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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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. 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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 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...

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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 ...

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Research

JoVE Journal

Developmental Biology

3.1K Views.  LV Prasad Eye Institute. 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.

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 and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

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