differentiation and maturation of human embryonic stem cells into neural organoids

Differentiation and Maturation of Human Embryonic Stem Cells into Neural Organoids

Dispense 1,000 thousand cells per microwell. Centrifuge the cells at 300 times g for 5 minutes, and check the homogeneous repartition of cells in the microwell plate under a microscope. Place the plate in the incubator at 37 degrees Celsius overnight.
The next day, examine the microwell plate to check the size of formed spheres in each microwell. Transfer the spheres to a six-well plate using the medium, supplemented with dual SMAD inhibition cocktail to promote fast neural induction. From this step forward, the spheres are cultured in rotation at 60 RPM to prevent them from sticking together or to the plate. On day 1 to 4, change the medium every two to three days before performing neural induction.
From day 4 to 11, promote proliferation of hESC-derived neural rosettes by adding EGF and bFGF at 10 nanograms per milliliter to the B27 medium, supplemented with dual SMAD cocktail. From day 11 to 13, culture the spheres in B27 medium, supplemented with 0.5 micromolar BMP inhibitor. From day 13 to 21, culture the spheres in B27 medium supplemented with GDNF and BDNF at 10 nanograms per milliliter and 1 micromolar gamma-secretase inhibitor.
On day 21, place one culture plate insert in one well of a new six-well plate. Afterward, add 1 milliliter of B27 medium, supplemented with growth factors and inhibitors to each well underneath the membrane insert. Subsequently, plate the spheres on a hydrophilic PTFE membrane deposited on a culture plate insert and change the medium every two to three days for the following three weeks of differentiation. Stop rotation from this step.
The presence of rosettes indicates the initiation of neural differentiation. From day 21 to 25, cultivate human neural organoids in the same neural maturation medium. Then, from day 25 to 28, only complement B27 medium with one micromolar gamma-secretase inhibitor. From day 28 to 39, stop adding the gamma-secretase inhibitor and continue human neural organoid culture in B27 medium only. After three weeks, neural organoids are ready to use for GIC implantation.

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1. Maintenance and culture of undifferentiated human embryonic stem cells (hESCs)
<ol>
	<li>Perform maintenance and expansion of hSECs on feeder-free conditions by pre-coating dishes with a specific extracellular matrix.
	<ol>
		<li>Thaw 300 &#181;L of extracellular matrix at 4 &#176;C (typical range concentration 18-22 mg/mL, keep on ice) and gently mix with 15 mL of cold DMEM(Dulbecco&#39;s Modified Eagle Medium )medium to avoid premature gelation of the extracellular matrix. Add 7.5 mL of the extracellular matrix to both T150 flasks.</li>
		<li>Incubate the dishes coated with extracellular matrix at 37 &#176;C for at least 1 h (maximum overnight).</li>
		<li>Remove the medium and seed hESCs to a density of 6.5 x 104 cell/cm2.</li>
	</ol>
	</li>
	<li>Maintain H1 (hESC cell line) in hESC medium and 1% penicillin/streptomycin.</li>
	<li>Pass the cells with enzymatic procedure: add 7.5 mL of enzymatic solution to a T75 cm2 flask over a period of 1-2 min at 37 &#176;C. Once cells are completely detached, add 7.5 mL of DMEM-F12 then centrifuge for 5 min at 300 x g. To allow for better survival, re-plate cells at the desired density onto extracellular matrix-coated dishes, in the same medium containing Rho-associated protein kinase (ROCK) inhibitor (10 &#181;M) for 24 h.</li>
</ol>
2. hESC-derived neural organoids 
<ol>
	<li>24 h before starting the 3D culture, replace the hESC medium with a serum-free medium supplemented with 10 &#181;M ROCK inhibitor (both components are necessary to support cell survival and spontaneous neurosphere formation during the aggregation phase in a microwell plate). The cells should be at 60% confluency. The next day (day 0), detach hESC colonies as single cells: remove the medium, rinse with PBS without Ca2+/Mg2+, add 5 mL of enzymatic dissolution solution, and incubate at 37 &#176;C for 1-2 min.</li>
	<li>Collect the cells in serum-free medium supplemented with 10 &#181;M of ROCK inhibitor and centrifuge them at 300 x g for 5 min. Remove the supernatant and count the cells in 10 mL of serum-free medium supplemented with 10 &#181;M of ROCK inhibitor.</li>
	<li>In parallel, rinse the microwell plate with 2 mL of serum-free medium per well and centrifuge the plate at 1200 x g for 5 min to remove all bubbles, which can prevent neurosphere formation.</li>
	<li>Prepare 28.2 x 106 of cells in 12.5 mL of serum-free medium supplemented with 10 &#181;M ROCK inhibitor. Dispense 1000 cells/microwell. Centrifuge the cells at 300 x g for 5 min and place the plate in the incubator at 37 &#176;C overnight (maximum 36 h). For example, to obtain 30 human neural organoids, use one T150 flask at 70%-80% of confluence (about 30 million cells).</li>
	<li>The next day (day 1), collect the spheres (with a P1000) and place them in a 6 well plate. In each well, add 2 mL of B27 medium and DMEM-F12 GlutaMAX and Neurobasal medium (mix at 1:1), supplemented with 1% B27 supplements and 1% non-essential amino acids (NEAA). To promote fast neural induction, supplement the medium with a dual-SMAD (Sma and Mad Related Protein) inhibition cocktail composed of 10 &#181;M TGF&#946;(Transforming Growth Factor-beta)/Activin/Nodal inhibitor and 0.5 &#181;M bone morphogenic protein (BMP) inhibitor. From this step forward, the spheres are cultured in rotation (60 rpm, orbital shaker). The rotation is critical to prevent the spheres from sticking together or to the plate.</li>
	<li>Change the medium every 2-3 days: bend the plate and let the spheres fall down for 5 min, remove half of the medium (2 mL), and add 2 mL of fresh B27 medium supplemented with growth factors and inhibitors. Do not centrifuge the spheres.</li>
	<li>Perform neural induction according to the following time course:
	<ol>
		<li>From days 1-4, culture the spheres in B27 medium supplemented with dual-SMAD. The dual-SMAD inhibition cocktail (10 &#181;M TGF&#946;/Activin/Nodal inhibitor and 0.5 &#181;M BMP inhibitor) promotes neural induction.</li>
		<li>From days 4-11, promote proliferation of hESC-derived neural rosettes (into the spheres), by adding 10 ng/mL epidermal growth factor (EGF) and 10 ng/mL basic fibroblast factor (bFGF) to the B27 medium supplemented with dual-SMAD cocktail. 
		NOTE: On day 11, most cells should be positive for Nestin.</li>
		<li>From days 11-13, culture the spheres in B27 medium supplemented with 0.5 &#181;M BMP inhibitor.</li>
		<li>From days 13-21, culture the spheres in B27 medium supplemented with 10 ng/mL glial derived neurotrophic factor (GDNF), 10 ng/mL brain derived neurotrophic factor (BDNF) and 1 &#181;M of &#947;-secretase inhibitor. GDNF and BDNF promote neuronal and glial differentiation. The &#947;-secretase inhibitor allows for greater neural maturation.</li>
		<li>On day 21, plate the spheres (about 1,000 spheres) on a hydrophilic polytetrafluoroethylene (PTFE) membrane (6 mm diameter, 0.4 &#181;m) deposited on a culture plate insert designed for 6 well plates. Stop any rotation from this step. The presence of rosettes, observed with a bright-field microscope, indicates the initiation of neural differentiation. The neural rosettes can be observed 2-3 days after plating spheres on the PTFE membrane.</li>
		<li>Add 1 mL of B27 medium supplemented with growth factors and inhibitors (as followed) to each well underneath the membrane insert, every 2-3 days (usually on Monday, Wednesday and Friday), for a following 3 weeks of differentiation.</li>
		<li>From days 21-25, cultivate human neural organoids in the same neural maturation medium.</li>
		<li>From days 25-28, only complement B27 medium with 1 &#181;M &#947;-secretase inhibitor.</li>
		<li>From days 28-39, stop adding the &#947;-secretase inhibitor and continue human neural organoid culture in the B27 medium only. 
		NOTE: After 3 weeks, neural organoids are ready to use for GIC implantation. Along the neural maturation, a decrease of neural immature marker Nestin and increase of mature neural markers &#946;3-tubulin and GFAP were observed.</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)
In this video, a stepwise protocol for Human embryonic stem cells into neural organoids is demonstrated. The method involves inducing neuronal rosette formation, followed by controlled maturation and final culturing on a hydrophobic membrane to achieve fully developed neural organoids.

1. Maintenance and culture of undifferentiated human embryonic stem cells (hESCs)

	Perform maintenance and expansion of hSECs on feeder-free conditions ...

Begin with human embryonic stem cells in a culture medium.
Incubate for cell proliferation and sphere formation.
Transfer these spheres to a new well with a medium containing differentiation factors.
Incubate with gentle shaking. The differentiation factors drive the stem cells towards the neural lineage.
Regularly replace half of the medium.
Replace the medium with a neural induction medium, then incubate to obtain neural progenitors.
Switch to a growth-factor-rich medium and incubate to promote cell proliferation and neural rosette formation.
Then, culture in a medium containing inhibitors to block undesired cell differentiation.
Replace this medium with a medium containing growth factors and inhibitors. Incubate to induce differentiation into glial cells and primary neurons.
Later, place the neural rosettes on a hydrophobic membrane in an insert-carrying well containing an inhibitor-rich maturation medium.
Incubate without shaking to allow neural and glial cell maturation at an air-liquid interface, forming neural organoids.

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Differentiation and Maturation of Human Embryonic Stem Cells into Neural Organoids

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