This study was supported by the National Key R&#38;D Program of China (2016YFC1101103, 2017YFA0104101), the Guangzhou Science and Technology Project Fund (201803010078), the Science &#38; Technology Project of Guangdong Province (2017B020230003), the Natural Science Foundation (NSF) of China (81570874, 81970842), Hundred talent program of Sun Yat-sen University (PT1001010), and the Fundamental Research Funds of the State Key Laboratory of Ophthalmology.

1. Culture and expansion of hPSCs
<ol>
	<li>HPSC culture
	<ol>
		<li>Coat two wells of a 6-well plate with extracellular matrix (ECM, hESC-qualified matrix). Prepare 50 mL of an ECM solution containing 8-12 &#181;g/mL of ECM in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM). In 49&#160;mL of DMEM, add 1 mL of the thawed ECM stock solution (50x). Add 1 mL of the ECM solution to each well of a 6-well plate. Incubate it for 1 h in an incubator at 37 &#176;C and 5% CO2.</li>
		<li>Prepare hPSC maintenance medium (MM) according to the manufacturer&#39;s instruction.</li>
		<li>Pre-warm MM at room temperature (RT) for 30 min.</li>
		<li>Thaw a cryogenic vial of hPSCs (hiPSCs or hESCs) (about 1 x 106) from a liquid nitrogen tank by incubation in a water bath at 37 &#176;C for 30 s.</li>
		<li>Take out the vial, and carefully disinfect it using a 75% disinfection alcohol spray. Put it in a biosafety cabinet.</li>
		<li>Transfer the cell suspension from the vial to a 15 mL tube, add 5 mL of pre-warmed MM drop by drop to the tube using a 5 mL pipette. Meanwhile, gently shake the tube to blend the hPSCs .</li>
		<li>Centrifuge the tube at 170 x g for 5 min. Remove most of the supernatant using a 1 mL pipette carefully and leave behind about 50 &#181;L of supernatant to avoid losing the cells.</li>
		<li>Add 1 mL of MM to the tube, and resuspend the pellet by gently pipetting up and down once or twice with a 1 mL pipette. 
		NOTE: The survival of single cells of hPSCs is low. Small cell clumps with 3-5 cells are preferred to keep the hPSCs growing in colonies.</li>
		<li>Remove ECM from the pre-coated wells (step 1.1.1), add 1.5 mL of&#160;MM to each well, and then distribute 0.5 mL of&#160;cell suspension per well.</li>
		<li>Gently shake the plate to distribute the hPSCs uniformly, and put the plate in an incubator at 37 &#176;C and 5% CO2. Do not move the plate for at least 24 h to promote cell adherence.</li>
		<li>Change MM every other day and passage the hPSCs when the confluence has reached about 80%.</li>
	</ol>
	</li>
	<li>Passaging of hPSCs 
	NOTE: The maintenance of the undifferentiated state in hPSCs is quite critical for further applications. Under the adherent conditions, hPSCs grow in colonies with a well-defined border. The cells should be passaged when the confluence of hPSCs reaches about 80%.
	<ol>
		<li>Observe the cells under a microscope. Mark and mechanically remove the clearly visible differentiated cells (&#60;5%) before passaging.</li>
		<li>Prepare the ECM-coated plate as described in step 1.1.1.</li>
		<li>Pre-warm MM and 1x phosphate buffer saline (PBS) without Ca2+ and Mg2+ at RT.</li>
		<li>Pre-warm the 0.5 mM EDTA (in 1x PBS) solution in a water bath at 37 &#176;C.</li>
		<li>Remove the medium from the culture plate using a vacuum-aspiration system, add 1 mL of&#160;1x PBS in each well to wash the cells using a 1 mL pipette and repeat twice.</li>
		<li>Add 1 mL of&#160;EDTA solution per well to dissociate the hPSCs in a cell culture incubator at 37 &#176;C and 5% CO2 for 5 min. Do not exceed the recommended incubation time in order to avoid dissociation to single cells.</li>
		<li>Take out the plate and check for the detachment of cells under a microscope. The confluent hPSCs loosen up and each cell border can be seen, but the cells cannot easily come off by gently shaking the cell plate.</li>
		<li>Remove the EDTA solution with a 1 mL pipette, and add 1 mL of MM to stop the dissociation. Gently pipette the hPSCs once or twice with a 1 mL pipette to resuspend the cells. There is no need to centrifuge to collect the&#160;cells. 
		&#8203;NOTE: If most of the cells come off from the plate after incubation with EDTA, cells can be collected by centrifuge.</li>
		<li>Remove ECM from the pre-coated wells (step 1.2.2), and add 1.5 mL of&#160;MM per well.</li>
		<li>Transfer 150-200 &#181;L of cell clumps to each well. Generally, hPSCs can be passaged at a ratio of 1:6. For example, cells from one well of a 6-well plate can be distributed to six new wells.</li>
		<li>Gently shake the plate to distribute the hPSCs uniformly and culture the hPSCs in the incubator at 37 &#176;C and 5% CO2 for at least 24 h without touching the plate.</li>
		<li>Change MM every other day as described in step 1.1.</li>
	</ol>
	</li>
</ol>
2. Retinal differentiation from hPSCs
NOTE: When the colonies reach ~80% confluence (Figure 1B), they can be guided to differentiate into retinal organoids following the protocol schematized in Figure 1A. To ensure the hPSCs have high quality and good yield, regularly evaluate the pluripotency with molecular markers such as OCT4 or NANOG using IFC or QPCR. HPSCs should be discarded if differentiated cells account for more than 5% of the total cells. Check for mycoplasma contamination with a mycoplasma detection kit according to the manufacturer&#39;s instructions. Use only mycoplasma-free hPSCs as mycoplasma can alter the differentiation capability of hPSCs.
<ol>
	<li>Prepare media and reagents
	<ol>
		<li>Prepare neural induction medium (NIM) by mixing the following: 500 mL of&#160;Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, 1:1), 5 mL of&#160;1% N2 supplement, 0.5 mL of&#160;0.1% heparin (2 mg/mL in 1x PBS), and 5 mL of&#160;1% MEM Non-Essential Amino Acids (NEAA).</li>
		<li>Prepare retinal differentiation medium (RDM) containing 300 mL of&#160;DMEM/F-12, 200 mL of&#160;DMEM basic, 10 mL of&#160;2% B27 supplement, 5 mL of&#160;1% Antibiotic Antimycotic, and 5 mL of&#160;1% MEM NEAA. 
		NOTE: Both NIM and RDM are not filtered but a sterility test is performed. Take out 1 mL of&#160;medium and add it into a 35 mm dish, and culture for 3-7 days in an incubator at 37 &#176;C and 5% CO2. The media can be stored at 4 &#176;C and should be used within 2 weeks to ensure the activity of the components.</li>
		<li>Prepare 10 mM Blebbistatin (1,000x) in DMSO. Add 1,710 &#181;L of&#160;DMSO to dissolve 5 mg of&#160;Blebbistatin to obtain 10 mM stock solution (1,000x), aliquot at 10 &#181;L/tube, and store at -20 &#176;C. 
		NOTE: All media and reagents should be warmed at RT for 30 min before use, unless otherwise mentioned.</li>
	</ol>
	</li>
	<li>Embryoid body (EB) formation
	<ol>
		<li>On day 0 (D0), initiate the differentiation. Take out one well of hPSCs from a 6-well plate, which has grown to ~80% confluence. Collect the cells with EDTA dissociation solution as described in steps 1.2.1 to 1.2.6.</li>
		<li>Remove the EDTA solution, add 1 mL of&#160;MM containing 10 &#181;M Blebbistatin to stop cell dissociation, and collect the cells with a 1 mL pipette. The size of cell clumps is one of the key factors impacting the yield of EBs. Approximately, five cells per clump&#160;are preferred to produce the right size of EBs on D5 to D7. 
		NOTE: This is a key step. Do not pipette the cells too many times since EB-like aggregates are hard to form from single cells of hPSCs.</li>
		<li>Transfer the cell suspension (about 2 x 106 cells) to a 100 mm ultra-low attachment Petri dish and add 9 mL of&#160;MM containing 10 &#181;M Blebbistatin to the dish.</li>
		<li>Gently shake the dish twice to distribute the cells uniformly, and put the dish in the incubator at 37 &#176;C and 5% CO2.</li>
		<li>On D1, after the cells are cultured for at least 24 h, take out the dish and observe it under the microscope. A large number of the small cell aggregates will be spontaneously&#160;formed by this time (Figure 1C).</li>
		<li>Prepare 12 mL of mixture with MM and NIM at a 3:1 ratio (9 mL of&#160;MM and 3 mL of&#160;NIM) in a 15 mL tube.</li>
		<li>Transfer the cell cultures to a 15 mL centrifuge tube with a 10 mL pipette perpendicularly, and add 10 mL of the pre-warmed mixture to the dish.</li>
		<li>Centrifuge the tube at 60 x g for 3 min to collect the aggregates, remove the supernatant using a 5 mL pipette and leave behind about 500 &#181;L to avoid losing cells.</li>
		<li>Add 2 mL of the mixture to the tube, and transfer the suspension to the same dish (step 2.2.7).</li>
		<li>Gently shake the dish to uniformly distribute the cell aggregates, and put the dish back&#160;in the incubator.</li>
		<li>On D2, prepare 12 mL of a new mixture with MM and NIM at a 1:1 ratio (6 mL of&#160;MM and 6 mL of&#160;NIM) in a 15 mL tube. Change cell medium with the fresh prepared mixture by repeating the steps from 2.2.5 to 2.2.10.</li>
		<li>On D3, change cell medium with 15 mL of&#160;NIM as described above. Culture the cells for at least 5 days under the suspension conditions. 
		NOTE: During D1 to D3, the medium should be changed each day, providing enough nutrition. Since D3, NIM can be changed every other day. Also, EBs can be divided into several dishes to provide abundant nutrition.</li>
	</ol>
	</li>
	<li>Seed the EBs 
	NOTE: On D5 to D7, choose an appropriate time point to plate the EBs on the ECM-coated dishes according to the size of EBs. EBs with an approximate diameter of 200 &#181;m&#160;is appropriate for the retinal differentiation. In general, one well of hPSCs in a 6-well plate can produce about 300 to 1,000 EBs. The variation of EB yield&#160;is varied by the hPSC lines.
	<ol>
		<li>On D4, prepare ECM-coated dishes for EBs adherent culture. Add 5 mL of&#160;ECM to each 100 mm tissue culture dish (surface treated), and put them in the incubator overnight.</li>
		<li>On D5, remove ECM from the pre-coated dishes, and add 10 mL of&#160;pre-warmed NIM to each dish.</li>
		<li>Take out the dish containing EBs. Check the quality of EBs under the microscope and ensure that they are quite bright and round in shape. The size of the EBs is approximate 200 &#181;m&#160;in diameter. Collect all EBs in a 15 mL tube. Transfer the EBs from the dishes to a 15 mL tube with a 5 mL pipette. Let the EBs settle down for 5 min. Remove most of the supernatant, leaving behind about 2 mL of medium.</li>
		<li>Distribute the EBs into the coated dishes containing 10 mL of&#160;NIM drop by drop with a 1 mL pipette. Seed the EBs at a density of approximately 2-3 EBs per cm2. For example, add about 120-180 EBs into a 100 mm dish. To roughly judge the EB number, place one drop of EB suspension onto a coverslip, and count the number of EBs under the microscope. 
		NOTE: The plating density of EBs is one of key factors impacting the efficiency of retinal induction. The density can be also adjusted by each hPSC line.</li>
		<li>Gently shake the dishes to distribute the EBs uniformly. Put them in the incubator at 37 &#176;C and 5% CO2. 
		NOTE:&#160;Do not move the dishes for at least 24 h to enhance the adherence of EBs.</li>
	</ol>
	</li>
	<li>Induction of optic vesicles (OVs) and retinal pigment epithelium (RPE) in adherent conditions 
	NOTE: After EBs are seeded on the ECM coated surface, hPSCs can develop OV-like structures, which can be observed as early as D20 after differentiation. In this protocol, specific growth factors or signaling molecules are not required to guide the hPSCs into the retinal fate except the addition of N2 and B27 supplements in the media.
	<ol>
		<li>On D8-D9, remove the dishes and observe the EBs under the microscope. All EBs will be attached and spread out on the dishes (Figure 1D). Add 10 mL&#160;of&#160; fresh NIM to each 100 mm dish containing 10 mL of&#160;old medium. Put them back in the incubator. 
		NOTE: Do not remove the old medium.</li>
		<li>On D12, change half of the medium with NIM using a 10 mL pipette. Keep the culture in the incubator.</li>
		<li>On D16, remove all NIM from the dishes using a vacuum-aspiration system. Add 20 mL of&#160;RDM to each dish. Keep culturing in RDM and change half of the medium every other day.</li>
		<li>During D10-D30, observe the morphological changes of the cells twice a week under a microscope and evaluate the efficiency of retinal differentiation. 
		&#8203;NOTE: Since D10, eye field (EF) domains are self-organized in the peripheral zones of adherent EBs. The OV-like structures appear between D20 to D25, gradually protrude from the dish, and self-form an optic cup, which is surrounded by the pigmented RPE (Figure 1E). The OVs can be easily recognized with the bright, refractive, and thick NR ring.</li>
	</ol>
	</li>
	<li>Detach and culture OVs and RPE in suspension to obtain retinal organoids (ROs)
	<ol>
		<li>On D28-D35, most of OVs appear in the dishes. Use a Tungsten needle or a needle with 1 mL syringe to mechanically detach the morphologically identifiable OVs along with the adjacent RPE. Culture them in suspension. 
		NOTE: The appearance and yield of OVs and RPE vary widely in different hPSC lines. Thus, the time point of detaching OV and RPE is flexible. Obvious OVs with the adjacent RPE can be detached, and then moved to a low attachment culture dish containing RDM. Keep culturing the rest of the cells until all OVs and RPEs are lifted up.</li>
		<li>Put 50-60 OVs into each 100 mm low attachment culture dish containing 15 mL of RDM for the ROs formation (Figure 1F).</li>
		<li>Change RDM every 2-3 days until D42, when the ROs are well round-shaped.</li>
	</ol>
	</li>
</ol>
3. Retinal development and maturation
NOTE: In this protocol, serum is required to keep the ROs grow and mature for long-term culture.
<ol>
	<li>Retinal lamination and specification in ROs
	<ol>
		<li>Prepare 10 mL of 100 mM taurine (1,000x) in 1x PBS. Weigh 125 mg of&#160;taurine, and dissolve in 10 mL of 1x PBS. Filter the solution with a 0.22 &#181;m syringe filter. Aliquot at 500 &#181;L/tube, and store at -20 &#176;C.</li>
		<li>Prepare retinal culture medium 1 (RC1). Mix the following components: 250 mL&#160;of&#160;DMEM/F-12, 175&#160;mL of&#160;DMEM basic, 50 mL of&#160;fetal bovine serum,10 mL of&#160;2% B27 supplement, 5 mL of&#160;1% Antibiotic Antimycotic, 5 mL of&#160;1% MEM NEAA, 0.5 mL of&#160;100 &#181;M taurine, and 5 mL of&#160;2 mM L-alanyl-L-glutamine.</li>
		<li>Prepare retinal culture medium 2 (RC2) containing 450 mL of&#160;DMEM/F-12, 50 mL of&#160;fetal bovine serum, 5 mL of&#160;1% N2 supplement, 5 mL of&#160;1% Antibiotic Antimycotic, 0.5 mL of&#160;100 &#181;M taurine, 5 mL of 1% MEM NEAA, and 5 mL of 2 mM L-alnyl-L-glutamine. 
		NOTE: The RC1 and RC2 are not filtered, but undergo a sterility test. Take out 1 mL of&#160;medium, add it to a 35 mm dish, and culture for 3-7 days in the incubator at 37 &#176;C and 5% CO2, to ensure sterility before use. The medium can be stored at 4 &#176;C and should be used within 2 weeks to ensure the activity of the components. All media and reagents should be pre-warmed at RT for 30 min before use.</li>
		<li>On D42, switch the culture medium from RDM to RC1.</li>
		<li>Tilt the dishes at about 30&#730; and let the ROs settle&#160;down for 30 s. Remove the old RDM with a 10 mL pipette leaving behind about 1 mL of&#160;medium to avoid losing ROs. Add 15 mL of&#160;fresh RC1 to each dish.</li>
		<li>Gently shake the dishes to distribute the ROs uniformly. Put the dishes back in the incubator. Change the whole medium twice a week thereafter.</li>
		<li>During D50-D90, select out high quality of ROs for long-term culture, which are&#160;round-shaped with a thick and bright NR. Place 30-40 ROs in a 100 mm low attachment dish with 20 mL of&#160;RC1,and&#160; change the whole medium twice a week.</li>
		<li>For the long-term suspension culture of ROs, pipette the ROs to avoid RO-RO reattaching using a pipette. Transfer ROs to new culture dishes once a month to avoid ROs sticking to the surface of the dishes. 
		NOTE: Under the suspension culture conditions, ROs are round-shaped, with a bright and thick NR ring attached with more or less RPE at one side. Laminated neural retina develop and retinal cell subtypes sequentially appear with retinal ganglion cells first generated, followed by photoreceptor cells, amacrine cells, and bipolar cells.</li>
	</ol>
	</li>
	<li>Human photoreceptor maturation with enrichment of cones in ROs
	<ol>
		<li>After D90, switch the medium from RC1 to RC2, which is suitable for photoreceptor maturation.</li>
		<li>Change the medium as described in steps 3.1.7-3.1.8. 
		NOTE: Under this culture condition, ROs can grow long-term (Figure 1G), up to D300 tested. Retinal cells in ROs become mature, and all cell subtypes of neural retina, including muller glial cells, rods and cones are also acquired. Without any addition of RA, cone photoreceptors are also rich in ROs.</li>
	</ol>
	</li>
</ol>

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C., Moosajee, M., Rainger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closing+the+Ggap:+Mechanisms+of+epithelial+fusion+during+optic+fissure+closure.">Closing the Ggap: Mechanisms of epithelial fusion during optic fissure closure.</a> Frontiers in Cell and Developmental Biology. 8, (2021).</li><li>Li, G., 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+retinal+organoids+with+mature+rods+and+cones+from+urine-derived+human+induced+pluripotent+stem+cells.">Generation of retinal organoids with mature rods and cones from urine-derived human induced pluripotent stem cells.</a> Stem Cells International. 2018, 4968658 (2018).</li><li>Liu, 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=Self-formation+of+RPE+spheroids+facilitates+enrichment+and+expansion+of+hiPSC-derived+RPE+generated+on+retinal+organoid+induction+platform.">Self-formation of RPE spheroids facilitates enrichment and expansion of hiPSC-derived RPE generated on retinal organoid induction platform.</a> Investigative Ophthalmology and Visual Science. 59 (13), 5659-5669 (2018).</li><li>Luo, Z., 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=An+optimized+system+for+effective+derivation+of+three-dimensional+retinal+tissue+via+Wnt+signaling+regulation.">An optimized system for effective derivation of three-dimensional retinal tissue via Wnt signaling regulation.</a> Stem Cells. 36 (11), 1709-1722 (2018).</li><li>Li, G., 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+and+characterization+of+induced+pluripotent+stem+cells+and+retinal+organoids+from+a+leber's+congenital+amaurosis+patient+with+novel+RPE65+mutations.">Generation and characterization of induced pluripotent stem cells and retinal organoids from a leber's congenital amaurosis patient with novel RPE65 mutations.</a> Frontiers in Molecular Neuroscience. 12, 212 (2019).</li><li>Matsa, E., Ahrens, J. H., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cells+as+a+platform+for+personalized+and+precision+cardiovascular+medicine.">Human induced pluripotent stem cells as a platform for personalized and precision cardiovascular medicine.</a> Physiology Reviews. 96 (3), 1093-1126 (2016).</li><li>Wahlin, K. 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=Photoreceptor+outer+segment-like+structures+in+long-term+3d+retinas+from+human+pluripotent+stem+cells.">Photoreceptor outer segment-like structures in long-term 3d retinas from human pluripotent stem cells.</a> Science Reports. 7 (1), 766 (2017).</li><li>Eiraku, M., 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-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472 (7341), 51-56 (2011).</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=Generation+of+storable+retinal+organoids+and+retinal+pigmented+epithelium+from+adherent+human+iPS+cells+in+xeno-free+and+feeder-free+conditions.">Generation of storable retinal organoids and retinal pigmented epithelium from adherent human iPS cells in xeno-free and feeder-free conditions.</a> Stem Cells. 35 (5), 1176-1188 (2017).</li><li>Lamba, D. A., Karl, M. O., Ware, C. B., Reh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+retinal+progenitor+cells+from+human+embryonic+stem+cells.">Efficient generation of retinal progenitor cells from human embryonic stem cells.</a> Proceedings of the National Academy of Science U. S. A. 103 (34), 12769-12774 (2006).</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> Nature Communication. 6, 6286 (2015).</li><li>da Silva, S., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf8+expression+and+degradation+of+retinoic+acid+are+required+for+patterning+a+high-acuity+area+in+the+retina.">Fgf8 expression and degradation of retinoic acid are required for patterning a high-acuity area in the retina.</a> Developmental Cell. 42 (1), 68-81 (2017).</li><li>Mitchell, D. M., 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=Retinoic+acid+signaling+regulates+differential+expression+of+the+tandemly-duplicated+long+wavelength-sensitive+cone+opsin+genes+in+zebrafish.">Retinoic acid signaling regulates differential expression of the tandemly-duplicated long wavelength-sensitive cone opsin genes in zebrafish.</a> PLoS Genetics. 11 (8), 1005483 (2015).</li><li>Stevens, C. B., Cameron, D. A., Stenkamp, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+photoreceptor-generating+retinal+progenitors+revealed+by+prolonged+retinoic+acid+exposure.">Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure.</a> BMC Developmental Biology. 11 (1), (2011).</li><li>Eldred, K. 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=Thyroid+hormone+signaling+specifies+cone+subtypes+in+human+retinal+organoids.">Thyroid hormone signaling specifies cone subtypes in human retinal organoids.</a> Science. 362 (6411), (2018).</li><li>Yang, F., Ma, H., Ding, X. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+hormone+signaling+in+retinal+development,+survival,+and+disease.">Thyroid hormone signaling in retinal development, survival, and disease.</a> Vitamins and Hormones. 106, 333-349 (2018).</li><li>Brzezinski, J. A., Reh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+cell+fate+specification+in+vertebrates.">Photoreceptor cell fate specification in vertebrates.</a> Development. 142 (19), 3263-3273 (2015).</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=Generation,+transcriptome+profiling,+and+functional+validation+of+cone-rich+human+retinal+organoids.">Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids.</a> Proceedings of the National Academy of Science U. S. A. 116 (22), 10824-10833 (2019).</li><li>Lowe, A., Harris, R., Bhansali, P., Cvekl, A., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+adhesion-dependent+cell+survival+and+ROCK-regulated+actomyosin-driven+forces+mediate+self-formation+of+a+retinal+organoid.">Intercellular adhesion-dependent cell survival and ROCK-regulated actomyosin-driven forces mediate self-formation of a retinal organoid.</a> Stem Cell Reports. 6 (5), 743-756 (2016).</li><li>Carcamo-Orive, I., 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=Analysis+of+transcriptional+variability+in+a+large+human+ipsc+library+reveals+genetic+and+non-genetic+determinants+of+heterogeneity.">Analysis of transcriptional variability in a large human ipsc library reveals genetic and non-genetic determinants of heterogeneity.</a> Cell Stem Cell. 20 (4), 518-532 (2017).</li><li>DeBoever, 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=Large-scale+profiling+reveals+the+influence+of+genetic+variation+on+gene+expression+in+human+induced+pluripotent+stem+cells.">Large-scale profiling reveals the influence of genetic variation on gene expression in human induced pluripotent stem cells.</a> Cell Stem Cell. 20 (4), 533-546 (2017).</li></ol>

Xiufeng Zhong is the patent inventor related to the generation of retinal cells from human pluripotent stem cells.

In this multi-step retinal induction protocol, hPSCs were guided step by step to gain the retinal fate, and self-organized into retinal organoids containing laminated NR and RPE. During the differentiation, hPSCs recapitulated all major steps of human retinal development in vivo, from EF, OV, and RPE, to retinal lamination, generating all subtypes of retinal cells, including retinal ganglion cells, amacrine cells, bipolar cells, rod, and cone photoreceptors, and muller glial cells in a spatial and temporal order...

Retinal degenerative diseases (RDs), such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are characterized by the dysfunction and death of photoreceptor cells and typically lead to irreversible vision loss without effective ways to cure1. The mechanism underlying these diseases is largely unknown partially due to lack of human disease models2. Over the past decades, significant advances have been accomplished in regenerative medicine through stem cell technology. Many researchers, including ourselves, have shown that human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), can differentiate into all types of retinal cells, even mini-retinal tissues through various differentiation approaches3,4,5,6,7,8,9,10,11, providing huge potential in disease modeling and cell therapy12,13,14.
However, the induction process from hPSCs to retinal cells is highly complicated and&#160;time-consuming with low repeatability, which requires researchers with rich experience and high skills. During the complex and dynamic induction process, a number of factors will impact the yield of retinal tissues15,16,17. Also, different induction methods often vary considerably in timing and robust expression of retinal markers, which might confound the sample collection and data interpretation3. Therefore, a straightforward protocol of retinal differentiation from hPSCs with step-by-step guidance would be in demand.
Here, based on our published studies18,19,20,21, an optimized retinal induction protocol to generate retinal organoids (ROs) with rich cone photoreceptors from hPSCs is described, which does not require the supplement of retinoic acid (RA). This protocol focuses on the description of the multi-step method to generate neural retina and RPE. EB formation is the essential part of the early induction stage. Both size and plating density of EBs are quantitatively optimized, which scientifically enhances the yield of retinal tissues and promotes repeatability. In the second part of the induction, optic vesicles (OVs) self-organize in the adherence culture and ROs form in the suspension culture; the time courses and efficiencies of this part vary considerably in different hPSC lines. The maturation and specification of retinal cells in ROs mainly occur in the middle and late stage of induction. Without the addition of RA, mature photoreceptors with both rich cones and rods can be produced.
The purpose of this protocol is to quantitatively describe and detail each step for inexperienced researchers to repeat. Various hPSC lines have been successfully induced into ROs by this protocol with a robust yield of cone-rich retinal tissues and high repeatability. HPSCs-derived ROs with this protocol can recapitulate the main steps of retinal development in vivo, and survive long-term, which facilitates downstream applications, such as disease modeling, drug screening, and cell therapy.

<table><tbody><tr><td>(&amp;minus;)-Blebbistatin</td><td>Sigma</td><td>B0560-5mg</td><td>ROCK-inhibitor</td></tr><tr><td>1 ml tips</td><td>Kirgen</td><td>KG1313</td><td>1 ml</td></tr><tr><td>10 ml pipette</td><td>Sorfa</td><td>3141001</td><td>Pipette</td></tr><tr><td>100 mm Tissue culture</td><td>BIOFIL</td><td>TCD000100</td><td>100 mm Petri dish</td></tr><tr><td>100 mm Tissue culture</td><td>Falcon</td><td>353003</td><td>100 mm Petri dish</td></tr><tr><td>15 ml Centrifuge tubes</td><td>BIOFIL</td><td>CFT011150</td><td>Centrifuge tubes</td></tr><tr><td>35 mm Tissue culture dishes</td><td>Falcon</td><td>353001</td><td>35 mm Petri dish</td></tr><tr><td>5 ml pipette</td><td>Sorfa</td><td>313000</td><td>Pipette</td></tr><tr><td>50 ml Centrifuge tubes</td><td>BIOFIL</td><td>CFT011500</td><td>Centrifuge tubes</td></tr><tr><td>6 wells tissue culture plates</td><td>Costar</td><td>3516</td><td>Culture plates</td></tr><tr><td>Anti-AP2&amp;alpha; Antibody</td><td>DSHB</td><td>3b5</td><td>Primary&amp;nbsp;antibody</td></tr><tr><td>ANTIBIOTIC ANTIMYCOTIC 100X</td><td>Gibco</td><td>15240062</td><td>Antibiotic-Antimycotic</td></tr><tr><td>Anti-ISL1 Antibody</td><td>Boster</td><td>BM4446</td><td>Primary&amp;nbsp;antibody</td></tr><tr><td>Anti-Ki67 Antibody</td><td>Abcam</td><td>ab15580</td><td>Primary&amp;nbsp;antibody</td></tr><tr><td>Anti-L/M opsin Antibody</td><td>gift from Dr. jeremy</td><td>/</td><td>Primary&amp;nbsp;antibody</td></tr><tr><td>Anti-PAX6 Antibody</td><td>DSHB</td><td>pax6</td><td>Primary&amp;nbsp;antibody</td></tr><tr><td>Anti-rabbit 555</td><td>Invitrogen</td><td>A31572</td><td>Donkey anti-Rabbit IgG (H+L)&lt;br/&gt; Secondary Antibody, Alexa Fluor 555</td></tr><tr><td>Anti-Recoverin Antibody</td><td>Millipore</td><td>ab5585</td><td>Primary&amp;nbsp;antibody</td></tr><tr><td>Anti-Rhodopsin Antibody</td><td>Abcam</td><td>ab5417</td><td>Primary&amp;nbsp;antibody</td></tr><tr><td>Anti-sheep 555</td><td>Invitrogen</td><td>A21436</td><td>Donkey anti-Sheep IgG (H+L)&lt;br/&gt; Secondary Antibody, Alexa Fluor 555</td></tr><tr><td>Anti-SOX9 Antibody</td><td>Abclonal</td><td>A19710</td><td>Primary&amp;nbsp;antibody</td></tr><tr><td>Anti-VSX2 Antibody</td><td>Millipore</td><td>ab9016</td><td>Primary&amp;nbsp;antibody</td></tr><tr><td>B-27 supplement W/O VIT A (50X)</td><td>Gibco</td><td>12587010</td><td>Supplement</td></tr><tr><td>Cryotube vial</td><td>Thermo scientific-NUNC</td><td>375418</td><td>1.8 ml</td></tr><tr><td>DAPI</td><td>DOJINDO</td><td>D532</td><td>4&#039;,6-Diamidino-2-phenylindole&lt;br/&gt; dihydrochloride; multiple suppliers</td></tr><tr><td>Dimethyl sulphoxide(DMSO) Hybri-max</td><td>Sigma</td><td>D2650-100ML</td><td>Multiple suppliers</td></tr><tr><td>DMEM</td><td>Gibco</td><td>C11995500BT</td><td>Medium</td></tr><tr><td>DMEM /F12</td><td>Gibco</td><td>C11330500BT</td><td>Medium</td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575-020</td><td>0.5 M PH 8.0</td></tr><tr><td>FBS</td><td>NATOCOR</td><td>SFBE</td><td>Serum</td></tr><tr><td>Filter</td><td>Millipore</td><td>SLGP033RB</td><td>0.22&amp;mu;m, sterile Millex filter</td></tr><tr><td>GlutaMax, 100X</td><td>Gibco</td><td>35050061</td><td>L-alanyl-L-glutamine</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3149</td><td>2 mg/ml in PBS to use</td></tr><tr><td>Matrigel, 100x</td><td>Corning</td><td>354277</td><td>Extracellular matrix (ECM)</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100X)</td><td>Gibco</td><td>11140050</td><td>MEM NEAA</td></tr><tr><td>mTeSR1</td><td>STEM CELL</td><td>85850</td><td>hPSCs maintenance medium (MM)</td></tr><tr><td>N2 supplement</td><td>Gibco</td><td>17502048</td><td>Supplement</td></tr><tr><td>Phosphate-buffered saline (PBS) buffer</td><td>GNM</td><td>GNM10010</td><td>Without Ca+,Mg+,PH7.2&amp;plusmn;0.1 0.1M</td></tr><tr><td>Taurine</td><td>Sigma</td><td>T0625</td><td>Supplement</td></tr><tr><td>Ultra-low attachment culture dishes 100mm petri dish, low-attachment</td><td>Corning</td><td>CLS3262-20EA</td><td>Petri dish</td></tr></tbody></table>

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.

The retinal induction process in this protocol mimics the development of human fetal retina. To initiate the retinal differentiation, hPSCs were dissociated into small clumps and cultured in suspension to induce the formation of EBs. On D1, the uniformed cell aggregates or EBs formed (Figure 1C). The culture medium was gradually transitioned into NIM. On D5, EBs were plated onto the ECM-coated culture dishes. Cells gradually migrated out of the EBs (Figure 1D). ...

Watch this Scientific Journal Video about Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells at JoVE.com

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

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 ...

retinal-organoid-induction-system-for-derivation-3d-retinal-tissues

Research

JoVE Journal

Medicine

5.8K Views.  Sun Yat-sen University, Guangzhou, China, 510060. 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.

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

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Developmental Biology

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 ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

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 ...

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 ...

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

Bioengineering

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.

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 ...

Microglia-Integrated Retinal Organoids: A Model for Studying Retinal Diseases

Assembling Retinal Organoids with Microglia

Due to the limited accessibility of the human retina, retinal organoids (ROs) are the best model for studying human retinal disease, which could reveal the mechanism of retinal development and the occurrence of retinal disease. Microglia (MG) are unique resident macrophages in the retina and central nervous system (CNS), serving crucial immunity functions. However, retinal organoids lack microglia since their differentiation origin is the yolk sac. The specific pathogenesis of microglia in these retinal diseases remains unclear; therefore, the establishment of a microglia-incorporated retinal organoid model turns out to be necessary. Here, we successfully constructed a co-cultured model of retinal organoids with microglia derived from human stem cells. In this article, we differentiated microglia and then co-cultured to retinal organoids in the early stage. As the incorporation of immune cells, this model provides an optimized platform for retinal disease modeling and drug screening to facilitate in-depth research on the pathogenesis and treatment of retinal and CNS-related diseases.

Generation of Human Microglia to Combine Them with Retinal Organoids for Improved Disease Modeling

Microglia are unique resident immune cells in the retina, playing crucial roles in various retinal degenerative diseases. Generating a co-culture model of retinal organoids with microglia can facilitate a better understanding of the pathogenesis and development progress of retinal diseases.

Due to the limited accessibility of the human retina, retinal organoids (ROs) are the best model for studying human retinal disease, which could reveal ...