The authors acknowledge the scientific and technical support from Dr. Chitra Kannabiran, Geneticist; Dr. Subhadra Jalali, Retinal Consultant; Dr. Milind Naik, Oculoplastic Surgeon; and Dr. Swathi Kaliki, Ocular Oncologist at the LV Prasad Eye Institute, Hyderabad toward the generation of normal and patient-specific iPSC lines. The authors acknowledge the R&#38;D grants from the Science and Engineering Research Board, Department of Science and Technology (IM), (SB/SO/HS/177/2013), Department of Biotechnology (IM), (BT/PR32404/MED/30/2136/2019) ,and Senior Research Fellowships from ICMR (S.M., D.P.), UGC (T.A.), and CSIR (V.K.P.), Government of India.

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RetNet - Retinal Information Network Available from: <a target="_blank" href="https://sph.uth.edu/retnet/">https://sph.uth.edu/retnet/</a> (2022)</li><li>Cicero, S. 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=Cells+previously+identified+as+retinal+stem+cells+are+pigmented+ciliary+epithelial+cells.">Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells.</a> Proceedings of the National Academy of Sciences. 106 (16), 6685-6690 (2009).</li><li>Guo, Y., 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=Modeling+retinitis+pigmentosa:+retinal+organoids+generated+from+the+iPSCs+of+a+patient+with+the+USH2A+mutation+show+early+developmental+abnormalities.">Modeling retinitis pigmentosa: retinal organoids generated from the iPSCs of a patient with the USH2A mutation show early developmental abnormalities.</a> Frontiers in Cellular Neuroscience. 13, 361 (2019).</li><li>Lane, 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=Modeling+and+rescue+of+RP2+Retinitis+pigmentosa+using+iPSC-derived+retinal+organoids.">Modeling and rescue of RP2 Retinitis pigmentosa using iPSC-derived retinal organoids.</a> Stem Cell Reports. 15 (1), 67-79 (2020).</li><li>Li, Y. P., Deng, W. L., Jin, Z. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+retinitis+pigmentosa+through+patient-derived+retinal+organoids.">Modeling retinitis pigmentosa through patient-derived retinal organoids.</a> STAR Protocols. 2 (2), 100438 (2021).</li><li>Gonzalez-Cordero, 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=Recapitulation+of+human+retinal+development+from+human+pluripotent+stem+cells+generates+transplantable+populations+of+cone+photoreceptors.">Recapitulation of human retinal development from human pluripotent stem cells generates transplantable populations of cone photoreceptors.</a> Stem Cell Reports. 9 (3), 820-837 (2017).</li><li>Meyer, J. 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=Optic+vesicle-like+structures+derived+from+human+pluripotent+stem+cells+facilitate+a+customized+approach+to+retinal+disease+treatment.">Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment.</a> Stem Cells. 29 (8), 1206-1218 (2011).</li><li>Zhu, J., Lamba, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule-based+retinal+differentiation+of+human+embryonic+stem+cells+and+induced+pluripotent+stem+cells.">Small molecule-based retinal differentiation of human embryonic stem cells and induced pluripotent stem cells.</a> Bio-Protocol. 8 (12), 2882 (2018).</li><li>Nakano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-formation+of+optic+cups+and+storable+stratified+neural+retina+from+human+ESCs.">Self-formation of optic cups and storable stratified neural retina from human ESCs.</a> Cell Stem Cell. 10 (6), 771-785 (2012).</li><li>Reichman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+confluent+human+iPS+cells+to+self-forming+neural+retina+and+retinal+pigmented+epithelium.">From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium.</a> Proceedings of the National Academy of Sciences. 111 (23), 8518-8523 (2014).</li><li>Zhong, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+three-dimensional+retinal+tissue+with+functional+photoreceptors+from+human+iPSCs.">Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.</a> Nature Communications. 5, 4047 (2014).</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> Scientific Reports. 7 (1), 766 (2017).</li><li>Capowski, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+and+staging+of+3D+human+retinal+organoids+across+multiple+pluripotent+stem+cell+lines.">Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.</a> Development. 146 (1), (2019).</li><li>Chichagova, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+retinal+organoids+from+human+pluripotent+stem+cells.">Differentiation of retinal organoids from human pluripotent stem cells.</a> Current Protocols in Stem Cell Biology. 50 (1), 95 (2019).</li><li>Kelley, R. A., Chen, H. Y., Swaroop, A., Li, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+development+of+rod+photoreceptors+in+retinal+organoids+derived+from+human+pluripotent+stem+cells+by+supplementation+with+9-cis+retinal.">Accelerated development of rod photoreceptors in retinal organoids derived from human pluripotent stem cells by supplementation with 9-cis retinal.</a> STAR Protocols. 1 (1), 100033 (2020).</li><li>Zhou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+embryonic+stem+cells+into+cone+photoreceptors+through+simultaneous+inhibition+of+BMP,+TGFbeta+and+Wnt+signaling.">Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGFbeta and Wnt signaling.</a> Development. 142 (19), 3294-3306 (2015).</li><li>Chambers, S. 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=Highly+efficient+neural+conversion+of+human+ES+and+iPS+cells+by+dual+inhibition+of+SMAD+signaling.">Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.</a> Nature Biotechnology. 27 (3), 275-280 (2009).</li><li>Mellough, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-1+signaling+plays+an+important+role+in+the+formation+of+three-dimensional+laminated+neural+retina+and+other+ocular+structures+from+human+embryonic+stem+cells.">IGF-1 signaling plays an important role in the formation of three-dimensional laminated neural retina and other ocular structures from human embryonic stem cells.</a> Stem Cells. 33 (8), 2416-2430 (2015).</li><li>Susaimanickam, P. 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All authors have no conflict of interest or financial disclosures.

hiPSCs are a powerful tool to study organ and tissue development in vitro. Recapitulating the disease phenotype by differentiating healthy versus disease-specific hiPSCs toward the retinal lineage can help in gaining newer insights into the pathophysiology of different forms of inherited retinal dystrophies. Several protocols have been described and adopted for the in vitro differentiation of PSCs into retinal cell types. Most of them involve the use of culture medium containing complex cocktails of recombinant growth factors, supplements, small molecules, and reagents, such as: N1, N2, and B27 supplements; BMP and TGF&#946; signaling blockers like Noggin, SB431542, LDN193189, and Follistatin, or inducers such as Activin A, Lefty, and IDE1; canonical Wnt signaling blockers such as DKK1, SFRP, IWP-2, and IWR-1-endo, or inducers such as CHIR99021, SB216763, and CKI-7; FGF receptor signaling blockers such as PD0325901 and PD173074 or inducers like bFGF; notch signaling inhibitors such as DAPT; other signaling molecules such as insulin-like growth factor (IGF-1); retinoic acid; growth hormones such as Triiodothyronine (T3) and Hydrocortisone; and anti-oxidants and other pro-survival factors such as Ascorbic acid, Nicotinamide, Taurine, Docosahexaenoic acid, 1-Thioglycerol, etc. These components are included in the culture medium at different stages of stem cell differentiation to stimulate or modulate various signaling cascades to induce eye and retinal lineage commitment11,12,13,14,15,16,17,18,19,20,21,22.
Here, a simpler, robust, and efficient method of generating retinal organoids directly from near-confluent, adherent cultures of hiPSCs is described. The simplified protocol involves using fewer supplements, growth factors, and small molecules that primarily trigger the initial differentiation of PSCs into the neuroectodermal lineage. Subsequent differentiation steps rely on the inherent ability of PSCs to synchronously differentiate into the cell type of related lineages, which then self-organize and mutually regulate the development and spatial organization of multiple cell types that contribute to the formation of complex tissues. The gradual withdrawal of bFGF and the addition of Noggin help in the successful induction of early neuro-ectodermal fate commitment within 3 days of differentiation. Continued maintenance of differentiation cultures in neural-inducing RDM, without adding any growth factors or small molecules, result in the induction of eye field primordial (EFP) structures with clear margins, within 3-4 weeks of differentiation. The EFPs contain multipotent progenitor cells, which upon undisturbed and continued maintenance, result in multi-lineage differentiation and self-assembly to form complex EFPs consisting of centrally positioned neuro-retinal cups or optic cups (OCs), surrounded by other related ocular cell types such as the retinal pigmented epithelium (RPE), neural crest epithelium, and ocular surface epithelium. Alternately, the PSCs can be grown as suspension cultures from day 1-3 to form EBs under identical culture conditions. The EBs can be further plated on day 4 and grown as adherent cultures on matrix-coated surfaces in RDM to initiate retinal lineage differentiation, as described above. Healthy differentiating cultures routinely give rise to about 20-30 EFPs per well of a 6-well plate. The OCs can be harvested from the EFPs at 3-4 weeks of retinal differentiation and are maintained in suspension cultures for a further 30-60 days, to enable the differentiation of neuro-retinal progenitors and to generate mature retinal organoids. After 4 weeks of suspension culture, about 70%-80% of the optic cups picked from EFPs remain intact, retain their lamination, and develop into mature retinal organoids (OC-2M), with RCVRN+ and CRX+ committed photoreceptor cells and inner segment-like extensions within the outermost layers.
The confluency of growing iPSC cultures is critical at the time of initiation of differentiation and shifting the cultures to DIM. Cultures with smaller colonies and confluence below 60%, and those that are precociously differentiating, result in significantly reduced EFP numbers. When the eye field clusters emerge, the central island of neuro-retinal cups should be harvested within 1 week. This can be done by gently nudging and lifting the intact cups using the angle or the curved region of the flame-pulled glass capillary tip, as described in the protocol section. Care must be taken not to damage the cups. Further delays in picking would result in flattening and a loss of 3D organization due to the proliferation and migration of neuro-retinal progenitor cells, which makes harvesting difficult and results in atypical retinal organoids.
The efficiency of eye field induction and maturation of retinal cups varies between different disease or patient-specific hiPSC lines, depending on the underlying genetic defects. For example, the retinal lineage commitment and EFP forming efficiencies of a&#160;RP disease-specific line was identical to that of the healthy control cells, whereas an RB1 null line failed to form EFPs (data not shown). Some lines carrying mutations linked to Leber Congenital Amaurosis&#160;formed atypical EFPs with defects in their size, self-assembly, lamination, and maturation of retinal progenitors (data not shown). Further molecular validations and gene expression profiling of patient-specific retinal organoids compared to healthy control tissues would be necessary to understand the pathophysiology of a disease condition. Considering the variability in patient genomes and the involvement of multi-gene networks in disease manifestations, it may also be important to study retinal organoids derived from isogenic iPSC lines to establish an absolute genotype-phenotype correlation in disease modeling studies. Such isogenic lines can be created either by targeted gene knockouts in healthy control lines or by correcting the pathogenic mutations in patient-specific iPSCs, using advanced genome editing techniques8,9.
Such intact retinal organoids derived from normal or disease-specific iPSC lines can be used in&#160;novel drug screening and testing. Photoreceptor precursors within the retinal organoids and other ocular cell types, such as the RPE and corneal epithelium within the EFP outgrowths, can be further isolated and enriched for their applications in basic research and regenerative medicine. The protocol described here can be easily adopted to GMP-compliant processes for preparing clinical grade cells meant for preclinical and clinical trial evaluations.

The retina is a light-sensitive tissue present at the back of the vertebrate eye that converts light signals into nerve impulses by a biochemical phenomenon known as the photo-transduction pathway. The initial nerve impulses generated in the photoreceptor cells of the retina get transduced to other retinal interneurons and retinal ganglion cells (RGCs) and reach the visual cortex of the brain, which helps in image perception and visual response.
According to the World Health Organization (WHO), an estimated 1.5 million children are blind, of which 1 million are in Asia. Inherited Retinal Dystrophy (IRD) is a major blinding disease that affects 1 in 4,000 individuals worldwide1,2,3, while the prevalence of blindness associated with age-related macular degeneration (AMD) ranges from 0.6%-1.1% in developing countries4. IRDs are caused by inherited genetic defects in over 300 different genes involved in retinal development and function5. Such genetic changes result in the disruption of normal retinal functions and gradual degeneration of retinal cells, namely the photoreceptor cells and the retinal pigmented epithelium (RPE), thus leading to severe vision loss and blindness. Enormous progress has been made in other blinding conditions involving the cornea, lens, etc. However, retinal dystrophies and optic nerve atrophies do not have any proven therapy to date. Since an adult human retina does not have stem cells6, alternate sources such as embryonic stem cells (ESCs) and patient-derived induced pluripotent stem cells (iPSCs) can provide an unlimited supply of desired cell types and hold a great promise for developing complex tissue organoids required for in vitro disease modeling studies and for developing regenerative therapies7,8,9,10.
Several years of retinal research have led to a better understanding of molecular events that orchestrate early retinal development. Most protocols to generate retinal cells and 3D organoids from PSCs aim to recapitulate these developmental events in vitro, by culturing the cells in a complex cocktail of growth factors and small molecules to modulate the known biological processes in a stepwise manner. The retinal organoids thus generated are comprised of major retinal cells: retinal ganglion cells (RGCs), interneurons, photoreceptors, and retinal pigmented epithelium (RPE)11,12,13,14,15,16,17,18,19. Despite successful attempts at modeling IRDs using retinal organoids, the requirement for the complex cocktail of growth factors and small molecules during differentiation and the relatively low efficiency of retinal organoid generation poses a major challenge with most protocols. They majorly include the formation of embryoid bodies, followed by their stepwise differentiation into retinal lineages using complex culture conditions at different stages of in vitro&#160;development20,21,22.
Here, a simplified and robust method of developing complex 3D neuro-retinal organoids from healthy control and retinal disease-specific hiPSCs is reported. The protocol described here utilizes direct differentiation of near-confluent hiPSC cultures without needing embryoid body formation. Also, the complexity of culture medium is simplified, making it a cost-effective and reproducible technique that can be easily adopted by new researchers. It involves 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 circular neuro-retinal islands within each EFP are manually picked and grown in suspension cultures for 1-2 weeks to prepare multilayered 3D retinal cups or organoids consisting of PAX6+ and CHX10+ proliferating neuro-retinal precursors. Extended culture of retinal organoids in 100 &#181;M Taurine-containing medium for a further 4 weeks resulted in the emergence of RCVRN+ and CRX+ photoreceptor precursors and mature cells with rudimentary inner segment-like extensions.

<table><tbody><tr><td>0.22 &amp;micro;m Syringe filters</td><td>TPP</td><td>99722&amp;nbsp;</td><td></td></tr><tr><td>15 mL centrifuge tube</td><td>TPP</td><td>91015</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>TPP</td><td>91050</td><td></td></tr><tr><td>6 well plates</td><td>TPP</td><td>92006</td><td></td></tr><tr><td>Anti-Chx10 Antibody; Mouse monoclonal</td><td>Santa Cruz</td><td>SC365519</td><td>1:50 dilution</td></tr><tr><td>Anti-CRX antibody; Rabbit monoclonal</td><td>Abcam</td><td>ab140603</td><td>1:300 dilution</td></tr><tr><td>Anti-MiTF antibody, Mouse monoclonal</td><td>Abcam</td><td>ab3201</td><td>1:250 dilution</td></tr><tr><td>Anti-Recoverin Antibody; Rabbit polyclonal&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Millipore</td><td>AB5585</td><td>1:300 dilution</td></tr><tr><td>B-27 Supplement (50x), serum free</td><td>Thermo Fisher</td><td>17504044</td><td></td></tr><tr><td>Basic Fibroblast growth factor (bFGF)</td><td>Sigma Aldrich</td><td>F0291</td><td></td></tr><tr><td>Centrifuge 5810R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Coplin Jar (50 mL)</td><td>Tarson</td><td></td><td></td></tr><tr><td>Corning Matrigel hESC-Qualified Matrix</td><td>Corning</td><td>354277</td><td></td></tr><tr><td>CryoTubes</td><td>Thermo Fisher</td><td>V7884</td><td></td></tr><tr><td>DMEM/F-12, GlutaMAX supplement (basal medium)</td><td>Thermo Fisher</td><td>10565-018</td><td></td></tr><tr><td>DreamTaq DNA polymerase</td><td>Thermo Fisher</td><td>EP0709</td><td></td></tr><tr><td>Dulbeco&amp;rsquo;s Phosphate Buffered Saline</td><td>Thermo Fisher</td><td>14190144</td><td></td></tr><tr><td>Essential 8 medium kit</td><td>Thermo Fisher</td><td>A1517001</td><td></td></tr><tr><td>Ethylene diamine tetraaceticacid disodium salt dihydrate (EDTA)</td><td>Sigma Aldrich</td><td>E5134</td><td></td></tr><tr><td>Falcon Not TC-treated Treated Petri Dish, 60 mm&amp;nbsp;</td><td>Corning</td><td>351007</td><td></td></tr><tr><td>Fetal&amp;nbsp;Bovine&amp;nbsp;Serum, qualified, United States&amp;nbsp;</td><td>Gibco</td><td>26140079</td><td></td></tr><tr><td>GelDocXR+ with Image lab software</td><td>BIO-RAD</td><td></td><td>Agarose Gel documentation system&amp;nbsp;</td></tr><tr><td>GlutaMAX Supplement</td><td>Thermo Fisher</td><td>35050061</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L), Alexa Fluor 488</td><td>Invitrogen</td><td>A11001</td><td>1:300 dilution</td></tr><tr><td>Goat anti-Mouse IgG (H+L), Alexa Fluor 546</td><td>Invitrogen</td><td>A11030</td><td>1:300 dilution</td></tr><tr><td>Goat anti-Rabbit IgG (H+L), Alexa Fluo 546</td><td>Invitrogen</td><td>A11035</td><td>1:300 dilution</td></tr><tr><td>Goat anti-Rabbit- IgG (H+L), Alexa Fluor 488</td><td>Invitrogen</td><td>A11008</td><td>1:300 dilution</td></tr><tr><td>HistoCore MULTICUT</td><td>Leica</td><td></td><td>For sectioning</td></tr><tr><td>KnockOut Serum Replacement</td><td>Thermo Fisher</td><td>10828028</td><td></td></tr><tr><td>L-Acsorbic acid</td><td>Sigma Aldrich</td><td>A92902</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)</td><td>Thermo Fisher</td><td>11140-050</td><td></td></tr><tr><td>N2 supplement (100x)</td><td>Thermo Fisher</td><td>17502048</td><td></td></tr><tr><td>NanoDrop 2000</td><td>Thermo Fisher</td><td></td><td>To quantify RNA</td></tr><tr><td>Paraformaldehyde</td><td>Qualigens</td><td>23995</td><td></td></tr><tr><td>Pasteur Pipets, 9 inch, Non-Sterile, Unplugged</td><td>Corning</td><td>7095D-9</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Thermo Fisher</td><td>15140-122</td><td></td></tr><tr><td>Recombinant Anti-Otx2 antibody , Rabbit monoclonal</td><td>Abcam</td><td>ab183951</td><td>1:300 dilution</td></tr><tr><td>Recombinant Anti-PAX6 antibody; Rabbit Monoclonal</td><td>Abcam</td><td>ab195045</td><td>1:300 dilution</td></tr><tr><td>Recombinant Anti-RPE65 antibody, Rabbit Monoclonal</td><td>Abcam</td><td>ab231782</td><td>1:300 dilution</td></tr><tr><td>Recombinant Human Noggin Protein</td><td>R&amp;amp;D Systems</td><td>6057-NG</td><td></td></tr><tr><td>SeaKem LE Agarose</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td>Serological pipettes 10 mL</td><td>TPP</td><td>94010</td><td></td></tr><tr><td>Serological pipettes 5 mL</td><td>TPP</td><td>94005</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium Citrate Tribasic dihydrate</td><td>Sigma Aldrich</td><td>S4641</td><td></td></tr><tr><td>Starfrost (silane coated) microscopic slides</td><td>Knittel</td><td></td><td></td></tr><tr><td>SuperScript III First-Strand Synthesis System</td><td>Thermo Fisher</td><td>18080051</td><td></td></tr><tr><td>SuperScript III First-Strand Synthesis System for RT-PCR</td><td>Invitrogen</td><td>18080051</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T8787</td><td></td></tr><tr><td>TRIzol&amp;nbsp;Reagent</td><td>Invitrogen</td><td>15596026</td><td></td></tr><tr><td>UltraPure 0.5 M EDTA, pH 8.0</td><td>Thermo Fisher</td><td>15575020</td><td></td></tr><tr><td>VECTASHIELD Antifade Mounting Medium with DAPI&amp;nbsp;</td><td>Vector laboratories</td><td>H-1200&amp;nbsp;</td><td></td></tr><tr><td>Vitronectin</td><td>Thermo Fisher</td><td>A27940</td><td></td></tr><tr><td>Y-27632 dihydrochloride (Rho-kinase inhibitor)</td><td>Sigma Aldrich</td><td>Y0503</td><td></td></tr><tr><td>Zeiss LSM 880</td><td>Zeiss</td><td></td><td>Confocal microscope</td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells at JoVE.com

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

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 ...

generation-retinal-organoids-from-healthy-retinal-disease-specific

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

3.3K Views.  LV Prasad Eye Institute. Dit protocol beschrijft een eenvoudige en efficiënte methode om menselijke IPC's te differentiëren in complexe driedimensionale neuro-retinale organoïden voor onderzoek en regeneratieve toepassingen. Deze techniek omvat zowel adherente als suspensieculturen, waardoor selectief plukken en verrijken van zowel retinale organoïden als RP-cellen mogelijk is. Het kan een levensvatbare en regelmatige toevoer van celbronnen bieden voor het ontwikkelen van celgebaseerde therapieën voor retinale d...

Video: Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced 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 ...

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

The objective of this report is to describe the protocols to derive the retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells using different sizes of embryoid bodies.

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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 ...

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 ...

Medicine

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

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

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

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

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

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