This study was conducted in accordance with the Tenets of the Declaration of Helsinki and approved by the Institutional Ethics Committee of the Chinese PLA General Hospital. The WA09 (H9) ESC line was obtained from the WiCell Research Institute.
1. Culture media and reagent preparation
<ol>
	<li>Human ESC culture medium and passage solution
	<ol>
		<li>Maintenance medium (MM): Prepare 500 mL of complete MM (Basal Medium + 5x supplement; see Table of Materials</strong...

Enhanced 3D Neural Retina Differentiation from Human Embryonic Stem Cells

<ol>
	<li>Hoon, M., Okawa, H., Della Santina, ., Wong, L., O, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+the+retina:+development+and+disease.">Functional architecture of the retina: development and disease.</a> Prog Retin Eye Res. 42, 44-84 (2014).</li><li>Steinmetz, J. D., 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=Causes+of+blindness+and+vision+impairment+in+2020+and+trends+over+30+years,+and+prevalence+of+avoidable+blindness+in+relation+to+VISION+2020:+the+Right+to+Sight:+an+analysis+for+the+Global+Burden+of+Disease+Study.">Causes of blindness and vision impairment in 2020 and trends over 30 years, and prevalence of avoidable blindness in relation to VISION 2020: the Right to Sight: an analysis for the Global Burden of Disease Study.</a> Lancet Glob Health. 9 (2), e144-e160 (2021).</li><li>Pascolini, D., Mariotti, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+visual+impairment:+2010.">Global estimates of visual impairment: 2010.</a> Br J Ophthalmol. 96 (5), 614-618 (2012).</li><li>Singh, H. 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=Developmental+stage-specific+proliferation+and+retinoblastoma+genesis+in+RB-deficient+human+but+not+mouse+cone+precursors.">Developmental stage-specific proliferation and retinoblastoma genesis in RB-deficient human but not mouse cone precursors.</a> Proc Natl Acad Sci U S A. 115 (40), e9391-e9400 (2018).</li><li>Slijkerman, R. W., 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=The+pros+and+cons+of+vertebrate+animal+models+for+functional+and+therapeutic+research+on+inherited+retinal+dystrophies.">The pros and cons of vertebrate animal models for functional and therapeutic research on inherited retinal dystrophies.</a> Prog Retin Eye Res. 48, 137-159 (2015).</li><li>Peng, Y. R., 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=Molecular+classification+and+comparative+taxonomics+of+foveal+and+peripheral+cells+in+primate+retina.">Molecular classification and comparative taxonomics of foveal and peripheral cells in primate retina.</a> Cell. 176 (5), 1222-1237 (2019).</li><li>Ribeiro, 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=Restoration+of+visual+function+in+advanced+disease+after+transplantation+of+purified+human+pluripotent+stem+cell-derived+cone+photoreceptors.">Restoration of visual function in advanced disease after transplantation of purified human pluripotent stem cell-derived cone photoreceptors.</a> Cell Rep. 35 (3), 109022 (2021).</li><li>Mehat, M. 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=Transplantation+of+human+embryonic+stem+cell-derived+retinal+pigment+epithelial+cells+in+macular+degeneration.">Transplantation of human embryonic stem cell-derived retinal pigment epithelial cells in macular degeneration.</a> Ophthalmology. 125 (11), 1765-1775 (2018).</li><li>Manafi, N., 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=Organoids+and+organ+chips+in+ophthalmology.">Organoids and organ chips in ophthalmology.</a> Ocul Surf. 19, 1-15 (2021).</li><li>Rossi, G., Manfrin, A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+potential+in+organoid+research.">Progress and potential in organoid research.</a> Nat Rev Genet. 19 (11), 671-687 (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>Kuwahara, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation`of+a+ciliary+margin-like+stem+cell+niche+from+self-organizing+human+retinal+tissue.">Generation`of a ciliary margin-like stem cell niche from self-organizing human retinal tissue.</a> Nat Commun. 6, 6286 (2015).</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> Nat Commun. 5, 4047 (2014).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+development+and+disease+with+organoids.">Modeling development and disease with organoids.</a> Cell. 165 (7), 1586-1597 (2016).</li><li>O'Hara-Wright, M., Gonzalez-Cordero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+organoids:+a+window+into+human+retinal+development.">Retinal organoids: a window into human retinal development.</a> Development. 147 (24), (2020).</li><li>Li, H., 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=Protective+effects+of+resveratrol+on+the+ethanol-induced+disruption+of+retinogenesis+in+pluripotent+stem+cell-derived+organoids.">Protective effects of resveratrol on the ethanol-induced disruption of retinogenesis in pluripotent stem cell-derived organoids.</a> FEBS Open Bio. 13 (5), 845-866 (2023).</li><li>Zou, 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=Organoid-derived+C-Kit(+)/SSEA4(-)+human+retinal+progenitor+cells+promote+a+protective+retinal+microenvironment+during+transplantation+in+rodents.">Organoid-derived C-Kit(+)/SSEA4(-) human retinal progenitor cells promote a protective retinal microenvironment during transplantation in rodents.</a> Nat Commun. 10 (1), 1205 (2019).</li><li>Mandai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cell-derived+Retinal+organoid/cells+for+retinal+regeneration+therapies:+A+review.">Pluripotent stem cell-derived Retinal organoid/cells for retinal regeneration therapies: A review.</a> Regen Ther. 22, 59-67 (2023).</li><li>Suarez-Martinez, E., Suazo-Sanchez, I., Celis-Romero, M., Carnero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+and+organoid+culture+in+research:+physiology,+hereditary+genetic+diseases+and+cancer.">3D and organoid culture in research: physiology, hereditary genetic diseases and cancer.</a> Cell Biosci. 12 (1), 39 (2022).</li><li>Bose, R., Banerjee, S., Dunbar, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+neurological+disorders+in+3D+organoids+using+human-derived+pluripotent+stem+cells.">Modeling neurological disorders in 3D organoids using human-derived pluripotent stem cells.</a> Front Cell Dev Biol. 9, 640212 (2021).</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), 171686 (2019).</li><li>Sanjurjo-Soriano, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RA+delays+initial+photoreceptor+differentiation+and+results+in+a+highly+structured+mature+Retinal+organoid.">RA delays initial photoreceptor differentiation and results in a highly structured mature Retinal organoid.</a> Stem Cell Res Ther. 13 (1), 478 (2022).</li><li>Li, X., Zhang, L., Tang, F., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+organoids:+cultivation,+differentiation,+and+transplantation.">Retinal organoids: cultivation, differentiation, and transplantation.</a> Front Cell Neurosci. 15, 638439 (2021).</li><li>Zerti, D., 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=Developing+a+simple+method+to+enhance+the+generation+of+cone+and+rod+photoreceptors+in+pluripotent+stem+cell-derived+Retinal+organoids.">Developing a simple method to enhance the generation of cone and rod photoreceptors in pluripotent stem cell-derived Retinal organoids.</a> Stem Cells. 38 (1), 45-51 (2020).</li><li>Kim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=transcriptome+profiling,+and+functional+validation+of+cone-rich+human+Retinal+organoids.">transcriptome profiling, and functional validation of cone-rich human Retinal organoids.</a> Proc Natl Acad Sci U S A. 116 (22), 10824-10833 (2019).</li><li>Yamasaki, 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=Addition+of+Chk1+inhibitor+and+BMP4+cooperatively+promotes+retinal+tissue+formation+in+self-organizing+human+pluripotent+stem+cell+differentiation+culture.">Addition of Chk1 inhibitor and BMP4 cooperatively promotes retinal tissue formation in self-organizing human pluripotent stem cell differentiation culture.</a> Regen Ther. 19, 24-34 (2022).</li><li>D&#246;pper, H., 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+protocol+for+3D+Retinal+organoids,+immunostaining+and+signal+quantitation.">Differentiation protocol for 3D Retinal organoids, immunostaining and signal quantitation.</a> Curr Protoc Stem Cell Biol. 55 (1), e120 (2020).</li><li>Norrie, J. 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Human ROs can spatially and temporally recapitulate the development of the fetal retina, and early ROs exhibit a high degree of similarity to the fetal retina at equivalent stages of development15. In terms of tissue morphology and molecular expression, human RO closely mirror the actual growth status of the retinal tissue, providing tremendous and unprecedented opportunities in the fields of disease modeling, drug screening, and regenerative medicine. Currently, several different methods have bee...

The eye serves as the primary source of information among human sensory organs, with the retina being the principal visual sensory tissue in mammalian eyes1. Retinopathy stands as one of the primary global causes of eye diseases, leading to blindness2. Approximately 2.85 million people worldwide suffer from varying degrees of vision impairment due to retinopathy3. Consequently, investigating its pathogenesis is crucial for early diagnosis and timely treatment. Most studies on human retinopathy have primarily focused on animal models4,5,6. However, the human retina is a complex, multi-layered tissue comprising various cell types. Traditional two-dimensional (2D) cell culture and animal model systems typically fail to faithfully recapitulate the normal spatiotemporal development and drug metabolism of the native human retina7,8.
Recently, 3D culture techniques have evolved to generate tissue-like organs from pluripotent stem cells (PSCs)9,10. Retinal organoids (ROs) generated from human PSCs in a 3D suspension culture system not only contain seven retinal cell types but also exhibit a distinct stratified structure similar to the human retina in vivo11,12,13. Human PSC-derived ROs have gained popularity and widespread availability and are currently the best in vitro models for studying the development and disease of the human retina14,15. Over the past few decades, numerous researchers have demonstrated that human PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can differentiate into ROs using various induction protocols. These advancements hold enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies16,17,18.
However, the generation of neural retina (NR) from human pluripotent stem cells (PSCs) is a complex, cumbersome, and time-consuming process. Furthermore, batch-to-batch variations in tissue organoids may lead to lower reproducibility of results19,20. Numerous intrinsic and extrinsic factors can influence the yield of retinal organoids (ROs), such as the number or species of starting cells and the use of transcription factors and small-molecule compounds21,22,23. Since the first human RO was generated by the Sasai laboratory11, multiple modifications have been proposed over the years to enhance the ease and effectiveness of the induction process13,21,24,25. Unfortunately, to date, no gold standard protocol has been established for generating ROs in all laboratories. Indeed, there is a certain degree of discrepancy in ROs resulting from different induction methods, as well as wide variation in the expression of retinal markers and the robustness of their structure22,26. These issues may severely complicate sample collection and the interpretation of study findings. Therefore, a more consolidated and robust differentiation protocol is needed to maximize efficiency with minimal heterogeneity of RO generation.
This study describes an optimized induction protocol based on a combination of Kuwahara et al.12 and D&#246;pper et al.27 with detailed instructions. The new method significantly reduces the probability of organoid vesiculation and fusion, increasing the success rate of generating NR. This development holds great promise for disease modeling, drug screening, and cell therapy applications for retinal disorders.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.01 M TPBS</td>
			<td>Servicebio</td>
			<td>G0002</td>
			<td>Washing slices</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Servicebio</td>
			<td>G1101-500ML</td>
			<td>Fix retinal organoids</td>
		</tr>
		<tr>
			<td>5 mL Pasteur pipette</td>
			<td>NEST Biotechnology</td>
			<td>318516</td>
			<td>Pipette retinal organoids</td>
		</tr>
		<tr>
			<td>96 V-bottomed conical wells</td>
			<td>Sumitomo Bakelit</td>
			<td>MS-9096VZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion Microscope Slides</td>
			<td>CITOTEST</td>
			<td>188105</td>
			<td>Fix slices</td>
		</tr>
		<tr>
			<td>AggreWell medium</td>
			<td>STEMCELL Technologies</td>
			<td>5893</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>SINOPHARM</td>
			<td>10009218</td>
			<td>Dehydrate&#160;</td>
		</tr>
		<tr>
			<td>Anti-CHX10</td>
			<td>Santa Cruz</td>
			<td>sc-365519</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Antifade Solution</td>
			<td>ZSGB-BIO</td>
			<td>ZLI-9556</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-KI67</td>
			<td>Abcam</td>
			<td>ab16667</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Anti-NESTIN</td>
			<td>Sigma</td>
			<td>N5413</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Anti-Neuronal Class III &#946;-Tubulin(TUJ1)</td>
			<td>Beyotime</td>
			<td>AT809</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Anti-PAX6</td>
			<td>Abcam</td>
			<td>ab195045</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Cell dissociation solution(CDS)</td>
			<td>STEMCELL Technologies</td>
			<td>7922</td>
			<td>Cell dissociation</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Selleckchem</td>
			<td>S2924</td>
			<td>GSK-3&#945;/&#946; inhibitor</td>
		</tr>
		<tr>
			<td>Cholesterol Lipid Concentrate</td>
			<td>Gibco</td>
			<td>12531018</td>
			<td>250&#215;</td>
		</tr>
		<tr>
			<td>Citrate Antigen Retrieval Solution</td>
			<td>Servicebio</td>
			<td>G1202-250ML</td>
			<td>20&#215;, pH 6.0</td>
		</tr>
		<tr>
			<td>CS10</td>
			<td>STEMCELL Technologies</td>
			<td>1001061</td>
			<td>Cell Freezing Medium</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td>Nuclear counterstain</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11330032</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>DMEM/F12-GlutaMAX</td>
			<td>Gibco</td>
			<td>10565018</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse Alexa Fluor Plus 488</td>
			<td>Invitrogen</td>
			<td>A32766</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A10042</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>Ethylene Diamine Tetraacetic Acid (EDTA)</td>
			<td>Biosharp</td>
			<td>BL518A</td>
			<td>0.5 M, pH 8.0, cell dissociation</td>
		</tr>
		<tr>
			<td>Extracellular matrix (ECM)</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Coating plates</td>
		</tr>
		<tr>
			<td>F12-Glutamax</td>
			<td>Gibco</td>
			<td>31765035</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>A5669701</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow-like tissue cell quantitative analyzer</td>
			<td>TissueGnostics</td>
			<td>TissueFAXS Plus</td>
			<td>Scan sections</td>
		</tr>
		<tr>
			<td>IMDM-GlutaMAX</td>
			<td>Gibco</td>
			<td>31980030</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>IWR1-endo</td>
			<td>Selleckchem</td>
			<td>S7086</td>
			<td>Wnt-inhibitor</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN-193189 2HCl</td>
			<td>Selleckchem</td>
			<td>S7507</td>
			<td>BMP-inhibitor</td>
		</tr>
		<tr>
			<td>Low-adhesion 24-well Plates</td>
			<td>Corning</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-adhesion 6-well Plates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Maintenance medium (MM)</td>
			<td>STEMCELL Technologies</td>
			<td>85850</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Solarbio</td>
			<td>SL050</td>
			<td>Blocking buffer</td>
		</tr>
		<tr>
			<td>Paraplast</td>
			<td>Leica</td>
			<td>39601006</td>
			<td>Tissue embedding</td>
		</tr>
		<tr>
			<td>PBS pH 7.4 basic (1x)</td>
			<td>Gibco</td>
			<td>C10010500BT</td>
			<td>Without Ca+,Mg+</td>
		</tr>
		<tr>
			<td>Reconbinant human bone morphogenetic protein-4(rhBMP4)</td>
			<td>R&#38;D</td>
			<td>314-BP</td>
			<td>Key protein factor</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Powder, keep out of light</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Selleckchem</td>
			<td>S1067</td>
			<td>ALK5-inhibitor</td>
		</tr>
		<tr>
			<td>SU5402</td>
			<td>Selleckchem</td>
			<td>S7667</td>
			<td>Tyrosine kinase inhibitor</td>
		</tr>
		<tr>
			<td>Super PAP Pen</td>
			<td>ZSGB-BIO</td>
			<td>ZLI-9305</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T0625-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioglycerol</td>
			<td>Sigma</td>
			<td>M1753</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>Permeabilization</td>
		</tr>
		<tr>
			<td>WA09 embryonic stem cell line</td>
			<td>WiCell Research Institute</td>
			<td></td>
			<td>Cell line</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>SINOPHARM</td>
			<td>10023418</td>
			<td>Dewaxing</td>
		</tr>
		<tr>
			<td>Y-27632 2HCL</td>
			<td>Selleckchem</td>
			<td>S1049</td>
			<td>ROCK-inhibitor</td>
		</tr>
	</tbody>
</table>

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

Generating Neural Retina from Human Pluripotent Stem Cells

Our group focuses on understanding the pathogenesis and the treatment of human retinal diseases. Our protocol describes a optimized induction technique to generate a neural retina that significantly reduce the probability of vasculation and fusion to increase the success rate of retinal production until day 60. The modified method is simpler and more efficient in generating neural retina, which can prove to be a better disease model for drug screening and cell therapy.In the future, we aim to study the effects of physicochemical factors on retinal development and underlying mechanism using our retinal organoid model.

A graphical overview of the modified protocol is shown in Figure 1. H9-ESCs were used to generate ROs when the cells were grown to a density of 70%-80%. Single-cell suspensions of H9-ESCs in 96 V-bottomed conical wells aggregated on day 1 and formed well-circumscribed round EBs by day 6. As the culture time increased, the volume of EBs gradually increased. On day 30, neuroepithelial-like structures were clearly formed and thickened during long-term NR differentiation.
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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 ...

generating-neural-retina-from-human-pluripotent-stem-cells

Research

JoVE Journal

Developmental Biology

Culturing H9-Embryonic Stem Cells for Generation of Human Neural Retina

To begin, add one milliliter of 1%extracellular matrix to each well of a six well plate. Incubate the plate at 37 degrees Celsius in a 5%carbon dioxide atmosphere. After one hour, add two milliliters of prewarm maintenance medium containing 0.4 micromolar Y-27632 to each well.To thaw the H9 embryonic stem cells, shake the cryovial stock in a 37 degree Celsius water bath for 30 seconds. Carefully sterilize the cryovial with a 75%disinfectant alcohol spray. Then transfer the thawed cells to a 15 milliliter tube containing the maintenance medium and Y-27632.Centrifuge the tube at 190 G for five minutes at room temperature. Then carefully remove most of the supernatant and resuspend the cells in one milliliter of maintenance medium containing Y-27632. Dispense 0.5 milliliters of the cell suspension to each well of the extracellular matrix coated plate containing maintenance medium.Shake the plate laterally and incubate the plate at 37 degrees Celsius under 5%carbon dioxide for at least 24 hours. Change the medium every day. Once the clone density reaches 70%remove the spent medium.After 2D PBS washes, incubate the cells with one milliliter of EDTA for four to seven minutes at room temperature and discard EDTA completely. Add one milliliter of maintenance medium to the cells and tap the plate gently. Then transfer the dislodged cells to a 15 milliliter tube and mix them three to five times.Add 20 to 100 microliters of cell suspension to each well of a previously prepared ECM-coated dish and mix well. Using a microscope, confirm the cell density and incubate the cells at 37 degrees Celsius under 5%carbon dioxide.

Inducing 3D Neural Retina Formation from Cultured H9-Embryonic Stem Cells for Stem-Cell Based Therapies

To begin, thaw and culture H9-embryonic stem cells in maintenance medium containing Y-27632. On day zero, once the colonies become 70%confluent, wash them with 2 milliliters of DPBS, then incubate the cells with 0.5 milliliters of cell dissociation solution containing 20 micromoles per liter Y-27632. To abort the digestion, add 3 milliliters of maintenance medium containing 20 micromoles per liter Y-27632.To pellet the cells, centrifuge the tube at 190 G for five minutes and discard the supernatant. Resuspend the pellet in one milliliter of growth factor-free chemically-defined medium with Y-27632. After counting the cells, add 1.2 million cells to 10 milliliters of growth factor-free chemically-defined medium and mix well.Then dispense 100 microliters of cell suspension to each well of a 96 well conical bottom plate. Incubate in a humidified 5%carbon dioxide atmosphere. To induce retina formation on day six, add medium containing BMP4 to each well of a 96 well V-bottom plate and incubate again.On day nine, 12, and 15, replace half of the spent medium with fresh medium without BMP4. On day 18, carefully transfer the formed embryoid bodies to a 15 milliliter centrifuge tube. After the natural precipitation of the embryoid bodies, gently remove the supernatant and rinse them with the neural retina differentiation medium.Then place the embryoid bodies in a low absorption six well plate and incubate the plate again. On day 21, Brightfield images showed continuous neuroepithelial structure in the neural retina generated using this method. The retinal induction success rate was significantly higher and retinal organoids generated using this protocol were similar in size and morphology compared to other methods.

707 Views.  Medical School of Chinese PLA.  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.

Enhanced 3D Neural Retina Differentiation from Human Embryonic Stem Cells

Summary