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.

All experiments involving hiPSCs were carried out aseptically, in adherence to the standard laboratory practices, ethical and biosafety guidelines, and with the approvals of regulatory bodies such as the Institutional Ethics Committee (IEC), Institutional Committee for Stem Cell Research (IC-SCR), and Institutional Bio-Safety Committee (IBSC).
1. Preparation of iPSC culture and retinal differentiation medium and reagents
<ol>
	<li>iPSC culture and maintenance medium
	<ol>
		...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinitis+pigmentosa.">Retinitis pigmentosa.</a> Lancet. 368 (9549), 1795-1809 (2006).</li><li>Sen, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+retinitis+pigmentosa+in+South+Indian+population+aged+above+40+years.">Prevalence of retinitis pigmentosa in South Indian population aged above 40 years.</a> Ophthalmic Epidemiology. 15 (4), 279-281 (2008).</li><li>Nazimul, H., Rohit, K., Anjli, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trend+of+retinal+diseases+in+developing+countries.">Trend of retinal diseases in developing countries.</a> Expert Review of Ophthalmology. 3 (1), 43-50 (2008).</li><li>. 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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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 rec...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m Syringe filters</td>
			<td>TPP</td>
			<td>99722&#160;</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&#160;&#160;&#160;&#160;&#160;</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&#8217;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&#160;</td>
			<td>Corning</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal&#160;Bovine&#160;Serum, qualified, United States&#160;</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&#160;</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&#160;</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&#38;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&#160;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&#160;</td>
			<td>Vector laboratories</td>
			<td>H-1200&#160;</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.

Differentiation of hiPSCs into eye lineages is achieved by culturing the cells in different cocktails of culture medium containing supplements and growth factors in sequential steps at different time points, as described in Figure 1. The hiPSC cultures are maintained in Essential 8 medium, the pluripotent stem cell maintenance medium. Once they reach 70%-80% confluency (Figure 2A), the medium is replaced with Differentiation Induction Medium (DIM) on day 0 (refe...

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 ...

This protocol describes a simple and efficient method of differentiating human IPCs into complex three-dimensional neuro retinal organoids for research and regenerative applications. This technique involves both adherent and suspension cultures, which allows selective picking and enrichment of both retinal organoids and RP cells. It can offer a viable and regular supply of cell sources for developing cell-based therapies for retinal degenerative diseases.Such stem cell-derived retinal organoids and RPE cells are useful as in vitro models to study eye development and inherited retinal diseases. This method can be easily replicated by research labs that are already familiar with handling human IPSC cultures. Visual demonstration greatly supports the identification of eye fields and the isolation of neuro retinal cups based on their spatial positioning and morphological features.To begin, aspirate the spent medium from 70 to 80%confluent human IPSC cultures in six-well plates. Add PBS to the wells, swirl them and aspirate out the wash buffer. Then add one milliliter of cell dissociation solution or CDS to each well and incubate the plate at 37 degrees Celsius for five to seven minutes until the cells round up.Aspirate the CDS and collect the cell suspension into a 15 milliliter centrifuge tube. Spin the tube at 1, 000 RPM for four minutes at room temperature. Discard the supernatant and resuspend the cell pellet in 1.2 milliliters of Essential 8 medium.Dispense 200 microliters of the cell suspension into each well of a matrix-coated six-well plate containing 1.5 milliliters of IPSC culture medium and 10 micromolar Y27632. After 12 to 24 hours, remove the spent medium and add pre-warmed complete ascension medium without Y27632 to maintain the cultures. Change the culture medium every 24 hours until the cultures reach 70 to 80%confluence.On day zero, aspirate out the human IPSC maintenance medium and add differentiation induction medium containing one nanogram per milliliter BFGF and one nanogram per milliliter Noggin to the six-well plate. Incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator. On day one, aspirate out the spent medium and add differentiation induction medium containing one nanogram per milliliter BFGF and 10 nanograms per milliliter Noggin.Incubate the cells again at 37 degrees Celsius in a 5%carbon dioxide incubator. On day two and three, remove the spent medium and add differentiation induction medium containing 10 nanograms per milliliter Noggin. After adding two milliliters per well medium to a six-well plate, incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator and change the medium every 24 hours.On day four, remove the spent medium and add the retinal differentiation medium or RDM. Follow the same procedure to add two milliliters per well medium to a six-well plate and to incubate the cultures. Change the medium every day.At around day 14 and 18, observe the cultures under a microscope at 10X magnification for the emergence of neural rosette-like domains consisting of early eye field progenitors. At around day 21 and 28, observe the cultures under a microscope at 4X and 10X magnification to observe the emergence of self-organized distinct EFPs with a central island of circular 3D neuro retinal structures surrounded by contiguous outgrowths of neuro epithelium and ocular surface epithelium. Take a sterile Pasteur pipette and hold the base in one hand and the capillary tip in the other.Flame sterilize and heat the region near the middle of the capillary tip with rotational movements until the glass becomes pliable. Then move away from the flame and swiftly pull the capillary tip outward to create a fine capillary tip with a closed lumen. Hold the fine tip horizontally in front of the flame and quickly pass it through the flame in an outward motion to create a smooth hook or an L-shaped capillary tip.Under a stereo microscope, manually pick the well-formed neuro retinal cups from the individual EFPs using the smooth outer curvature zone of the capillary hook as a fine scoop. At days 25 and 30, add four milliliters of pre-warmed retinal differentiation medium in a low attachment 60 millimeter dish to culture and maintain retinal cups for generating 3D retinal organoids. This should be done prior to harvesting the neuro retinal cups.Set a one millimeter micropipette to aspirate 100 microliters and use one milliliter micro tips with wide bore openings to aspirate and transfer the floating retinal cups into the fresh low adherent culture dishes prepared earlier. Maintain the retinal cups in RDM as non-adherent suspension cultures and incubate them at 37 degrees Celsius in a 5%carbon dioxide incubator. On days 30 and 45, following a partial feeding method, remove half the volume of the spent medium and replace it with an equal volume of fresh medium.On day 45 and 60, culture the retinal organoids for a further two weeks in RDM containing 100 micromolar taurine to support better survival and lineage differentiation of neuro retinal progenitors and the development of mature retinal cell types. Retinal organoids are characterized at different stages of maturation for the expression of several retinal progenitor markers using RT-PCR and immunohistochemistry. RT-PCR results confirmed the induction and expression of neuro retinal markers in one-month-old retinal organoids and two-month-old retinal organoids.The confocal images of immuno-labeled sections of immature retinal organoids using antibodies against the neuro retinal progenitor markers Chx10, PAX6, and Otx2, and mature retinal organoids using antibodies against the photoreceptor precursor markers recoverin and CRX are shown here. The marked outermost layer of the retinal organoids with differentiating photoreceptor cells are zoomed and shown in these images. The rudimentary inner segment-like extensions are marked by white arrows.EFP clusters with the neuro retinal cup at the center and migrating RPE outgrowths show pigmentation along the leading edges. Well-differentiated pigmented epithelial outgrowths from multiple EFPs all around the neuro retinal island are shown here. Higher magnification of an EFP shows the migratory zone of RPE progenitors and ocular surface epithelium surrounding a neuro retinal cup.Extended adherent cultures that developed monolayers of immature RPE cells containing both pigmented and non-pigmented cells are shown here. Monolayer cultures of fully mature and pigmented RPE cells show cobblestone morphology at day 60. RPE cells expressing PAX6, MITF, and RPE65 are shown in this figure.These images represent EFPs with abnormal aggregates of retinal progenitors with distorted lamination and lack of striations. EFP with the miniature neuro retinal cup, but lacking the surrounding zone of RPE and ocular surface epithelium are shown in this figure. The representative image shows irregular neuro retinal aggregates formed in the suspension culture.It is very critical to start the differentiation process using near confluent growing cultures of IPCs to achieve efficient induction of eye fields within three to four weeks. Retinal organoids and RPE cells generated from normal and patient-specific IPCs can be used as retinal disease models and for testing novel therapeutic drugs.

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JoVE Journal

Developmental Biology

2.9K vistas.  LV Prasad Eye Institute. Este protocolo describe un método simple y eficiente para diferenciar las IPC humanas en organoides neuroretinianos tridimensionales complejos para investigación y aplicaciones regenerativas. Esta técnica involucra cultivos adherentes y en suspensión, lo que permite la recolección selectiva y el enriquecimiento tanto de organoides retinianos como de células RP. Puede ofrecer un suministro viable y regular de fuentes celulares para desarrollar terapias basadas en células para enfermedad...