generation of dopaminergic neuronal organoids from human embryonic stem cells

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Replace the stem cell medium used to maintain pluripotency features of hESCs, with a serum-free medium containing dual SMAD inhibition cocktail to start neural induction, and 10 micromolar ROCK inhibitor to increase the survival rate of cells during passage the next day. On day one, prepare the microwell plate with 2.5 milliliters per well of serum-free medium containing the same concentration of ROCK inhibitor and dual SMAD inhibition molecules.
To differentiate cells towards the neural tube ventral pattern, add SHH and FGF8 at 100 nanograms per milliliter and two micromolar smoothened agonist. After adding the medium, centrifuge the plate at 1,200 times g for five minutes to remove air bubbles from the microwells. Once the microwell plate is ready to use with half differentiating medium, remove the medium of hESCs, and quickly wash with calcium and magnesium chloride-free PBS.
Dissociate the colonies into a single-cell suspension by adding 7.5 mL of recombinant enzymatic solution into a T75 flask. Incubate the cells for two minutes at 37 degrees Celsius, and then add 7.5 of DMEM F-12. Subsequently, collect the cell suspension, and centrifuge at 300 times g for five minutes. Then, remove the supernatant, and count the cells in the same medium used to prepare the microwell plate.
Adjust the medium volume to obtain a cell suspension, allowing to form neurospheres containing 1,000 cells per microwell, by preparing 4.7 million cells in 2.5 milliliters of medium, and adding it to the previous 2.5 milliliters of medium already placed in the plate. In order to correctly distribute the cells in each microwell, gently shake the plate, and centrifuge it at 300 times g for five minutes. Next, incubate the plate at 37 degrees Celsius for 24 hours to generate spheres.
On day two, gently flush the microwells with medium, and transfer the spheres in a tissue-treated six well-plate. Place the spheres in rotation at 37 degrees Celsius, and change half the medium with fresh medium every two to three days. On day 3 to 13, to enhance neural induction and convert to neural progenitors with a midbrain identity, supplement the medium with three micromolar GSK-3-beta inhibitor. Split the sample into two new tissue-treated six well-plates to reduce the sphere density and avoid sphere aggregation.
On day eight, start the neural maturation by switching to new medium with growth factors, and change the medium every two to three days. On day 21, place one culture plate insert in one well of a new six-well plate, and add 1.2 milliliters of neural maturation medium used for neurosphere differentiation underneath the membrane insert. Then, depose the PTFE membrane on a culture plate insert.
Finally, to generate the neural organoid, seed around 100 neurospheres under air-liquid interface conditions on the PTFE membrane. Stop rotation from this step and change the medium every two to three days until the required differentiation time-point is achieved.

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1. hESC-derived dopaminergic organoids for Parkinson&#39;s Disease (PD) studies
<ol>
	<li>Day 0: Amplify hESCs in 2D culture up to 60% confluency (day 0), then replace stem cell media used to maintain pluripotency features of hESCs with a serum-free medium. Start neural induction by supplementing culture medium with 0.5 &#181;M bone morphogenetic protein (BMP) inhibitor and 10 &#181;M transforming growth factor beta (TGF&#946;)/Activin/Nodal inhibitor (dual-SMAD inhibition cocktail), then add 10 &#181;M Rho-associated protein kinase (ROCK) inhibitor for 24 h to increase the survival rate of cells during passage.</li>
	<li>Day 1: Prepare the microwell plate with 2.5 mL per well of serum-free medium supplemented with 0.5 &#181;M BMP inhibitor, 10 &#181;M TGF&#946;/Activin/Nodal inhibitor, and 10 &#181;M ROCK inhibitor. To specify cells towards the ventral pattern of the neural tube, add 100 ng/mL sonic hedgehog (SHH), 100 ng/mL fibroblast growth factor 8 (FGF8), and 2 &#181;M smoothened agonist. Centrifuge the plate (only with medium and without cells) at 1200 x&#160;g&#160;for 5 min to remove air bubbles from the microwells.
	<ol>
		<li>After 1 day of neural induction in 2D, remove the medium and quickly wash with phosphate-buffered saline (PBS) without Ca2+/MgCl2+. Dissociate the colonies in single cells suspension by adding 7.5 mL of recombinant enzymatic solution in a T75 cm2&#160;flask. Incubate for 2 min at 37 &#176;C then complete with 7.5 mL of Dulbecco&#39;s modified Eagle medium/nutrient mixture F-12 (DMEM-F12).</li>
		<li>Collect the cell suspension and centrifuge at 300 x&#160;g&#160;for 5 min. Remove the supernatant and count the cells in the same medium used to prepare the microwell plate.</li>
		<li>Adjust the medium volume to obtain a cell suspension allowing to form neurospheres containing 1000 cells per microwell (for example, the microwell plate used here contains 4,700 microwells per well). So, prepare 4.7 million cells in 2.5 mL of medium and add it to the previous 2.5 mL of medium already placed in the plate.</li>
		<li>To correctly distribute the cells in each microwell, gently shake the plate, and centrifuge the microwell plate 300 x&#160;g&#160;for 5 min. Incubate the plate at 37 &#176;C for 24 h to generate spheres.</li>
	</ol>
	</li>
	<li>Day 2: Gently flush the microwells with the medium and collect then transfer the spheres in tissue-treated six-well plate. Replace medium with Neurobasal medium supplemented with 1% B27, 1x non-essential amino acids (NEAA), 2 mM L-glutamine, and 1% of penicillin/ streptomycin. Additionally, add regionalization factors SHH, FGF8, smoothened agonist, and dual-SMAD inhibition small molecules.
	<ol>
		<li>Place spheres in rotation at 37 &#176;C (60 rpm, orbital shaker) and change half-medium freshly supplemented every 2-3 days.</li>
	</ol>
	</li>
	<li>Day 3: To enhance neural induction and convert to neural progenitors with a midbrain identity, supplement the medium with 3 &#181;M Glycogen synthase kinase-3 beta (GSK-3&#946;) inhibitor, which activates the Wnt/&#946;-catenin pathway. Maintain GSK-3&#946; inhibitor in the medium up to day 13. Split into two new tissue-treated 6 well plates to reduce both sphere density per well and avoid sphere aggregation. 
	NOTE: At Day 8, most of the cells should be positive for Nestin.</li>
	<li>Day 8: Start the neural maturation: replace regionalization factors SHH, FGF8, smoothened agonist, and dual-SMAD inhibition cocktail with 0.5 mM dibutyryl cyclic adenosine monophosphate (cAMP) (to favor maturation), 20 nM inhibitor of histone deacetylase (for cell cycle exit), 1 &#181;M &#947;-secretase inhibitor and growth factors, 10 ng/mL glial cell line-derived neurotrophic factor (GDNF), 10 ng/mL brain-derived neurotrophic factor (BDNF), 1 ng/mL transforming growth factor &#946;3 (TGF&#946;3), and 5 ng/mL fibroblast growth factor 20 (FGF20) (both favor dopaminergic [DA] progenitor survival). Change the medium every 2-3 days.</li>
	<li>Day 21: Generate the neural organoid: seed around 100 neurospheres under air-liquid interface conditions on polytetrafluoroethylene (PTFE membrane, 6 mm diameter). Transfer the membrane to a culture plate insert (0.4 mm) and add 1.2 mL of neural maturation medium used for neurosphere differentiation as previously described.
	<ol>
		<li>Stop any rotation from this step. Change the medium every 2-3 days until the required differentiation time point is achieved.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well plate (6-well plate)</td>
			<td>Falcon / Corning</td>
			<td>07-201-588</td>
			<td></td>
		</tr>
		<tr>
			<td>Aggrewell 400 (Microwell culture plates )</td>
			<td>StemCell Technologies</td>
			<td>34421</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplements (B27)</td>
			<td>Life Technologies / Invitrogen</td>
			<td>1238</td>
			<td>For both protocol, stock solution 100x, final solution 1x</td>
		</tr>
		<tr>
			<td>Brain-derived neurotrophic factor (BDNF)</td>
			<td>Cell Guidance</td>
			<td>GFH1-2</td>
			<td>For both protocol,&#160; stock solution 100 &#181;g/mL in pure H2O, final solution 20 ng/mL</td>
		</tr>
		<tr>
			<td>CHIR-99021 (GSK-3&#946; inhibitor )</td>
			<td>Axon Medchem</td>
			<td>ct99021</td>
			<td>For Dopaminergic protocol,&#160; stock solution 7.5 mM in DMSO, final solution 3 &#181;M</td>
		</tr>
		<tr>
			<td>Compound E a &#947;&#8211;secretase inhibitor (&#947;&#8211;secretase inhibitor)</td>
			<td>Calbiochem</td>
			<td>CAS 209986-17-4</td>
			<td>For both protocol (gamma- secretase inhibitor XXI),&#160; stock solution 5 mM in DMSO, final solution 1 &#181;M</td>
		</tr>
		<tr>
			<td>Dibutyryl cyclic-AMP (Dibutyryl cAMP)</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>For Dopaminergic protocol,&#160; stock solution 0.5 M in DMSO, final solution 0.5 mM</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium Mixture F-12 (DMEM-F12)</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td>For cell culture, ready to use</td>
		</tr>
		<tr>
			<td>EDTA 0.1 mM (EDTA)</td>
			<td>Life Technologies</td>
			<td>AM9912</td>
			<td>For cell culture, ready to use</td>
		</tr>
		<tr>
			<td>Fibroblast Growth Factor 20 (FGF20)</td>
			<td>Peprotech</td>
			<td>100-41</td>
			<td>For Dopaminergic protocol,&#160; stock solution 100 &#181;g/mL in pure H2O, final solution 5 ng/mL</td>
		</tr>
		<tr>
			<td>fibroblast growth factor 8&#160; (FGF8)</td>
			<td>Peprotech</td>
			<td>GFH176-5</td>
			<td>For Dopaminergic protocol,&#160; stock solution 100 &#181;g/mL in pure H2O, final solution 100 ng/mL</td>
		</tr>
		<tr>
			<td>Glial cell-derived neurotrophic factor (GDNF)</td>
			<td>Cell Guidance</td>
			<td>GFH2-2</td>
			<td>For both protocol,&#160; stock solution 100 &#181;g/mL in pure H2O, final solution 20 ng/mL</td>
		</tr>
		<tr>
			<td>Hydrophilic polytetrafluoroethylene membrane (PTFE membrane)</td>
			<td>BioCell-Interface</td>
			<td>Discontinued</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN-193189 (BMP inhibitor)</td>
			<td>Axon Medchem /Stemgen</td>
			<td>04-0072-02 /1509</td>
			<td>Dual/Smad,&#160; stock solution 5 mM in DMSO, final solution 0.5 &#181;M</td>
		</tr>
		<tr>
			<td>L-glutamine (L-glutamine)</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td>L-Glutamine (200 mM), stock solution 200 mM, final solution 2 mM</td>
		</tr>
		<tr>
			<td>Millicell-CM Culture plate insert (0.4 &#181;m) (Culture plate insert)</td>
			<td>Millipore</td>
			<td>PICM03050</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Orbital Shaker, MS-NOR-30 (Orbital shaker)</td>
			<td>Major Science</td>
			<td>MS-NRC-30</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplements (N2)</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>For GIC culture, stock solution 100x, final solution 1x</td>
		</tr>
		<tr>
			<td>Neurobasal (Neurobasal)</td>
			<td>Life Technologies / Gibco</td>
			<td>21103049</td>
			<td>Maintenance and maturation embryonic neuronal cell populations , ready to use</td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids (NEAA)</td>
			<td>Gibco</td>
			<td>11140</td>
			<td>Non-essential Amino Acids 100X, stock solution 100x, final solution 1x</td>
		</tr>
		<tr>
			<td>Nutristem&#160; (hESC medium )</td>
			<td>Biological Industries</td>
			<td>05-100-1A</td>
			<td>Stem cell media, ready to use</td>
		</tr>
		<tr>
			<td>Penicilin / Streptomycin&#160; (Penicilin / Streptomycin )</td>
			<td>Life Technologies / Gibco</td>
			<td>15140122</td>
			<td>For cell culture, stock solution 5 mg/mL, final solution 50 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline without Ca2+/Mg2+&#160; (PBS without Ca2+/ Mg2+ )</td>
			<td>Life Technologies</td>
			<td>14190250</td>
			<td>For cell culture, ready to use</td>
		</tr>
		<tr>
			<td>Purmorphamin (smoothened agonist)</td>
			<td>Calbiochem</td>
			<td>SML0868</td>
			<td>For Dopaminergic protocol,&#160; stock solution 10 mM in DMSO, final solution 2 &#181;M</td>
		</tr>
		<tr>
			<td>Rho-associated Kinase Y-27632 (ROCK)</td>
			<td>Abcam Biochemicals</td>
			<td>ab120129-1</td>
			<td>Rock Inhibitor,&#160; stock solution 50 mM in DMSO, final solution 10 &#181;M</td>
		</tr>
		<tr>
			<td>SB-431542 (TGF&#946;/Activin/Nodal inhibitor )</td>
			<td>Ascent</td>
			<td>Asc- 163</td>
			<td>Dual-Smad,&#160; stock solution 50 mM in DMSO, final solution 10 &#181;M</td>
		</tr>
		<tr>
			<td>Sonic Hedgehog (SHH)</td>
			<td>Cell Guidance</td>
			<td>GFH168-5</td>
			<td>For Dopaminergic protocol,&#160; stock solution 100 &#181;g/mL in pure H2O, final solution 100 ng/mL</td>
		</tr>
		<tr>
			<td>StemPro Accutase (hESC enzymatic solution)</td>
			<td>Gibco</td>
			<td>A11105-01</td>
			<td>hESC enzymatic solution, ready to use</td>
		</tr>
		<tr>
			<td>T150 flask (T150 flask)</td>
			<td>Falcon</td>
			<td>08-772-1F</td>
			<td></td>
		</tr>
		<tr>
			<td>Transforming Growth Factors beta 3 (TGF&#946;3)</td>
			<td>Cell Guidance</td>
			<td>GFH109-2</td>
			<td>For Dopaminergic protocol, stock solution 100 &#181;g/mL in pure ethanol , final solution 1 ng/mL</td>
		</tr>
		<tr>
			<td>Trichostatine A (inhibitor of histone deacetylase )</td>
			<td>Sigma</td>
			<td>T8552</td>
			<td>For Dopaminergic protocol,&#160; stock solution 100 &#181;M in DMSO, final solution 20 nM</td>
		</tr>
		<tr>
			<td>TrypLE (recombinant enzymatic solution)</td>
			<td>Invitrogen</td>
			<td>12604021</td>
			<td>recombinant enzymatic solution, ready to use</td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (enzymatic solution)</td>
			<td>Life Technologies</td>
			<td>15050065</td>
			<td>enzymatic solution, ready to use</td>
		</tr>
		<tr>
			<td>X-vivo (serum free medium)</td>
			<td>Lonza</td>
			<td>BE04-743Q</td>
			<td>serum free medium, ready to use</td>
		</tr>
	</tbody>
</table>

Source: Cosset, E. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59682">Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases.</a> J. Vis. Exp. (2019)
This video demonstrates the differentiation of human embryonic stem cells (hESCs) into dopaminergic neurons, to provide a model for studying Parkinson&#39;s disease. The hESCs are cultured in a neural induction medium in a microwell plate to form 3D neurospheres. These are then cultured in a neural induction medium to promote the differentiation into dopaminergic progenitor neurons. The final maturation of the neurons occurs in a culture insert under air-liquid interface conditions, resulting in 3D organoids consisting of mature dopaminergic neurons.

1. hESC-derived dopaminergic organoids for Parkinson&#39;s Disease (PD) studies

	Day 0: Amplify hESCs in 2D culture up to 60% confluency (day 0), then ...

Take human embryonic stem cells, or hESCs, in an extracellular matrix-coated flask.
Replace the medium with a neural induction medium and incubate, differentiating hESCs into neural progenitor cell, or NPC, colonies.
Add enzymatic solution to dissociate the colonies.
Centrifuge, remove the supernatant, and resuspend cells in an induction medium with growth factors to support NPC differentiation.
Add the cell suspension onto inverse pyramidal-shaped microwells to form three-dimensional neurospheres.
Transfer neurospheres to an extracellular matrix-coated plate containing a differentiation medium with growth factors and small molecules. Incubate under agitation to promote differentiation into dopaminergic progenitor neurons.
Replace the medium with neural maturation medium and place neurospheres on a membrane disc in a culture-well insert, allowing diffusion of air and nutrients.
Incubate under static conditions to form three-dimensional organoids consisting of mature dopaminergic neurons.
These organoids, mimicking a developing brain, are useful for studying dopaminergic neuron degeneration, a characteristic of Parkinson&#39;s disease.

28 vistas. Generación de organoides neuronales dopaminérgicos a partir de células madre embrionarias humanas

Video: Generación de organoides neuronales dopaminérgicos a partir de células madre embrionarias humanas - Experimento

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Un método autodirigido estático para generar organoides cerebrales a partir de células madre embrionarias humanas

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Investigación

JoVE Journal

Biología del Desarrollo

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

Generación robusta y altamente reproducible de organoides cerebrales corticales para modelar la senescencia neuronal cerebral in vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...

Neurociencias

Cultivo de neuronas dopaminérgicas del mesencéfalo humano para el perfil fenotípico

Phenotypic Profiling of Human Stem Cell-Derived Midbrain Dopaminergic Neurons

Parkinson&#39;s disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro PD cellular models lack complexity and do not take multiple phenotypes into account. Phenotypic profiling in human induced pluripotent stem cell (iPSC)-derived mDA neurons can address these shortcomings by simultaneously measuring a range of neuronal phenotypes in a PD-relevant cell type in parallel. Here, we describe a protocol to obtain and analyze phenotypic profiles from commercially available human mDA neurons. A neuron-specific fluorescent staining panel is used to visualize the nuclear, &#945;-synuclein, Tyrosine hydroxylase (TH), and Microtubule-associated protein 2 (MAP2) related phenotypes. The described phenotypic profiling protocol is scalable as it uses 384-well plates, automatic liquid handling and high-throughput microscopy. The utility of the protocol is exemplified using healthy donor mDA neurons and mDA neurons carrying the PD-linked G2019S mutation in the Leucine-rich repeat kinase 2 (LRRK2) gene. Both cell lines were treated with the LRRK2 kinase inhibitor PFE-360 and phenotypic changes were measured. Additionally, we demonstrate how multidimensional phenotypic profiles can be analyzed using clustering or machine learning-driven supervised classification methods. The described protocol will particularly interest researchers working on neuronal disease modeling or studying chemical compound effects in human neurons.

Autor destacado: Generación de perfiles fenotípicos neuronales: un protocolo para cultivar e imagen de neuronas dopaminérgicas del mesencéfalo humano 

This protocol describes the cell culturing of human midbrain dopaminergic neurons, followed by immunological staining and the generation of neuronal phenotypic profiles from acquired microscopic high-content images allowing the identification of phenotypic variations due to genetic or chemical modulations.

Perfil fenotípico de neuronas dopaminérgicas del mesencéfalo derivadas de células madre humanas

Parkinson&#39;s disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro ...

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Transcripción

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells