This study was conducted in accordance with the Tenets of the Declaration of Helsinki and approved by the Institutional Ethics Committee of the Chinese PLA General Hospital. The WA09 (H9) ESC line was obtained from the WiCell Research Institute.
1. Culture media and reagent preparation
<ol>
	<li>Human ESC culture medium and passage solution
	<ol>
		<li>Maintenance medium (MM): Prepare 500 mL of complete MM (Basal Medium + 5x supplement; see Table of Materials) aseptically. Thaw 5x supplement at room temperature (RT) or overnight at 2-8 &#176;C. Prewarm to RT. Stir well before use until the supplement is free of cloudiness.</li>
		<li>1% extracellular matrix (ECM): Use a pre-chilled pipet tip and sterile tubes to dispense 200 &#181;L of ECM (see Table of Materials) per tube on ice. Store the tubes in a -20 &#176;C freezer. Melt ECM on ice and dilute with pre-cooled DMEM/F12 at 1:100.</li>
		<li>0.5 mM pH = 8.0 EDTA: To prepare 500 mL of 0.5 mM EDTA, add 500 &#181;L of 0.5 M EDTA and 0.9 g of NaCl to 500 mL of 1x Dulbecco&#39;s phosphate-buffered saline (DPBS) (see Table of Materials). Mix thoroughly and store at 2-8 &#176;C.</li>
	</ol>
	</li>
	<li>Retinal differentiation medium
	<ol>
		<li>Growth factor-free chemically defined medium (gfCDM): Prepare the gfCDM by combining 45% Iscove&#39;s modified Dulbecco&#39;s medium-GlutaMAX (IMDM-GlutaMAX), 45% Ham&#39;s F12-GlutaMAX (F12-Glutamax), 10% knockout serum replacement (KSR), 1% cholesterol lipid concentrate, and 450 &#181;M thioglycerol (see Table of Materials).</li>
	</ol>
	</li>
	<li>Small molecule compounds
	<ol>
		<li>Y-27632 2HCl: Add 50 mg of Y-27632 powder to 3.122 mL of dimethyl sulfoxide (DMSO). Dispense and store Y-27632 (see Table of Materials) at a concentration of 50 mM at -80 &#176;C for 2 years, keeping away from light. Dilute the stock 2,500 times (20 &#181;M) for use, equivalent to 0.4 &#181;L of Y-27632 per mL of gfCDM.</li>
		<li>IWR1-endo: Add 2.4424 mL of DMSO to dissolve 10 mg of IWR1-endo (see Table of Materials) to obtain a 10 mM stock solution. Dispense aliquots and store them at -80 &#176;C for up to 2 years. Add 0.3 &#181;L of 10 mM IWR1-endo per mL of gfCDM for the 3 &#181;M concentration for induction.</li>
		<li>SB431542: To prepare 50 mM SB431542, add 10 mg of SB431542 (see Table of Materials) to 0.5203 mL of DMSO and mix thoroughly. Store at -80 &#176;C in solvent for a maximum of 2 years. For the preparation of SB431542 at a working concentration of 10 &#181;M, add 2 &#181;L of the 50 mM stock solution to 10 mL of gfCDM.</li>
		<li>LDN-193189 2HCl: Dissolve 5 mg of LDN-193189 2HCl (see Table of Materials) in 10.4297 mL of DMSO to get a 1 mM of stock solution. Store at -20 &#176;C or -80 &#176;C (as recommended by the manufacturer). Add 0.1 &#181;L of the stock to every 10 mL of gfCDM, resulting in 100 nM of LDN-193189 for induction.</li>
		<li>Recombinant Human bone morphogenetic protein 4 (BMP4): Reconstitute at 50-200 &#181;g/mL in 4 mM HCl (see Table of Materials). Store at 2 &#176;C to 8 &#176;C for 1 month or -20 &#176;C for 1 year after reconstitution. Perform induction of NR using 1.5 nM BMP4.</li>
	</ol>
	</li>
	<li>Long-term NR culture medium
	<ol>
		<li>Retinoic acid (RA): Measure 6 mg of RA powder (see Table of Materials) and add to 3.9941 mL of DMSO. Store in aliquots of 5 mM at &#8722;80 &#176;C and use within 3 months. To achieve a working concentration of 0.5 &#181;M, add 10 &#181;L of the master mix to 100 mL of neural retina differentiation medium (NRDM). Add just before use. 
		NOTE: Keep away from light during preparation and storage.</li>
		<li>Taurine: Add taurine powder (see Table of Materials) weighing 200 mg to 7.9904 mL of DMSO to obtain a 200 mM stock solution. Dispense and store at 2-8 &#176;C. A working concentration of 0.1 mM taurine is achieved by adding 50 &#181;L of the stock solution per 100 mL of NRDM.</li>
		<li>NRDM: Compose NRDM with DMEM/F12-GlutaMAX medium, 1% N2 supplement, 10% fetal bovine serum, 0.5 mM RA and 0.1 mM taurine (see Table of Materials). Store at 2-8 &#176;C for up to 2 weeks or 6 months at -20 &#176;C to ensure the activity of the components.</li>
	</ol>
	</li>
	<li>Blocking buffer: Dilute 1:9 with DPBS to obtain a 10% working solution for use (see Table of Materials). Store at -20 &#176;C for up to 5 years. 
	&#8203;NOTE: Perform all other procedures in a Class II Biosafety Cabinet to ensure sterility, except for weighing. If adding weighed reagents during the preparation process is necessary, use 0.22 &#181;m filters for filtration.</li>
</ol>
2. Culturing of H9-ESCs
<ol>
	<li>Thawing of H9-ESCs
	<ol>
		<li>Add 1 mL of 1% ECM to each well of a 6-well plate. Incubate for 1 h in an incubator at 37 &#176;C in a 5% CO2 atmosphere. 
		NOTE: Avoid the addition of ECM along the well walls, because it may stick to the surface.</li>
		<li>Take an H9-ESC cryovial stock from the liquid nitrogen tank and quickly shake it in 37 &#176;C water for 30 s. 
		NOTE: Do not allow the vial to thaw completely.</li>
		<li>Remove the vial and carefully sterilize it using 75% disinfectant alcohol spray. Add the thawed H9-ESC from the cryovial to a 15 mL tube containing 9 mL MM with 10 &#181;M Y-27632.</li>
		<li>Centrifuge the tube at 190 &#215; g for 5 min at RT. Carefully remove most of the supernatant with a 1 mL pipette, leaving approximately 50 &#181;L of supernatant to avoid cell loss.</li>
		<li>Add 1 mL of MM containing 10 &#181;M of Y27632 to the cell sediment and gently resuspend the cell sediment with a 1 mL pipette by pipetting up and down 5-10 times.</li>
		<li>Remove the ECM coating after 1 h of incubation. Add 2 mL of pre-warmed MM containing 10 &#181;M Y-27632 to each well.</li>
		<li>Dispense 0.5 mL of cell suspension per well. Gently shake the plate laterally to ensure even distribution of cells.</li>
		<li>Incubate the plate at 37 &#176;C under 5% CO2 for at least 24 h without touching it.</li>
		<li>Change the medium daily. When clone density reaches 70% and above, passaging is required.</li>
	</ol>
	</li>
	<li>Passaging of H9-ESCs
	<ol>
		<li>Prepare the ECM-coated plate as described above (step 2.1.1) and add 2 mL of MM per well.</li>
		<li>Remove the spent medium from the 6-well plates. Wash each well twice with 1 mL of DPBS.</li>
		<li>Wash each well twice by slowly adding 1 mL of EDTA, and incubating with 1 mL of EDTA for 4-7 min at RT. 
		NOTE: During the incubation, examine the 6-well plate for 3 min under a microscope. Proceed immediately to the next step if most cells are close to detaching from the dish. Avoid long time incubation to reduce negative impact on the cells.</li>
		<li>Discard EDTA and add 1 mL of MM to abort digestion.</li>
		<li>Gently tap the well plate until the majority of the cells are detached.</li>
		<li>Carefully transfer the cell suspension into a 15 mL centrifuge tube with a 5 mL pipette. Gently resuspend the H9-ESC colonies up and down 3-5 times to mix them with a Pasteur pitette. Transfer 20-100 &#181;L of cell suspension in a new ECM-coated 6-well culture dish and shake it back and forth.</li>
		<li>When the cell density is observed as sufficient under a microscope, incubate the plate at 37 &#176;C under 5% CO2 for at least 24 h without touching it.</li>
		<li>Change the media daily. At 70% clone density and above, passage is required.</li>
	</ol>
	</li>
</ol>
3. Generation of human NRs
NOTE: Once the colonies achieve approximately 70% confluence, they can be directed towards differentiation into retinal organoids (ROs) using the procedural steps outlined in Figure 1.
<ol>
	<li>Day 0 - Embryoid body (EB) formation
	<ol>
		<li>After washing the cells with 2 mL of DPBS, add 0.5 mL of CDS (see Table of Materials) containing 20 &#181;M Y27632 to the well. Incubate for 3 min at 37 &#176;C in a humidified 5% CO2 incubator. 
		NOTE: Check under a microscope. Reduce the time of incubation as much as possible to avoid harmful effects on the cells.</li>
		<li>Abort digestion by adding 3 mL of MM containing 20 &#181;M Y27632. Centrifuge at 190 &#215; g for 5 min and discard supernatant.</li>
		<li>Resuspend the cell pellet in 1 mL of gfCDM containing 20 &#181;M Y27632, and count the cells. Add the corresponding volume for 1.2 &#215; 106 cells (1.2 &#215; 104 cells per well) to 10 mL of gfCDM, which is pre-incorporated with 20 &#181;M Y-27632, 3 &#181;M IWR1-endo, 10 &#181;M SB431542, and 100 nM LDN-193189 (see Table of Materials).</li>
		<li>Add 100 &#181;L of cell suspension to each well of 96 V-bottomed conical wells. Place the plate in a humidified 5% CO2 incubator until day 6. 
		NOTE: Do not move the dishes for at least 24 h to enhance the adherence of EBs.</li>
	</ol>
	</li>
	<li>Day 6 - Retina induction
	<ol>
		<li>On day 6, add 10 mL of gfCDM containing 55 ng/mL BMP4 to allow a complete change of culture medium on the EBs. Return the plate to the incubator. 
		NOTE: Pipette toward the walls of the 96 V-bottomed conical well to avoid air bubbles. Avoid taking up any organoids when changing the medium.</li>
		<li>Carry out half-medium change on day 9, day 12 and day 15. Replace half of the medium with fresh gfCDM to gradually dilute the BMP4.</li>
	</ol>
	</li>
	<li>Day 18 - Long term NR culture
	<ol>
		<li>On day 18, carefully transfer the formed EBs to 15 mL centrifuge tubes using 5 mL Pasteur pipettes and gently rinse again with NRDM. Transfer the EBs to low-adsorption 6-well or 24-well plates (see Table of Materials). Return the plate to the incubator.</li>
		<li>Replace the medium with fresh NRDM every 3 days. 
		&#8203;NOTE: Switch off the light during the medium change because the RA is light-sensitive. Remove badly differentiated organoids, and separate adherent organoids under a microscope.</li>
	</ol>
	</li>
</ol>
4. Analysis of human NRs
<ol>
	<li>Mounting of the ROs
	<ol>
		<li>Select different generations of H9-ESCs to induce three batches of ROs. 
		NOTE: Three plates of the number of forming NRs for each of the three assessed methods (Kuwahara et al.12, D&#246;pper et al.27, and the modified method in the present study) are calculated to assess the induction success rate. Induction is considered successful if light microscopy reveals the formation of neuroepithelial-like structures on day 30.</li>
	</ol>
	</li>
	<li>Immunofluorescent staining of ROs
	<ol>
		<li>Transfer 3-5 ROs to 1.5 mL tubes. Pipette off excess medium and wash the ROs once with 1 mL of DPBS at RT. Allow the ROs to sink and carefully remove the DPBS. Add 1 mL of 4% paraformaldehyde (see Table of Materials) per 1.5 mL tube and fix at 4 &#176;C. 
		NOTE: ROs on day 6 and day 18, fix 2 h. Fix for 12 h on day 30 and 14 h on day 60.</li>
		<li>After fixation, dehydrate using gradient alcohol. Leave the ROs to stand for 15 min each in 50%, 60%, and 70% alcohol, and subsequently for 10 min each in 80%, 90%, 95%, 100%, and 100% alcohol. Then, add a 1:1 mix of 100% alcohol and xylene for 10 min, followed by xylene twice for 10 min each.</li>
		<li>Place the ROs in metal molds filled with melted paraplast (see Table of Materials) for 40 min, and then quench the molds on ice to fix the ROs. Place embedding boxes into the molds and freeze them at -20 &#176;C overnight. Subsequently, carefully detach the embedding boxes. Finally, store them at RT.</li>
		<li>Create continuous sections (5 &#181;m thickness) with a paraffin slicer. Fix slices on adhesion microscope slides, dry, and store at RT.</li>
		<li>Perform dewaxing and rehydration before antigen repair12,27.</li>
		<li>Add 10 mL of 20x pH 6.0 Citrate Antigen Retrieval Solution (see Table of Materials) to 190 mL of ddH2O (double distilled H2O). Heat in a microwave on high mode for 4 min until boiling. After adding paraffin sections, heat the sections on low mode for 20 min to complete antigen repair.</li>
		<li>Allow paraffin sections to cool naturally in a ventilated area and then put them into a humid chamber.</li>
		<li>Use a pap pen (see Table of Materials) to outline the sections. Incubate with 0.2% Triton X-100 at RT for 30 min to break the membranes, followed by washing the slides three times with TPBS, for 5 min each.</li>
		<li>Block with 10% donkey serum diluted in DPBS at RT for 1 h in a humid chamber with 10 &#181;L per staining area.</li>
		<li>Add primary antibodies diluted in 10% donkey serum. Incubate overnight at 4 &#176;C in a humidity chamber. Wash samples thrice with TPBS for 10 min each to remove the unbound antibody. 
		NOTE: Primary antibodies are as follows: anti-PAX6 (1: 250), anti-SOX2 (1: 200), anti-KI67 (1: 200), anti-CHX10 (1: 200), anti-&#946; tubulin III (1: 250) and anti-NESTIN (1: 200) (see Table of Materials).</li>
		<li>Incubate with secondary antibodies diluted in DPBS for 1 h at RT in a humidified chamber. Repeat the wash step with TPBS three times for 10 min each. 
		NOTE: Keep in the dark to prevent quenching of the fluorescent secondary antibody. Secondary antibodies are Alexa Fluor 488 conjugated with donkey anti-mouse IgG and Alexa Fluor 568 conjugated with donkey anti-rabbit IgG at a dilution of 1:400 (see Table of Materials).</li>
		<li>Incubate with DPBS containing DAPI (1: 500; see Table of Materials) for 10 min at RT in the dark. Wash three times with TPBS for 10 min each.</li>
		<li>Drop an appropriate amount of anti-fluorescent sealer (see Table of Materials) onto the slide and cover with cover slips.</li>
		<li>Visualize using a flow-like tissue cell quantitative analyzer (see Table of Materials), or similar.</li>
		<li>Store slides at -20 &#176;C in the dark after microscopic analysis.</li>
	</ol>
	</li>
</ol>

Enhanced 3D Neural Retina Differentiation from Human Embryonic Stem Cells

<ol>
	<li>Hoon, M., Okawa, H., Della Santina, ., Wong, L., O, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+the+retina:+development+and+disease.">Functional architecture of the retina: development and disease.</a> Prog Retin Eye Res. 42, 44-84 (2014).</li><li>Steinmetz, J. 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=Causes+of+blindness+and+vision+impairment+in+2020+and+trends+over+30+years,+and+prevalence+of+avoidable+blindness+in+relation+to+VISION+2020:+the+Right+to+Sight:+an+analysis+for+the+Global+Burden+of+Disease+Study.">Causes of blindness and vision impairment in 2020 and trends over 30 years, and prevalence of avoidable blindness in relation to VISION 2020: the Right to Sight: an analysis for the Global Burden of Disease Study.</a> Lancet Glob Health. 9 (2), e144-e160 (2021).</li><li>Pascolini, D., Mariotti, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+visual+impairment:+2010.">Global estimates of visual impairment: 2010.</a> Br J Ophthalmol. 96 (5), 614-618 (2012).</li><li>Singh, H. P., 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=Developmental+stage-specific+proliferation+and+retinoblastoma+genesis+in+RB-deficient+human+but+not+mouse+cone+precursors.">Developmental stage-specific proliferation and retinoblastoma genesis in RB-deficient human but not mouse cone precursors.</a> Proc Natl Acad Sci U S A. 115 (40), e9391-e9400 (2018).</li><li>Slijkerman, R. W., 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=The+pros+and+cons+of+vertebrate+animal+models+for+functional+and+therapeutic+research+on+inherited+retinal+dystrophies.">The pros and cons of vertebrate animal models for functional and therapeutic research on inherited retinal dystrophies.</a> Prog Retin Eye Res. 48, 137-159 (2015).</li><li>Peng, Y. R., 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=Molecular+classification+and+comparative+taxonomics+of+foveal+and+peripheral+cells+in+primate+retina.">Molecular classification and comparative taxonomics of foveal and peripheral cells in primate retina.</a> Cell. 176 (5), 1222-1237 (2019).</li><li>Ribeiro, J., 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=Restoration+of+visual+function+in+advanced+disease+after+transplantation+of+purified+human+pluripotent+stem+cell-derived+cone+photoreceptors.">Restoration of visual function in advanced disease after transplantation of purified human pluripotent stem cell-derived cone photoreceptors.</a> Cell Rep. 35 (3), 109022 (2021).</li><li>Mehat, M. 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=Transplantation+of+human+embryonic+stem+cell-derived+retinal+pigment+epithelial+cells+in+macular+degeneration.">Transplantation of human embryonic stem cell-derived retinal pigment epithelial cells in macular degeneration.</a> Ophthalmology. 125 (11), 1765-1775 (2018).</li><li>Manafi, N., 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=Organoids+and+organ+chips+in+ophthalmology.">Organoids and organ chips in ophthalmology.</a> Ocul Surf. 19, 1-15 (2021).</li><li>Rossi, G., Manfrin, A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+potential+in+organoid+research.">Progress and potential in organoid research.</a> Nat Rev Genet. 19 (11), 671-687 (2018).</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>Kuwahara, A., 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=Generation`of+a+ciliary+margin-like+stem+cell+niche+from+self-organizing+human+retinal+tissue.">Generation`of a ciliary margin-like stem cell niche from self-organizing human retinal tissue.</a> Nat Commun. 6, 6286 (2015).</li><li>Zhong, X., 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=Generation+of+three-dimensional+retinal+tissue+with+functional+photoreceptors+from+human+iPSCs.">Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.</a> Nat Commun. 5, 4047 (2014).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+development+and+disease+with+organoids.">Modeling development and disease with organoids.</a> Cell. 165 (7), 1586-1597 (2016).</li><li>O'Hara-Wright, M., Gonzalez-Cordero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+organoids:+a+window+into+human+retinal+development.">Retinal organoids: a window into human retinal development.</a> Development. 147 (24), (2020).</li><li>Li, 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=Protective+effects+of+resveratrol+on+the+ethanol-induced+disruption+of+retinogenesis+in+pluripotent+stem+cell-derived+organoids.">Protective effects of resveratrol on the ethanol-induced disruption of retinogenesis in pluripotent stem cell-derived organoids.</a> FEBS Open Bio. 13 (5), 845-866 (2023).</li><li>Zou, 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=Organoid-derived+C-Kit(+)/SSEA4(-)+human+retinal+progenitor+cells+promote+a+protective+retinal+microenvironment+during+transplantation+in+rodents.">Organoid-derived C-Kit(+)/SSEA4(-) human retinal progenitor cells promote a protective retinal microenvironment during transplantation in rodents.</a> Nat Commun. 10 (1), 1205 (2019).</li><li>Mandai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cell-derived+Retinal+organoid/cells+for+retinal+regeneration+therapies:+A+review.">Pluripotent stem cell-derived Retinal organoid/cells for retinal regeneration therapies: A review.</a> Regen Ther. 22, 59-67 (2023).</li><li>Suarez-Martinez, E., Suazo-Sanchez, I., Celis-Romero, M., Carnero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+and+organoid+culture+in+research:+physiology,+hereditary+genetic+diseases+and+cancer.">3D and organoid culture in research: physiology, hereditary genetic diseases and cancer.</a> Cell Biosci. 12 (1), 39 (2022).</li><li>Bose, R., Banerjee, S., Dunbar, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+neurological+disorders+in+3D+organoids+using+human-derived+pluripotent+stem+cells.">Modeling neurological disorders in 3D organoids using human-derived pluripotent stem cells.</a> Front Cell Dev Biol. 9, 640212 (2021).</li><li>Capowski, E. E., 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=Reproducibility+and+staging+of+3D+human+Retinal+organoids+across+multiple+pluripotent+stem+cell+lines.">Reproducibility and staging of 3D human Retinal organoids across multiple pluripotent stem cell lines.</a> Development. 146 (1), 171686 (2019).</li><li>Sanjurjo-Soriano, C., 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=RA+delays+initial+photoreceptor+differentiation+and+results+in+a+highly+structured+mature+Retinal+organoid.">RA delays initial photoreceptor differentiation and results in a highly structured mature Retinal organoid.</a> Stem Cell Res Ther. 13 (1), 478 (2022).</li><li>Li, X., Zhang, L., Tang, F., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+organoids:+cultivation,+differentiation,+and+transplantation.">Retinal organoids: cultivation, differentiation, and transplantation.</a> Front Cell Neurosci. 15, 638439 (2021).</li><li>Zerti, 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=Developing+a+simple+method+to+enhance+the+generation+of+cone+and+rod+photoreceptors+in+pluripotent+stem+cell-derived+Retinal+organoids.">Developing a simple method to enhance the generation of cone and rod photoreceptors in pluripotent stem cell-derived Retinal organoids.</a> Stem Cells. 38 (1), 45-51 (2020).</li><li>Kim, 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=transcriptome+profiling,+and+functional+validation+of+cone-rich+human+Retinal+organoids.">transcriptome profiling, and functional validation of cone-rich human Retinal organoids.</a> Proc Natl Acad Sci U S A. 116 (22), 10824-10833 (2019).</li><li>Yamasaki, 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=Addition+of+Chk1+inhibitor+and+BMP4+cooperatively+promotes+retinal+tissue+formation+in+self-organizing+human+pluripotent+stem+cell+differentiation+culture.">Addition of Chk1 inhibitor and BMP4 cooperatively promotes retinal tissue formation in self-organizing human pluripotent stem cell differentiation culture.</a> Regen Ther. 19, 24-34 (2022).</li><li>D&#246;pper, 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=Differentiation+protocol+for+3D+Retinal+organoids,+immunostaining+and+signal+quantitation.">Differentiation protocol for 3D Retinal organoids, immunostaining and signal quantitation.</a> Curr Protoc Stem Cell Biol. 55 (1), e120 (2020).</li><li>Norrie, J. 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=Retinoblastoma+from+human+stem+cell-derived+Retinal+organoids.">Retinoblastoma from human stem cell-derived Retinal organoids.</a> Nat Commun. 12 (1), 4535 (2021).</li></ol>

All authors declare that they have no conflicts of interest.

Human ROs can spatially and temporally recapitulate the development of the fetal retina, and early ROs exhibit a high degree of similarity to the fetal retina at equivalent stages of development15. In terms of tissue morphology and molecular expression, human RO closely mirror the actual growth status of the retinal tissue, providing tremendous and unprecedented opportunities in the fields of disease modeling, drug screening, and regenerative medicine. Currently, several different methods have bee...

The eye serves as the primary source of information among human sensory organs, with the retina being the principal visual sensory tissue in mammalian eyes1. Retinopathy stands as one of the primary global causes of eye diseases, leading to blindness2. Approximately 2.85 million people worldwide suffer from varying degrees of vision impairment due to retinopathy3. Consequently, investigating its pathogenesis is crucial for early diagnosis and timely treatment. Most studies on human retinopathy have primarily focused on animal models4,5,6. However, the human retina is a complex, multi-layered tissue comprising various cell types. Traditional two-dimensional (2D) cell culture and animal model systems typically fail to faithfully recapitulate the normal spatiotemporal development and drug metabolism of the native human retina7,8.
Recently, 3D culture techniques have evolved to generate tissue-like organs from pluripotent stem cells (PSCs)9,10. Retinal organoids (ROs) generated from human PSCs in a 3D suspension culture system not only contain seven retinal cell types but also exhibit a distinct stratified structure similar to the human retina in vivo11,12,13. Human PSC-derived ROs have gained popularity and widespread availability and are currently the best in vitro models for studying the development and disease of the human retina14,15. Over the past few decades, numerous researchers have demonstrated that human PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can differentiate into ROs using various induction protocols. These advancements hold enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies16,17,18.
However, the generation of neural retina (NR) from human pluripotent stem cells (PSCs) is a complex, cumbersome, and time-consuming process. Furthermore, batch-to-batch variations in tissue organoids may lead to lower reproducibility of results19,20. Numerous intrinsic and extrinsic factors can influence the yield of retinal organoids (ROs), such as the number or species of starting cells and the use of transcription factors and small-molecule compounds21,22,23. Since the first human RO was generated by the Sasai laboratory11, multiple modifications have been proposed over the years to enhance the ease and effectiveness of the induction process13,21,24,25. Unfortunately, to date, no gold standard protocol has been established for generating ROs in all laboratories. Indeed, there is a certain degree of discrepancy in ROs resulting from different induction methods, as well as wide variation in the expression of retinal markers and the robustness of their structure22,26. These issues may severely complicate sample collection and the interpretation of study findings. Therefore, a more consolidated and robust differentiation protocol is needed to maximize efficiency with minimal heterogeneity of RO generation.
This study describes an optimized induction protocol based on a combination of Kuwahara et al.12 and D&#246;pper et al.27 with detailed instructions. The new method significantly reduces the probability of organoid vesiculation and fusion, increasing the success rate of generating NR. This development holds great promise for disease modeling, drug screening, and cell therapy applications for retinal disorders.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.01 M TPBS</td>
			<td>Servicebio</td>
			<td>G0002</td>
			<td>Washing slices</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Servicebio</td>
			<td>G1101-500ML</td>
			<td>Fix retinal organoids</td>
		</tr>
		<tr>
			<td>5 mL Pasteur pipette</td>
			<td>NEST Biotechnology</td>
			<td>318516</td>
			<td>Pipette retinal organoids</td>
		</tr>
		<tr>
			<td>96 V-bottomed conical wells</td>
			<td>Sumitomo Bakelit</td>
			<td>MS-9096VZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion Microscope Slides</td>
			<td>CITOTEST</td>
			<td>188105</td>
			<td>Fix slices</td>
		</tr>
		<tr>
			<td>AggreWell medium</td>
			<td>STEMCELL Technologies</td>
			<td>5893</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>SINOPHARM</td>
			<td>10009218</td>
			<td>Dehydrate&#160;</td>
		</tr>
		<tr>
			<td>Anti-CHX10</td>
			<td>Santa Cruz</td>
			<td>sc-365519</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Antifade Solution</td>
			<td>ZSGB-BIO</td>
			<td>ZLI-9556</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-KI67</td>
			<td>Abcam</td>
			<td>ab16667</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Anti-NESTIN</td>
			<td>Sigma</td>
			<td>N5413</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Anti-Neuronal Class III &#946;-Tubulin(TUJ1)</td>
			<td>Beyotime</td>
			<td>AT809</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Anti-PAX6</td>
			<td>Abcam</td>
			<td>ab195045</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Cell dissociation solution(CDS)</td>
			<td>STEMCELL Technologies</td>
			<td>7922</td>
			<td>Cell dissociation</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Selleckchem</td>
			<td>S2924</td>
			<td>GSK-3&#945;/&#946; inhibitor</td>
		</tr>
		<tr>
			<td>Cholesterol Lipid Concentrate</td>
			<td>Gibco</td>
			<td>12531018</td>
			<td>250&#215;</td>
		</tr>
		<tr>
			<td>Citrate Antigen Retrieval Solution</td>
			<td>Servicebio</td>
			<td>G1202-250ML</td>
			<td>20&#215;, pH 6.0</td>
		</tr>
		<tr>
			<td>CS10</td>
			<td>STEMCELL Technologies</td>
			<td>1001061</td>
			<td>Cell Freezing Medium</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td>Nuclear counterstain</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11330032</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>DMEM/F12-GlutaMAX</td>
			<td>Gibco</td>
			<td>10565018</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse Alexa Fluor Plus 488</td>
			<td>Invitrogen</td>
			<td>A32766</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A10042</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>Ethylene Diamine Tetraacetic Acid (EDTA)</td>
			<td>Biosharp</td>
			<td>BL518A</td>
			<td>0.5 M, pH 8.0, cell dissociation</td>
		</tr>
		<tr>
			<td>Extracellular matrix (ECM)</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Coating plates</td>
		</tr>
		<tr>
			<td>F12-Glutamax</td>
			<td>Gibco</td>
			<td>31765035</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>A5669701</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow-like tissue cell quantitative analyzer</td>
			<td>TissueGnostics</td>
			<td>TissueFAXS Plus</td>
			<td>Scan sections</td>
		</tr>
		<tr>
			<td>IMDM-GlutaMAX</td>
			<td>Gibco</td>
			<td>31980030</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>IWR1-endo</td>
			<td>Selleckchem</td>
			<td>S7086</td>
			<td>Wnt-inhibitor</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN-193189 2HCl</td>
			<td>Selleckchem</td>
			<td>S7507</td>
			<td>BMP-inhibitor</td>
		</tr>
		<tr>
			<td>Low-adhesion 24-well Plates</td>
			<td>Corning</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-adhesion 6-well Plates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Maintenance medium (MM)</td>
			<td>STEMCELL Technologies</td>
			<td>85850</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Solarbio</td>
			<td>SL050</td>
			<td>Blocking buffer</td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Leica</td>
			<td>39601006</td>
			<td>Tissue embedding</td>
		</tr>
		<tr>
			<td>PBS pH 7.4 basic (1x)</td>
			<td>Gibco</td>
			<td>C10010500BT</td>
			<td>Without Ca+,Mg+</td>
		</tr>
		<tr>
			<td>Reconbinant human bone morphogenetic protein-4(rhBMP4)</td>
			<td>R&#38;D</td>
			<td>314-BP</td>
			<td>Key protein factor</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Powder, keep out of light</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Selleckchem</td>
			<td>S1067</td>
			<td>ALK5-inhibitor</td>
		</tr>
		<tr>
			<td>SU5402</td>
			<td>Selleckchem</td>
			<td>S7667</td>
			<td>Tyrosine kinase inhibitor</td>
		</tr>
		<tr>
			<td>Super PAP Pen</td>
			<td>ZSGB-BIO</td>
			<td>ZLI-9305</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T0625-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioglycerol</td>
			<td>Sigma</td>
			<td>M1753</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>Permeabilization</td>
		</tr>
		<tr>
			<td>WA09 embryonic stem cell line</td>
			<td>WiCell Research Institute</td>
			<td></td>
			<td>Cell line</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>SINOPHARM</td>
			<td>10023418</td>
			<td>Dewaxing</td>
		</tr>
		<tr>
			<td>Y-27632 2HCL</td>
			<td>Selleckchem</td>
			<td>S1049</td>
			<td>ROCK-inhibitor</td>
		</tr>
	</tbody>
</table>

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

Generating Neural Retina from Human Pluripotent Stem Cells

Our group focuses on understanding the pathogenesis and the treatment of human retinal diseases. Our protocol describes a optimized induction technique to generate a neural retina that significantly reduce the probability of vasculation and fusion to increase the success rate of retinal production until day 60. The modified method is simpler and more efficient in generating neural retina, which can prove to be a better disease model for drug screening and cell therapy.In the future, we aim to study the effects of physicochemical factors on retinal development and underlying mechanism using our retinal organoid model.

A graphical overview of the modified protocol is shown in Figure 1. H9-ESCs were used to generate ROs when the cells were grown to a density of 70%-80%. Single-cell suspensions of H9-ESCs in 96 V-bottomed conical wells aggregated on day 1 and formed well-circumscribed round EBs by day 6. As the culture time increased, the volume of EBs gradually increased. On day 30, neuroepithelial-like structures were clearly formed and thickened during long-term NR differentiation.
<p class="jove_cont...

Watch this Scientific Journal Video about Generating Neural Retina from Human Pluripotent Stem Cells at JoVE.com

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 ...

generating-neural-retina-from-human-pluripotent-stem-cells

Research

JoVE Journal

Developmental Biology

Culturing H9-Embryonic Stem Cells for Generation of Human Neural Retina

To begin, add one milliliter of 1%extracellular matrix to each well of a six well plate. Incubate the plate at 37 degrees Celsius in a 5%carbon dioxide atmosphere. After one hour, add two milliliters of prewarm maintenance medium containing 0.4 micromolar Y-27632 to each well.To thaw the H9 embryonic stem cells, shake the cryovial stock in a 37 degree Celsius water bath for 30 seconds. Carefully sterilize the cryovial with a 75%disinfectant alcohol spray. Then transfer the thawed cells to a 15 milliliter tube containing the maintenance medium and Y-27632.Centrifuge the tube at 190 G for five minutes at room temperature. Then carefully remove most of the supernatant and resuspend the cells in one milliliter of maintenance medium containing Y-27632. Dispense 0.5 milliliters of the cell suspension to each well of the extracellular matrix coated plate containing maintenance medium.Shake the plate laterally and incubate the plate at 37 degrees Celsius under 5%carbon dioxide for at least 24 hours. Change the medium every day. Once the clone density reaches 70%remove the spent medium.After 2D PBS washes, incubate the cells with one milliliter of EDTA for four to seven minutes at room temperature and discard EDTA completely. Add one milliliter of maintenance medium to the cells and tap the plate gently. Then transfer the dislodged cells to a 15 milliliter tube and mix them three to five times.Add 20 to 100 microliters of cell suspension to each well of a previously prepared ECM-coated dish and mix well. Using a microscope, confirm the cell density and incubate the cells at 37 degrees Celsius under 5%carbon dioxide.

Inducing 3D Neural Retina Formation from Cultured H9-Embryonic Stem Cells for Stem-Cell Based Therapies

To begin, thaw and culture H9-embryonic stem cells in maintenance medium containing Y-27632. On day zero, once the colonies become 70%confluent, wash them with 2 milliliters of DPBS, then incubate the cells with 0.5 milliliters of cell dissociation solution containing 20 micromoles per liter Y-27632. To abort the digestion, add 3 milliliters of maintenance medium containing 20 micromoles per liter Y-27632.To pellet the cells, centrifuge the tube at 190 G for five minutes and discard the supernatant. Resuspend the pellet in one milliliter of growth factor-free chemically-defined medium with Y-27632. After counting the cells, add 1.2 million cells to 10 milliliters of growth factor-free chemically-defined medium and mix well.Then dispense 100 microliters of cell suspension to each well of a 96 well conical bottom plate. Incubate in a humidified 5%carbon dioxide atmosphere. To induce retina formation on day six, add medium containing BMP4 to each well of a 96 well V-bottom plate and incubate again.On day nine, 12, and 15, replace half of the spent medium with fresh medium without BMP4. On day 18, carefully transfer the formed embryoid bodies to a 15 milliliter centrifuge tube. After the natural precipitation of the embryoid bodies, gently remove the supernatant and rinse them with the neural retina differentiation medium.Then place the embryoid bodies in a low absorption six well plate and incubate the plate again. On day 21, Brightfield images showed continuous neuroepithelial structure in the neural retina generated using this method. The retinal induction success rate was significantly higher and retinal organoids generated using this protocol were similar in size and morphology compared to other methods.

816 Views.  Medical School of Chinese PLA.  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.

Enhanced 3D Neural Retina Differentiation from Human Embryonic Stem Cells

Summary