12 with N2 supplement (1:200), B27 supplement (1:100), glucose (0.2 mg/mL), cyclic adenosine monophosphate (cAMP; 0.15 &#181;g/mL), 0.5% non-essential amino acids (NEAA), 1% L-alanyl-L-glutamine, heparin (10 &#181;g/mL), and the compounds LDN-193189 (180 nM), A83-01 (500 nM), and inhibitor of Wnt response-1 (IWR-1) (10 &#181;g/mL). Feed the cell aggregates by changing the cortical induction medium 3 days after transferring them to the 6-cm dish.</li>
	<li>Closely monitor morphological changes during cortical induction under the tissue culture microscope using a 4X power lens. 
	NOTE: After 4-5 days in cortical induction medium, edges of the cell aggregates should begin to brighten at the surface, indicating neuroectodermal differentiation. At this stage, radial organization of a pseudostratified epithelium emerges. Proceed to Section 3 to embed the cell aggregates in a BME matrix.</li>
</ol>
3. Embedding of Neuroectodermal Aggregates in a Matrix Scaffold
<ol>
	<li>Thaw BME on ice at 4 &#176;C for 2-3 h. Aliquot undiluted BME in sufficient amounts.</li>
	<li>Prepare a plastic paraffin film sheet for the embedding procedure. Cut the plastic paraffin film using sterile scissors in 4 cm x 4 cm large pieces, put a piece of the plastic paraffin film over an empty 100 &#181;L tip tray for 100 &#181;L tips, and press with a gloved finger so that small dimples in the plastic paraffin film sheet are created (1 dimple/cell aggregate needed). Clean the plastic paraffin film with 70% ethanol and irradiate it with UV light (power: 15 watts, wavelength: 435 nm) under the closed sterile bench for 30 min.</li>
	<li>Transfer each cell aggregate to one dimple of the plastic paraffin film sheet using a 100 &#181;L cut tip with 1.5-2 mm opening in diameter. In the case that two cell aggregates are fused, do not separate them but transfer them together to one dimple.</li>
	<li>Gently aspirate the medium surrounding the cell aggregates using an uncut 100 &#181;L pipette tip. 
	NOTE: Be careful not to suck the cell aggregates into the tip, as this will damage the aggregates.</li>
	<li>Add 40 &#181;L of undiluted BME to each cell aggregate.</li>
	<li>Position each cell aggregate in the middle of the BME drop using an uncut 100 &#181;L pipette tip. 
	NOTE: Be very careful not to harm the developing neuroepithelium with the pipette tip.</li>
	<li>Carefully transfer the plastic paraffin film sheet using sterile forceps into a 10 cm Petri dish (or another sufficient cell culture dish) and place the dish in the incubator for 15-20 min to allow the BME to solidify.</li>
	<li>Meanwhile, prepare a low-attachment 6 cm dish containing 5 mL of cortical induction medium.</li>
	<li>After the polymerization of BME, remove the droplets containing the cell aggregates from the plastic paraffin film sheet. Using sterile forceps, turn the plastic paraffin film over and gently squeeze the cell aggregates into the slightly tilted (about 30 &#176;) low-attachment 6 cm dish until the droplets fall off the plastic paraffin film sheet. Transfer a maximum of 16 cell aggregates into one 6 cm dish.</li>
	<li>Continue to incubate the cell aggregates at 37 &#176;C.</li>
</ol>
4. Generation of Forebrain-type Organoids from Neuroectodermal Aggregates
<ol>
	<li>One day after embedding in a BME matrix, place organoid culture dishes on a rocking cell culture shaker with a tilting angle of 5 &#176; and 14 rpm, installed in a cell culture incubator.</li>
	<li>Monitor organoids every day. Homogeneous neuroepithelial loop-like structures gradually develop after embedding in the BME matrix.</li>
	<li>When neuroepithelial loop structures are visible, change the medium to organoid differentiation medium containing DMEM-F12 with N2 supplement (1:200), B27 supplement (1:100), glucose (0.2 mg/mL), cAMP (0.15 &#181;g/mL), 0.5% NEAA, 1% L-alanyl-L-glutamine, insulin (2.5 &#181;g/mL)</li>
	<li>Replace the organoid differentiation medium every 3-4 days until the desired differentiation time point is reached. Then, fix organoids (see Section 5). 
	NOTE: Occasionally, the tissue may exhibit buds of optically translucent tissue without loop structures. Although this is not ideal, this is not affecting the development of cortical structures. The organoids can be cultured in organoid differentiation medium for up to 40 days. For extended culture periods (40-100 days) the organoid differentiation medium can be supplemented with 1:50 BME to increase tissue complexity and with BDNF and GDNF to allow neuronal survival and maturation.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A83-01</td>
			<td>StemGent</td>
			<td>130-106-274</td>
			<td>500 nM</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>1 to 100</td>
		</tr>
		<tr>
			<td>Basement membrane extract (e.g. Geltrex)</td>
			<td>Gibco</td>
			<td>A14132-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclic adenosine monophosphate (cAMP)</td>
			<td>Sigma-Aldrich</td>
			<td>A9501</td>
			<td>0.15 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Cell-dissociation reagent (TrypLE Express)</td>
			<td>Gibco</td>
			<td>12605028</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting chamber e.g. Fuchs-Rosenthal</td>
			<td>Karl Hecht</td>
			<td>40449001</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Carl Roth</td>
			<td>HN06.3</td>
			<td>0.2 mg/mL</td>
		</tr>
		<tr>
			<td>DMEM-F12 L-Glutamin</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding molds (Tissue-Tek Cryomold)</td>
			<td>Sakura Finetek</td>
			<td>4565</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>E6511</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>Gelantin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-25KU</td>
			<td>10 ug/mL</td>
		</tr>
		<tr>
			<td>Inhibitor of WNT response (IWR-1)</td>
			<td>Enzo Life Science</td>
			<td>BML-WN103-0005</td>
			<td>10 ug/mL</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>91077C</td>
			<td>2.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>L-alanyl-L-glutamine (GlutaMax)</td>
			<td>Gibco</td>
			<td>35050038</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Low-adhesion 6 cm plates</td>
			<td>Labomedic</td>
			<td>2081646L</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-adhesion 10 cm plates</td>
			<td>Labomedic</td>
			<td>2081646O</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN-193189</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-171</td>
			<td>180 nM</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td>1 to 200</td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Gibco</td>
			<td>11140035</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>4.00%</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic paraffin film (Parafilm)</td>
			<td>BRAND GMBH + CO KG</td>
			<td>701606</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y-27632</td>
			<td>Cell Guidance Systems</td>
			<td>SM02-100</td>
			<td>5 &#181;M or 50 &#181;M</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes 15 mL</td>
			<td>Corning Life Sciences</td>
			<td>734-0451</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides e.g. Superfrost Plus Microscope Slides</td>
			<td>Thermo Scientific</td>
			<td>4951PLUS4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture 6 well plate</td>
			<td>Falcon</td>
			<td>734-0019</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture 24 well plate</td>
			<td>Falcon</td>
			<td>734-0949</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-low-binding 96 well lipidure-coat plate A-U96</td>
			<td>Amsbio</td>
			<td>AMS.51011610</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Krefft, O. et al., <a href="https://app.jove.com/v/56768">Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video outlines the creation of forebrain-type organoids from human induced pluripotent stem cells (iPSCs), demonstrating the progression from stem cell aggregates to complex structures that mimic the brain&#39;s cortex and differentiate into various neuron types.

1. Generation of iPSC Aggregates

	Generation of single-cell monolayer cultures from iPSC colonies
	
		Prepare a basement membrane extract (BME) coated ...

Begin with a low-attachment well plate containing human-induced pluripotent stem cells or iPSCs in a stem cell medium supplemented with a Rho-kinase inhibitor.
The inhibitors enter the cells, block the Rho-kinase enzymes, and maintain cell viability. This promotes the formation of iPSC aggregates.
Replace the medium with a stem cell medium to allow aggregates to grow. Transfer them into a low-attachment culture dish with a cortical induction medium.
The components of this medium cause the iPSCs to differentiate into neuroectodermal cells, a neural precursor.
Transfer each neuroectodermal aggregate onto a film with shallow wells.
Replace the medium with a basement membrane matrix and incubate to allow matrix solidification and entrapment of aggregates.
Transfer these matrix-embedded aggregates to a dish containing a cortical induction medium and incubate with agitation.
Over time, the neuroectodermal cells utilize the differentiation factors and develop into neuroepithelial cells.
Change the medium to an organoid differentiation medium, which allows the cells to mature into more specialized cell types, forming a cerebral organoid that resembles the forebrain cortex.

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비디오: 인간 유도 만능 줄기 세포에서 전뇌형 대뇌 오가노이드 생성 - 실험

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

인간의 다 능성 줄기 세포에서 도파민 신경 세포의 분화를 감독

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

연구

JoVE Journal

신경과학

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

집에서 만든 미니 생물 반응기에서 유도 만능 줄기 세포에서 뇌 오르간성 생성

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

발생학

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Neisseria meningitidis 유도만능줄기세포 유래 뇌내피세포의 감염

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...

Generating Forebrain-Type Cerebral Organoids from Human-Induced Pluripotent Stem Cells

내레이션 대본

Generating Forebrain-Type Cerebral Organoids from Human-Induced Pluripotent Stem Cells