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>

Source: Cosset, E. et al. <a href="https://app.jove.com/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.

11 Views. Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

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