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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(&#8722;)-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&#945; Antibody</td>
			<td>DSHB</td>
			<td>3b5</td>
			<td>Primary&#160;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&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-Ki67 Antibody</td>
			<td>Abcam</td>
			<td>ab15580</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-L/M opsin Antibody</td>
			<td>gift from Dr. jeremy</td>
			<td>/</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-PAX6 Antibody</td>
			<td>DSHB</td>
			<td>pax6</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-rabbit 555</td>
			<td>Invitrogen</td>
			<td>A31572</td>
			<td>Donkey anti-Rabbit IgG (H+L) 
			Secondary Antibody, Alexa Fluor 555</td>
		</tr>
		<tr>
			<td>Anti-Recoverin Antibody</td>
			<td>Millipore</td>
			<td>ab5585</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-Rhodopsin Antibody</td>
			<td>Abcam</td>
			<td>ab5417</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-sheep 555</td>
			<td>Invitrogen</td>
			<td>A21436</td>
			<td>Donkey anti-Sheep IgG (H+L) 
			Secondary Antibody, Alexa Fluor 555</td>
		</tr>
		<tr>
			<td>Anti-SOX9 Antibody</td>
			<td>Abclonal</td>
			<td>A19710</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-VSX2 Antibody</td>
			<td>Millipore</td>
			<td>ab9016</td>
			<td>Primary&#160;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&#39;,6-Diamidino-2-phenylindole 
			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&#956;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&#177;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

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  ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

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 ...