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 manufacturer&#39;s instructions. Thaw an aliquot of 1x106 hESCs. 
		NOTE: Prepare 3 mL of pre-warmed reagent B (Table of Materials) and transfer the cryopreserved cells (1 mL) into 3 mL of fresh medium. Do not pipette the hESCs to single cells.</li>
		<li>Centrifuge at 200 x g for 5 min and remove the supernatant.</li>
		<li>Seed the cells into the reagent A-coated plate with 2 mL of reagent B and change 2 mL of reagent B daily. Passage cells when reaching approximately 80% confluency (usually around 4 days).</li>
	</ol>
	</li>
	<li>Day 0
	<ol>
		<li>Dissociate hESCs to a single-cell suspension using medium I (Table 1). Prepare medium I by mixing the following: 20% (v/v) KnockOut serum replacement (KSR), 0.1 mM MEM non-essential amino acids solution (NEAA), 1 mM pyruvate,3 &#181;M IWR-1-endo (IWR1e), 30 ng/mL COCO, 100 U/mL penicillin, 100 &#181;g/mL streptomycin (PS), 0.1 mM &#946;-mercaptoethanol, and Glasgow&#39;s Eagle&#39;s minimal essential medium (GMEM). 
		NOTE: Before the start of the dissociation, prepare medium I and transfer 12 mL of medium I with 20 &#181;M Y-27632 into a 10 cm Petri dish, 500 &#181;L of reagent C (Table of Materials) containing 0.05 mg/mL of reagent D (Table of Materials) and 20 &#181;M Y-27632 in a 1.5 mL tube. Perform the above steps in the dark, as component IWR1e in medium I is light-sensitive.</li>
		<li>Wash the hESCs with pre-warmed 1x Dulbecco&#39;s phosphate-buffered saline (DPBS) buffer.</li>
		<li>Add the prepared 500 &#181;L reagent (in the 1.5 mL) tube and incubate the hESCs for 3.5 min at 37 &#176;C and 5% CO2.</li>
		<li>Detach the cells by flicking the side and bottom of the plate for a few seconds and add 500 &#181;L of prepared medium I from the Petri dish into the hESC plate. 
		NOTE: Prepare a 1.5 mL tube with 900 &#181;L of 1x DPBS and use a hemocytometer for cell counting.</li>
		<li>Harvest the cells in a new 1.5 mL tube and pipette the cell suspension up and down and then take out 100 &#181;L from the tube and then add into the tube with 900 &#181;L of DPBS for cell counting.</li>
		<li>Disperse the left 900 &#181;L cell suspension and then dilute the cells with prepared medium I in the Petri dish to 9 x 104 cells/mL. 
		NOTE: The whole volume of the medium is 12 mL; 1.08 x 106 cells are needed in total.</li>
		<li>Add 100 &#181;L of cell suspension to each well of a non-adherent, V-bottom, 96-well plate (Table of Materials). 
		NOTE: Use a multichannel pipette to shorten the time and ensure that each well contains an equivalent cell number. Shake the Petri dish each time before removing 100 &#181;L portions. It is important that the cells are uniformly distributed.</li>
		<li>Lightly spin down the 96-well plate in a low-speed shaker for 5 min and then incubate at 37 &#176;C and 5% CO2. 
		NOTE: Keep the plate in dark. Set the day as day 0.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Add 1% reagent A (Table of Materials). Prepare reagent A (protein concentration of 10 mg/mL) by adding 133.4 &#181;L of reagent A into 2 mL of medium I. 
		NOTE: Maintain reagent A at 4 &#176;C overnight before use to achieve complete and uniform melting. Please note the product information and search the catalog number the and the lot number in the official website of the company to have the protein concentration of reagent A as each bottle of reagent A is at a different protein concentration. If the protein concentration is low, an increased volume of reagent A would be helpful.</li>
		<li>Add 20 &#181;L of prepared reagent A to each well and pipette twice in the center to scatter the dead cells. 
		NOTE: Maintain the reagent A and the 96-well plate under cool conditions and complete all the steps on ice. Place the plate in dark.</li>
	</ol>
	</li>
	<li>Day 2-12
	<ol>
		<li>Clean the bottom of the 96-well plate and incubate at 37 &#176;C and 5% CO2 until day 6. At day 6, change half of the medium by removing 58 &#181;L of the medium from each well and adding 60 &#181;L of medium I. 
		NOTE: Use a multichannel pipette to complete the half medium change. Conduct this step gently to ensure that no cell pellets are removed from the well. Aspirate the medium into a clean 10 cm Petri dish. If there are cell pellets inside, add them back to the 96-well plate.</li>
	</ol>
	</li>
	<li>Day 12- 18
	<ol>
		<li>Change the medium to medium II (Table 1) at day 12. Prepare medium II using 1% (v/v) reagent A by mixing the following: 10% fetal bovine serum (FBS), 0.1 mM NEAA, 1 mM pyruvate,100 U/mL penicillin, 100 &#181;g/mL streptomycin, 0.1 mM &#946;-mercaptoethanol, 100 nM SAG dihydrochloride and GMEM. Store it under cool and dark conditions.</li>
		<li>Harvest the cell pellets in a 15 mL conical tube from the 96-well plate and allow the pellets to settle naturally for 5 min at room temperature. 
		NOTE: Remove the medium and pellets gently under the surface to avoid bubbles.</li>
		<li>Remove the supernatant while taking care of the organoids. Transfer the cell aggregates to a 10 cm suspension dish containing 18 mL of prepared medium II with reagent A. 
		NOTE: Do not add the prepared medium II into the 10 cm dish in advance, as the reagent A should be at 4 &#176;C.</li>
		<li>Incubate at 37 &#176;C and 5% CO2 until day 18.</li>
	</ol>
	</li>
	<li>From day 18 onwards
	<ol>
		<li>Change the medium to medium III (Table 1). Prepare medium III under dark conditions by mixing the following: 10% FBS, 1xsupplement 1, 0.5 &#181;M retinoic acid (RA), 100 &#181;M taurine, 100 U/mL penicillin, 100 &#181;g/mL streptomycin and 1:1 mixture of Dulbecco&#39;s modified eagle medium (DMEM)/nutrient mixture F-12.</li>
		<li>On day 18, when a semitransparent optic vesicle is generated, cut the organoids using a microsurgical knife. 
		NOTE: Cleave the organoid, usually into four pieces, in a Petri dish with medium II. Each piece should grow into an intact optic cup in the following weeks.</li>
		<li>Aggregate all the pellets to the center of the dish. Harvest the cells into a 15 mL conical falcon tube and allow natural settling. Then gently remove the supernatant. 
		&#8203;NOTE: Due to their sizes, organoids can be aggregated at the center by rotating the Petri dish in one direction for 90&#176; on a horizontal plane a few times.</li>
		<li>Suspend and disperse the pellets in two 10 cm Petri dishes, with 18 mL of medium III per dish, and gently transfer the dishes to the incubator to avoid cell aggregation. Continue culturing in medium III at 37 &#176;C and 5% CO2 and change the medium weekly. The CRX expressed after day 45 and until day 120, we could detect the out-segment of the photoreceptor.</li>
	</ol>
	</li>
</ol>
2. Analyzing human ROs
<ol>
	<li>FACS analysis
	<ol>
		<li>Assemble three CRX-tdTomato and three H9 ES-derived organoids from the dishes by using the cut 1 mL tips. Wash the organoids with 1 mL of pre-warmed (room temperature) DPBS.</li>
		<li>Prepare the digestion buffer by mixing 0.25% trypsin-EDTA Solution with 0.05 mg/mL of reagent D.</li>
		<li>Dissociate the organoids into single cells by using the prepared digestion buffer for 8 min at 37 &#176;C. Then add the same volume of DPBS with 10% FBS and 0.05 mg/mL of reagent D to inactivate the reaction.</li>
		<li>Lightly suspend and scatter the cells and then filter using a 100 &#181;m cell strainer and use a Fluorescence-Activated Cell Sorting (FACS) system at 561 nm excitation laser line and 780/60 filter to analyze the CRX-tdTomato positive signals. 
		NOTE: CRX initially expresses at day 45 and increases with the maturation of organoids. Ten thousand cells are used for each test, for each timepoint, at least three repeats are completed.</li>
	</ol>
	</li>
	<li>Fluorescence intensity quantification of ROs
	<ol>
		<li>Prepare the viable organoids randomly for imaging. 
		NOTE: Set the parameters and use the same filters and parameters for all the fluorescence intensity monitors.</li>
		<li>Capture images and analyze the mean fluorescence intensity using suitable software (e.g., ImageJ).</li>
		<li>Import the images and then convert to 8-bit using Image | Type.</li>
		<li>Adjust the threshold by using the default parameters by Image | Adjust | Threshold.</li>
		<li>Determine the measured area and then evaluate the gray value by using Analyze | Measure. 
		NOTE: The mean values in the result panel are the mean fluorescence intensity of the measured area.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Xie, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+accessibility+analysis+reveals+regulatory+dynamics+of+developing+human+retina+and+hiPSC-derived+retinal+organoids.">Chromatin accessibility analysis reveals regulatory dynamics of developing human retina and hiPSC-derived retinal organoids.</a> Science Advances. 6 (6), 5247 (2020).</li><li>Lu, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Analysis+of+Human+Retina+Identifies+Evolutionarily+Conserved+and+Species-Specific+Mechanisms+Controlling+Development.">Single-Cell Analysis of Human Retina Identifies Evolutionarily Conserved and Species-Specific Mechanisms Controlling Development.</a> Developmental Cell. 53 (4), 473-491 (2020).</li><li>Cowan, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Types+of+the+Human+Retina+and+Its+Organoids+at+Single-Cell+Resolution.">Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.</a> Cell. 182 (6), 1623-1640 (2020).</li><li>Jin, Z. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stemming+retinal+regeneration+with+pluripotent+stem+cells.">Stemming retinal regeneration with pluripotent stem cells.</a> Progress in Retinal and Eye Research. 69, 38-56 (2019).</li><li>Nakano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-formation+of+optic+cups+and+storable+stratified+neural+retina+from+human+ESCs.">Self-formation of optic cups and storable stratified neural retina from human ESCs.</a> Cell Stem Cell. 10 (6), 771-785 (2012).</li><li>Pan, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COCO+enhances+the+efficiency+of+photoreceptor+precursor+differentiation+in+early+human+embryonic+stem+cell-derived+retinal+organoids.">COCO enhances the efficiency of photoreceptor precursor differentiation in early human embryonic stem cell-derived retinal organoids.</a> Stem Cell Research and Therapy. 11 (1), 366 (2020).</li><li>Zhou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+embryonic+stem+cells+into+cone+photoreceptors+through+simultaneous+inhibition+of+BMP,+TGFbeta+and+Wnt+signaling.">Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGFbeta and Wnt signaling.</a> Development. 142 (19), 3294-3306 (2015).</li><li>Deng, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Correction+Reverses+Ciliopathy+and+Photoreceptor+Loss+in+iPSC-Derived+Retinal+Organoids+from+Retinitis+Pigmentosa+Patients.">Gene Correction Reverses Ciliopathy and Photoreceptor Loss in iPSC-Derived Retinal Organoids from Retinitis Pigmentosa Patients.</a> Stem Cell Reports. 10 (4), 1267-1281 (2018).</li><li>Gao, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-Specific+Retinal+Organoids+Recapitulate+Disease+Features+of+Late-Onset+Retinitis+Pigmentosa.">Patient-Specific Retinal Organoids Recapitulate Disease Features of Late-Onset Retinitis Pigmentosa.</a> Frontiers in Cell and Developmental Biology. 8, 128 (2020).</li><li>Zhang, C. J., Ma, Y., Jin, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+road+to+restore+vision+with+photoreceptor+regeneration.">The road to restore vision with photoreceptor regeneration.</a> Experimental Eye Research. 202, 108283 (2020).</li><li>Reichman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+confluent+human+iPS+cells+to+self-forming+neural+retina+and+retinal+pigmented+epithelium.">From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium.</a> Proceedings of the National Academy of Sciences of the U. S. 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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 to 180 days). However, for photoreceptor transplantation, or studying cone-rod or rod-cone dystrophy, it is advantageous to obtain a relatively high percentage of photoreceptors in the 3D culturing system10. 
It is also challenging to monitor the organoids' development without interrupting the normal developmental processes. Therefore, we used CRX, a cone-rod homeobox protein, predominantly expressed in photoreceptor precursors, as a target gene to trace photoreceptor precursor cells during their 3D differentiation. With the tdTomato system, CRX-expressing cells can be spatio-temporally tracked by the red fluorescence without interrupting retinalization during their 3D differentiation. The utilization of CRX-tdTomato system could accelerate the process of drug screening for photoreceptor precursor differentiation in the ROs.
Using our method, around 70% organoids could develop into retinal organoids, which display the vesicle-like structures. Importantly, with the incision of superior organoids, we usually could harvest around 100 retinal organoids from a 96-well plate. Additionally, abundant photoreceptor precursors are generated in the early stage of RO maturation with the COCO culture, which helps progression toward direct differentiation of certain cells through pathway regulation11,12. The protein concentration of reagent A is crucial for the differentiation. Altogether, sufficient reagent A with the aggregates in the early days as well as the cutting of the organoids on day 18 are important to harvest abundant ROs with high quality. This method also promotes the development of directional differentiation of photoreceptor cells in 3D organoids and contributes to the transplantation of photoreceptor cells.

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><tbody><tr><td>2-mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>COCO</td><td>R&amp;amp;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;m in diameter in a 96-well plate, with dense connections inside and bright rim (Figure 2A). On day 12, the optic vesicle-like structures initially generate (Figure 2B). From day 12 to day 18, the presence of optic vesicle structures is clear, and they continued to grow after day 18. The organoids without the vesicle-like architecture are discarded (Figure 2C). By day 30, the vesicle-like architecture is more obvious, and it is easier to distinguish the superior ROs from the inferior ones (Figure 2D-E). The organoids that lose the translucent structure (asterisks in Figure 2D-E), should be removed in the following days.
The organoids express the CRX, which is a marker of photoreceptor precursors, from day 45 onwards (Figure 2F,2I). Other photoreceptor precursor markers, such as RCVRN and OTX2, were also positively detected at day 45 (Figure 2G-H). The addition of COCO promotes the generation of photoreceptor precursors.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62298/62298fig01.jpg" /> 
Figure 1. Timetable for stepwise treatment for RO differentiation from hESC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62298/62298fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62298/62298fig02.jpg" /> 
Figure 2. Human retinal organoid generation. (A-B) Early-stage optic vesicle-like structure formed in a 96-well plate. The black arrows indicate the optic vesicle-like structures. (C) The first day of suspension culture in a Petri dish on day 18. (D-E) Optic cup structures are observed at this stage. The stars show the inferior organoids (F) The bright field image on day 45. Scale bars = 400&#160;&#181;m.&#160;(G-H) Immunostaining results of RCVRN (G) and OTX2 (H) on day 45. Scale bars = 50&#160;&#181;m.&#160;(I) TdTomato-positive signals indicate the expression of CRX on day 45 in (F). Scale bars = 400 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62298/62298fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Medium I (50 mL)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>KSR</td>
			<td>G-MEM</td>
			<td>NEAA</td>
			<td>Pyruvate</td>
			<td>b-ME</td>
			<td>IWR1e</td>
			<td>COCO</td>
			<td>PS</td>
		</tr>
		<tr>
			<td>Percentage % OR&#160; final concentration</td>
			<td>20</td>
			<td>78</td>
			<td>0.1 mM</td>
			<td>1 mM</td>
			<td>0.1 mM</td>
			<td>3 &#181;M</td>
			<td>30 ng/mL</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Volume</td>
			<td>10 mL</td>
			<td>39 mL</td>
			<td>0.5 mL</td>
			<td>0.5 mL</td>
			<td>90.9 &#181;L</td>
			<td>5 &#181;L</td>
			<td>10 &#181;L</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td colspan="8">The store concentration of IWR1e is 30 mM. COCO is 150&#160;&#181;g/mL.&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Medium II (50 mL)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>FBS</td>
			<td>G-MEM</td>
			<td>NEAA</td>
			<td>Pyruvate</td>
			<td>b-ME</td>
			<td>SAG</td>
			<td>PS</td>
			<td></td>
		</tr>
		<tr>
			<td>Percentage % OR&#160; final concentration</td>
			<td>10</td>
			<td>88</td>
			<td>0.1 mM</td>
			<td>1 mM</td>
			<td>0.1 mM</td>
			<td>100 nM</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>Volume</td>
			<td>5 mL</td>
			<td>44 mL</td>
			<td>0.5 mL</td>
			<td>0.5 mL</td>
			<td>90.9 &#181;L</td>
			<td>2.5&#160; &#181;L</td>
			<td>0.5 mL</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="8">The store concentration of SAG is 2 mM.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Medium III (50 mL)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>FBS</td>
			<td>DMEM/F12- Glutamax</td>
			<td>Supplement 1</td>
			<td>RA</td>
			<td>Taurine</td>
			<td>PS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Percentage % OR&#160; final concentration</td>
			<td>10</td>
			<td>88</td>
			<td>1</td>
			<td>0.5 &#181;M</td>
			<td>100 &#181;M</td>
			<td>1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Volume</td>
			<td>5 mL</td>
			<td>44 mL</td>
			<td>0.5 mL</td>
			<td>5 &#181;L</td>
			<td>50 &#181;L</td>
			<td>0.5 mL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="7">The store concentration of RA is 5 mM, and Taurine is 100 mM.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Medium I, II, and II recipes

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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

2.8K Views.  Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Science Key Laboratory. Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Video: Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Developmental Biology

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.

In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Enhanced 3D Neural Retina Differentiation from Human Embryonic Stem Cells

Generating Neural Retina from Human Pluripotent Stem Cells

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of retinopathy. Unfortunately, ethical barriers hinder the collection of evidence from humans. Recently, numerous studies have shown that human pluripotent stem cells (PSCs) can be differentiated into retinal organoids (ROs) using different induction protocols, which have enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies. This study describes an optimized induction protocol to generate neural retina (NR) that significantly reduces the probability of vesiculation and fusion, increasing the success rate of production until day 60. Based on the ability of PSCs to self-reorganize after dissociation, combined with certain complementary factors, this new method can specifically drive NR differentiation. Furthermore, the approach is uncomplicated, cost-effective, exhibits notable repeatability and efficiency, presents encouraging prospects for personalized models of retinal diseases, and supplies a plentiful cell reservoir for applications such as cell therapy, drug screening, and gene therapy testing.

Author Spotlight: Simple and Efficient Neural Retina Organoid Production for Disease Modeling

The present protocol describes an optimized 3D neural retina induction system that reduces the adhesion and fusion of retinal organoids with high repeatability and efficiency.

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of ...

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human vascular networks. This three-dimensional (3D) model holds substantial potential for vascular pathology modeling and in vitro drug screening. Despite recent advances, a key technical challenge remains in reproducibly generating organoids with consistent quality, which is crucial for downstream assays and applications. Here, a modified protocol is presented that improves both the homogeneity and reproducibility of vascular organoid generation. The modified protocol incorporates the use of microwells and the CEPT cocktail (chroman 1, emricasan, polyamines, and the integrated stress response inhibitor, trans-ISRIB) to improve embryoid body formation and cell survival. Differentiated, mature vascular organoids generated using this protocol are characterized by whole-mount 3D immunofluorescence microscopy to analyze their morphology and complex vasculature. This protocol enables the production of high-quality vascular organoids in a scalable manner, potentially facilitating their use in disease modeling and drug screening applications.

Author Spotlight: Improving Reproducibility in Vascular Organoids Using ROCK Inhibitors and Microwell Confinement

This protocol presents a method for generating vascular organoids using human pluripotent stem cells.

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human ...

Bioengineering

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...