Name: directed induction of retinal organoids from human pluripotent stem cells
Uploaded: 2021-04-21T13:30:00
Description: Watch this Scientific Journal Video about Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells at JoVE.com

We thank members of 502 laboratory for their technical supports and helpful comments regarding the manuscript. This work was partly supported by the Beijing Municipal Natural Science Foundation (Z200014) and National Key R&#38;D Program of China (2017YFA0105300).

This study was approved by the institutional Ethics Committee of Beijing Tongren Hospital, Capital Medical University. H9 hESCs were obtained from the WiCell Research Institute and genetically engineered to tdTomato-tagged cell line.
1. Generation of human ROs
<ol>
	<li>Culture the hESCs under feeder-free conditions.
	<ol>
		<li>Coat one well of a 6-well plate with 1 mL of 0.1 mg/mL reagent A (Table of Materials) at 37 &#176;C for at least half an hour following the manufact...

<ol>
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Retinal organoid differentiation is a desirable method for the generation of ample functional retinal cells. The RO is a composite of different retinal cells, such as ganglion cells, bipolar cells, and photoreceptors, generated by pluripotent stems cells toward the neural retina4,5,8,9. Although confluent ROs could be harvested, it is time-consuming, which may require long culturing periods (up...

Pluripotent stem cells (PSCs) are characterized by their self-renewal and ability to differentiate into all kinds of somatic cells. Thus, organoids derived from PSCs have become an important resource in regenerative medicine research. Retinal degeneration is characterized by the loss of photoreceptors (rods and cones) and retinal pigment epithelium. Retinal cell replacement could be an encouraging treatment for this disease. However, it is not feasible to obtain human retinas for disease research and therapy. Therefore, retinal organoids (ROs) derived from PSCs, which effectively and successfully recapitulate multi-layered native retinal cells, are beneficial for basic and translational research1,2,3. Our research focuses on RO differentiation to provide sufficient and quality cells for studying retinal degeneration4.
Methods for differentiating ROs are continuously emerging, with three-dimensional (3D) suspension differentiation pioneered by the Sasai laboratory in 20125. We introduced the CRX-tdTomato tag in the human embryonic stem cells (hESCs) to specifically track the photoreceptor precursor cells and modified the method with the addition of COCO, a multifunctional antagonist of the Wnt, TGF-&#946;, and BMP pathways6. COCO has been shown to efficiently improve the differentiation efficiency of photoreceptor precursors and cones6,7.
Altogether, by modifying the classical differentiation method, we have developed an accessible protocol to harvest abundant photoreceptor precursors and cones from human ROs for analyzing the retinal disease associated with the photoreceptors through laboratory investigations and for further clinical application/transplantation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>COCO</td>
			<td>R&#38;D Systems</td>
			<td>3047-CC-050</td>
			<td>DAN Domain family of BMP antagonists</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>10565-042</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>C141905005BT</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermo</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), Qualified for Human Embryonic Stem Cells</td>
			<td>Biological Industry</td>
			<td>04-002-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>GMEM</td>
			<td>Gibco</td>
			<td>11710-035</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement-Multi-Species</td>
			<td>Gibco</td>
			<td>A3181502</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acid Solution (100X)</td>
			<td>sigma</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Primesurface 96 V-plate</td>
			<td>Sbio</td>
			<td>MS9096SZ</td>
			<td>Cell aggregation in 1.2.7</td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent A</td>
			<td>BD</td>
			<td>356231</td>
			<td>Matrigel in 1.1.1</td>
		</tr>
		<tr>
			<td>Reagent B</td>
			<td>StemCell</td>
			<td>5990</td>
			<td>mTeSR- E8 , PSCs basal medium in 1.1.2</td>
		</tr>
		<tr>
			<td>Reagent C</td>
			<td>Gibco</td>
			<td>12563-011</td>
			<td>TrypLE Express in 1.2</td>
		</tr>
		<tr>
			<td>Reagent D</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td>DNase I , in 1.2</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Enzo Life Science</td>
			<td>ALX-270-426-M001</td>
			<td></td>
		</tr>
		<tr>
			<td>Supplement 1</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>N-2 Supplement (100X), Liquid, supplemet in medum III</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T-8691-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>organoids dissociation in 2.1.3</td>
		</tr>
		<tr>
			<td>Wnt Antagonist I, IWR-1-endo - Calbiochem</td>
			<td>Sigma</td>
			<td>681669</td>
			<td>Wnt inhibitor</td>
		</tr>
		<tr>
			<td>Y-27632 2HCl</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

directed induction of retinal organoids from human pluripotent stem cells

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

The schematic illustration depicts the differentiation protocol to improve precursor cells with COCO (Figure 1). From PSC to ROs, numerous details could cause result variations. It is recommended to record every step and even the catalog number and lot number of every medium to track the entire procedure.
Herein, we provide bright field images for days 6, 12, 18, and 45 (Figure 2). On day 6, the organoids are usually around 600 &#181;...

Watch this Scientific Journal Video about Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells at JoVE.com

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...

This is a model for retinal disease and emulates the in vivo retina. To some extent, it could replace animal experiments. The protocol is optimized for the quality and quantity of the photoreceptor precursors in the retinal organoids, and the photoreceptor is the key to several irreversible blindness conditions.Begin by culturing the human embryotic stem cells, or hESCs, under feeder-free conditions. To do so, coat one well of a six-well plate with one milliliter of 100 micrograms per milliliter of reagent A, and incubate the plate at 37 degrees Celsius for at least half an hour. Thaw an aliquot of one million hESCs, and centrifuge it at 200 xg for five minutes.After removing the supernatant, seed the cells into the previously coated plate, along with two milliliters of reagent B.Passage the cells after reaching approximately 80%confluency at around four days. After reaching confluency, wash the cells with pre-warmed DPBS, and transfer them into 500 microliters of freshly prepared reagent. Then, incubate the cells for 3.5 minutes at 37 degrees Celsius and 5%carbon dioxide.Detach the cells by flicking the side and bottom of the plate for a few seconds, and add 500 microliters of prepared medium one. Harvest the detached cells into a new 1.5-milliliter tube, and pipette the cell suspension up and down. For cell counting, add 100 microliters of cell suspension to 900 microliters of DPBS.Dilute the 900 microliters of the remaining cell suspension with medium one in the Petri dish to achieve nine times 10 to the fourth power cells per milliliter. Add 100 microliters of diluted cell suspension to each well of a non-adherent, V-bottom, 96-well plate. Lightly spin down the plate on a low-speed shaker for five minutes, and incubate at 37 degrees Celsius and 5%carbon dioxide until day two.On day two, add 20 microliters of 1%reagent A to each well of the 96-well plate, and pipette twice in the center to scatter the dead cells. Clean the bottom of the plate before incubating it at 37 degrees Celsius and 5%carbon dioxide until day six. On day six, replace 58 microliters of the medium with 60 microliters of fresh medium one, and continue the incubation.On day 12, harvest the cell pellets from the 96-well plate in a 15-milliliter conical tube, and allow the pellets to settle naturally for five minutes at room temperature. When pellets settle, remove the supernatant without disturbing the organoids, and transfer the cell aggregates to a 10-centimeter suspension dish containing 18 milliliters of medium two with reagent A.Incubate the dish at 37 degrees Celsius and 5%carbon dioxide until day 18. On day 18, confirm the generation of a semi-transparent optic vesicle in the dish, and cut the generated organoids using a microsurgical knife.Aggregate all the cut pellets to the center of the dish. Then, harvest the cells into a 15-milliliter conical Falcon tube. When the cells settle, gently remove the supernatant, and resuspend the pellets in two 10-centimeter Petri dishes with 18 milliliters of medium, three per dish.Gently transfer the dishes to the incubator to avoid cell aggregation. Continue culturing the cells, changing the medium weekly. Analyze the CRX expression after day 45 until day 120.In this representative analysis, various stages of human retinal generation are displayed. On day six, organoids appeared as an aggregate of dense connections inside a bright rim with a diameter of around 600 micrometers. On day 12, the initial generation of the optic vesicle-like structures took place.The organoids without proper vesicle-like architecture were discarded before culturing in a Petri dish on day 18. By day 30 of the culture, the vesicle-like architecture was more obvious, and inferior ROs could be easily distinguished and removed. Starting on day 45, the organoids expressed various markers of photoreceptor precursors, such as CRX, RCVRN, and OTX2.By cleaving the superior retinal organoids, the differentiation efficiency is significantly improved, and more than 100 superior organoids from 96 plates could be harvested.

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