Name: retinal organoid induction system for derivation of 3d retinal tissues from human pluripotent stem cells
Uploaded: 2021-04-12T14:00:00
Description: Watch this Scientific Journal Video about Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells at JoVE.com

This study was supported by the National Key R&#38;D Program of China (2016YFC1101103, 2017YFA0104101), the Guangzhou Science and Technology Project Fund (201803010078), the Science &#38; Technology Project of Guangdong Province (2017B020230003), the Natural Science Foundation (NSF) of China (81570874, 81970842), Hundred talent program of Sun Yat-sen University (PT1001010), and the Fundamental Research Funds of the State Key Laboratory of Ophthalmology.

1. Culture and expansion of hPSCs
<ol>
	<li>HPSC culture
	<ol>
		<li>Coat two wells of a 6-well plate with extracellular matrix (ECM, hESC-qualified matrix). Prepare 50 mL of an ECM solution containing 8-12 &#181;g/mL of ECM in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM). In 49&#160;mL of DMEM, add 1 mL of the thawed ECM stock solution (50x). Add 1 mL of the ECM solution to each well of a 6-well plate. Incubate it for 1 h in an incubator at 37 &#176;C and 5% CO2.</li>
		<li>Prepare hPS...

<ol>
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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>Brooks, M. 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=Improved+retinal+organoid+differentiation+by+modulating+signaling+pathways+revealed+by+comparative+transcriptome+analyses+with+development+in+vivo.">Improved retinal organoid differentiation by modulating signaling pathways revealed by comparative transcriptome analyses with development in vivo.</a> Stem Cell Reports. 13 (5), 891-905 (2019).</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>Osakada, F., 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=Toward+the+generation+of+rod+and+cone+photoreceptors+from+mouse,+monkey+and+human+embryonic+stem+cells.">Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.</a> Nature Biotechnology. 26 (2), 215-224 (2008).</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>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 Communication. 5, 4047 (2014).</li><li>Liu, C., Oikonomopoulos, A., Sayed, N., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+human+diseases+with+induced+pluripotent+stem+cells:+from+2D+to+3D+and+beyond.">Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond.</a> Development. 145 (5), (2018).</li><li>Lamba, D. A., Gust, J., Reh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+embryonic+stem+cell-derived+photoreceptors+restores+some+visual+function+in+Crx-deficient+mice.">Transplantation of human embryonic stem cell-derived photoreceptors restores some visual function in Crx-deficient mice.</a> Cell Stem Cell. 4 (1), 73-79 (2009).</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>Maeda, A., Mandai, M., Takahashi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+and+Induced+Pluripotent+Stem+Cell+Therapy+for+Retinal+Diseases.">Gene and Induced Pluripotent Stem Cell Therapy for Retinal Diseases.</a> Annual Review Genomics and Human Genetics. 20, 201-216 (2019).</li><li>Kruczek, K., Swaroop, A. <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+organoids+for+disease+modeling+and+development+of+therapies.">Pluripotent stem cell-derived retinal organoids for disease modeling and development of therapies.</a> Stem Cells. 38 (10), 1206-1215 (2020).</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>Eckert, P., Knickmeyer, M. D., Schutz, L., Wittbrodt, J., Heermann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+axis+specification+occur+in+parallel+during+optic+cup+and+optic+fissure+formation,+differentially+modulated+by+BMP+and+Wnt.">Morphogenesis and axis specification occur in parallel during optic cup and optic fissure formation, differentially modulated by BMP and Wnt.</a> Open Biology. 9 (2), 180179 (2019).</li><li>Patel, A., Sowden, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+pathways+in+optic+fissure+closure.">Genes and pathways in optic fissure closure.</a> Seminals in Cell and Development Biology. 91, 55-65 (2019).</li><li>Chan, B. H. C., Moosajee, M., Rainger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closing+the+Ggap:+Mechanisms+of+epithelial+fusion+during+optic+fissure+closure.">Closing the Ggap: Mechanisms of epithelial fusion during optic fissure closure.</a> Frontiers in Cell and Developmental Biology. 8, (2021).</li><li>Li, G., 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+retinal+organoids+with+mature+rods+and+cones+from+urine-derived+human+induced+pluripotent+stem+cells.">Generation of retinal organoids with mature rods and cones from urine-derived human induced pluripotent stem cells.</a> Stem Cells International. 2018, 4968658 (2018).</li><li>Liu, 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=Self-formation+of+RPE+spheroids+facilitates+enrichment+and+expansion+of+hiPSC-derived+RPE+generated+on+retinal+organoid+induction+platform.">Self-formation of RPE spheroids facilitates enrichment and expansion of hiPSC-derived RPE generated on retinal organoid induction platform.</a> Investigative Ophthalmology and Visual Science. 59 (13), 5659-5669 (2018).</li><li>Luo, Z., 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=An+optimized+system+for+effective+derivation+of+three-dimensional+retinal+tissue+via+Wnt+signaling+regulation.">An optimized system for effective derivation of three-dimensional retinal tissue via Wnt signaling regulation.</a> Stem Cells. 36 (11), 1709-1722 (2018).</li><li>Li, G., 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+and+characterization+of+induced+pluripotent+stem+cells+and+retinal+organoids+from+a+leber's+congenital+amaurosis+patient+with+novel+RPE65+mutations.">Generation and characterization of induced pluripotent stem cells and retinal organoids from a leber's congenital amaurosis patient with novel RPE65 mutations.</a> Frontiers in Molecular Neuroscience. 12, 212 (2019).</li><li>Matsa, E., Ahrens, J. H., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cells+as+a+platform+for+personalized+and+precision+cardiovascular+medicine.">Human induced pluripotent stem cells as a platform for personalized and precision cardiovascular medicine.</a> Physiology Reviews. 96 (3), 1093-1126 (2016).</li><li>Wahlin, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+outer+segment-like+structures+in+long-term+3d+retinas+from+human+pluripotent+stem+cells.">Photoreceptor outer segment-like structures in long-term 3d retinas from human pluripotent stem cells.</a> Science Reports. 7 (1), 766 (2017).</li><li>Eiraku, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472 (7341), 51-56 (2011).</li><li>Reichman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+storable+retinal+organoids+and+retinal+pigmented+epithelium+from+adherent+human+iPS+cells+in+xeno-free+and+feeder-free+conditions.">Generation of storable retinal organoids and retinal pigmented epithelium from adherent human iPS cells in xeno-free and feeder-free conditions.</a> Stem Cells. 35 (5), 1176-1188 (2017).</li><li>Lamba, D. A., Karl, M. O., Ware, C. B., Reh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+retinal+progenitor+cells+from+human+embryonic+stem+cells.">Efficient generation of retinal progenitor cells from human embryonic stem cells.</a> Proceedings of the National Academy of Science U. S. A. 103 (34), 12769-12774 (2006).</li><li>Kuwahara, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+ciliary+margin-like+stem+cell+niche+from+self-organizing+human+retinal+tissue.">Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue.</a> Nature Communication. 6, 6286 (2015).</li><li>da Silva, S., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf8+expression+and+degradation+of+retinoic+acid+are+required+for+patterning+a+high-acuity+area+in+the+retina.">Fgf8 expression and degradation of retinoic acid are required for patterning a high-acuity area in the retina.</a> Developmental Cell. 42 (1), 68-81 (2017).</li><li>Mitchell, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+signaling+regulates+differential+expression+of+the+tandemly-duplicated+long+wavelength-sensitive+cone+opsin+genes+in+zebrafish.">Retinoic acid signaling regulates differential expression of the tandemly-duplicated long wavelength-sensitive cone opsin genes in zebrafish.</a> PLoS Genetics. 11 (8), 1005483 (2015).</li><li>Stevens, C. B., Cameron, D. A., Stenkamp, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+photoreceptor-generating+retinal+progenitors+revealed+by+prolonged+retinoic+acid+exposure.">Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure.</a> BMC Developmental Biology. 11 (1), (2011).</li><li>Eldred, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+hormone+signaling+specifies+cone+subtypes+in+human+retinal+organoids.">Thyroid hormone signaling specifies cone subtypes in human retinal organoids.</a> Science. 362 (6411), (2018).</li><li>Yang, F., Ma, H., Ding, X. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+hormone+signaling+in+retinal+development,+survival,+and+disease.">Thyroid hormone signaling in retinal development, survival, and disease.</a> Vitamins and Hormones. 106, 333-349 (2018).</li><li>Brzezinski, J. A., Reh, T. 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In this multi-step retinal induction protocol, hPSCs were guided step by step to gain the retinal fate, and self-organized into retinal organoids containing laminated NR and RPE. During the differentiation, hPSCs recapitulated all major steps of human retinal development in vivo, from EF, OV, and RPE, to retinal lamination, generating all subtypes of retinal cells, including retinal ganglion cells, amacrine cells, bipolar cells, rod, and cone photoreceptors, and muller glial cells in a spatial and temporal order...

Retinal degenerative diseases (RDs), such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are characterized by the dysfunction and death of photoreceptor cells and typically lead to irreversible vision loss without effective ways to cure1. The mechanism underlying these diseases is largely unknown partially due to lack of human disease models2. Over the past decades, significant advances have been accomplished in regenerative medicine through stem cell technology. Many researchers, including ourselves, have shown that human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), can differentiate into all types of retinal cells, even mini-retinal tissues through various differentiation approaches3,4,5,6,7,8,9,10,11, providing huge potential in disease modeling and cell therapy12,13,14.
However, the induction process from hPSCs to retinal cells is highly complicated and&#160;time-consuming with low repeatability, which requires researchers with rich experience and high skills. During the complex and dynamic induction process, a number of factors will impact the yield of retinal tissues15,16,17. Also, different induction methods often vary considerably in timing and robust expression of retinal markers, which might confound the sample collection and data interpretation3. Therefore, a straightforward protocol of retinal differentiation from hPSCs with step-by-step guidance would be in demand.
Here, based on our published studies18,19,20,21, an optimized retinal induction protocol to generate retinal organoids (ROs) with rich cone photoreceptors from hPSCs is described, which does not require the supplement of retinoic acid (RA). This protocol focuses on the description of the multi-step method to generate neural retina and RPE. EB formation is the essential part of the early induction stage. Both size and plating density of EBs are quantitatively optimized, which scientifically enhances the yield of retinal tissues and promotes repeatability. In the second part of the induction, optic vesicles (OVs) self-organize in the adherence culture and ROs form in the suspension culture; the time courses and efficiencies of this part vary considerably in different hPSC lines. The maturation and specification of retinal cells in ROs mainly occur in the middle and late stage of induction. Without the addition of RA, mature photoreceptors with both rich cones and rods can be produced.
The purpose of this protocol is to quantitatively describe and detail each step for inexperienced researchers to repeat. Various hPSC lines have been successfully induced into ROs by this protocol with a robust yield of cone-rich retinal tissues and high repeatability. HPSCs-derived ROs with this protocol can recapitulate the main steps of retinal development in vivo, and survive long-term, which facilitates downstream applications, such as disease modeling, drug screening, and cell therapy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(&#8722;)-Blebbistatin</td>
			<td>Sigma</td>
			<td>B0560-5mg</td>
			<td>ROCK-inhibitor</td>
		</tr>
		<tr>
			<td>1 ml tips</td>
			<td>Kirgen</td>
			<td>KG1313</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>10 ml pipette</td>
			<td>Sorfa</td>
			<td>3141001</td>
			<td>Pipette</td>
		</tr>
		<tr>
			<td>100 mm Tissue culture</td>
			<td>BIOFIL</td>
			<td>TCD000100</td>
			<td>100 mm Petri dish</td>
		</tr>
		<tr>
			<td>100 mm Tissue culture</td>
			<td>Falcon</td>
			<td>353003</td>
			<td>100 mm Petri dish</td>
		</tr>
		<tr>
			<td>15 ml Centrifuge tubes</td>
			<td>BIOFIL</td>
			<td>CFT011150</td>
			<td>Centrifuge tubes</td>
		</tr>
		<tr>
			<td>35 mm Tissue culture dishes</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>35 mm Petri dish</td>
		</tr>
		<tr>
			<td>5 ml pipette</td>
			<td>Sorfa</td>
			<td>313000</td>
			<td>Pipette</td>
		</tr>
		<tr>
			<td>50 ml Centrifuge tubes</td>
			<td>BIOFIL</td>
			<td>CFT011500</td>
			<td>Centrifuge tubes</td>
		</tr>
		<tr>
			<td>6 wells tissue culture plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td>Culture plates</td>
		</tr>
		<tr>
			<td>Anti-AP2&#945; Antibody</td>
			<td>DSHB</td>
			<td>3b5</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>ANTIBIOTIC ANTIMYCOTIC 100X</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td>Antibiotic-Antimycotic</td>
		</tr>
		<tr>
			<td>Anti-ISL1 Antibody</td>
			<td>Boster</td>
			<td>BM4446</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-Ki67 Antibody</td>
			<td>Abcam</td>
			<td>ab15580</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-L/M opsin Antibody</td>
			<td>gift from Dr. jeremy</td>
			<td>/</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-PAX6 Antibody</td>
			<td>DSHB</td>
			<td>pax6</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-rabbit 555</td>
			<td>Invitrogen</td>
			<td>A31572</td>
			<td>Donkey anti-Rabbit IgG (H+L) 
			Secondary Antibody, Alexa Fluor 555</td>
		</tr>
		<tr>
			<td>Anti-Recoverin Antibody</td>
			<td>Millipore</td>
			<td>ab5585</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-Rhodopsin Antibody</td>
			<td>Abcam</td>
			<td>ab5417</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-sheep 555</td>
			<td>Invitrogen</td>
			<td>A21436</td>
			<td>Donkey anti-Sheep IgG (H+L) 
			Secondary Antibody, Alexa Fluor 555</td>
		</tr>
		<tr>
			<td>Anti-SOX9 Antibody</td>
			<td>Abclonal</td>
			<td>A19710</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>Anti-VSX2 Antibody</td>
			<td>Millipore</td>
			<td>ab9016</td>
			<td>Primary&#160;antibody</td>
		</tr>
		<tr>
			<td>B-27 supplement W/O VIT A (50X)</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td>Supplement</td>
		</tr>
		<tr>
			<td>Cryotube vial</td>
			<td>Thermo scientific-NUNC</td>
			<td>375418</td>
			<td>1.8 ml</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>DOJINDO</td>
			<td>D532</td>
			<td>4&#39;,6-Diamidino-2-phenylindole 
			dihydrochloride; multiple suppliers</td>
		</tr>
		<tr>
			<td>Dimethyl sulphoxide(DMSO) Hybri-max</td>
			<td>Sigma</td>
			<td>D2650-100ML</td>
			<td>Multiple suppliers</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>DMEM /F12</td>
			<td>Gibco</td>
			<td>C11330500BT</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15575-020</td>
			<td>0.5 M PH 8.0</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>NATOCOR</td>
			<td>SFBE</td>
			<td>Serum</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td>0.22&#956;m, sterile Millex filter</td>
		</tr>
		<tr>
			<td>GlutaMax, 100X</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>L-alanyl-L-glutamine</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td>2 mg/ml in PBS to use</td>
		</tr>
		<tr>
			<td>Matrigel, 100x</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Extracellular matrix (ECM)</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td>MEM NEAA</td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>STEM CELL</td>
			<td>85850</td>
			<td>hPSCs maintenance medium (MM)</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>Supplement</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) buffer</td>
			<td>GNM</td>
			<td>GNM10010</td>
			<td>Without Ca+,Mg+,PH7.2&#177;0.1 0.1M</td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma</td>
			<td>T0625</td>
			<td>Supplement</td>
		</tr>
		<tr>
			<td>Ultra-low attachment culture dishes 100mm petri dish, low-attachment</td>
			<td>Corning</td>
			<td>CLS3262-20EA</td>
			<td>Petri dish</td>
		</tr>
	</tbody>
</table>

retinal organoid induction system for derivation of 3d retinal tissues from human pluripotent stem cells

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

The retinal induction process in this protocol mimics the development of human fetal retina. To initiate the retinal differentiation, hPSCs were dissociated into small clumps and cultured in suspension to induce the formation of EBs. On D1, the uniformed cell aggregates or EBs formed (Figure 1C). The culture medium was gradually transitioned into NIM. On D5, EBs were plated onto the ECM-coated culture dishes. Cells gradually migrated out of the EBs (Figure 1D). ...

Watch this Scientific Journal Video about Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells at JoVE.com

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

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

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

The induction process from hPSCs to retinal cells is complicated and time-consuming. This optimized protocol can generate retinal tissues with high reproducibility and no cost. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction from hPSCs.With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate disease modeling and cell therapy for retinal degenerative diseases. Demonstrating the procedure, we are with Yuanyuan Guan, a PhD student, and Bingbing Xie, a technician from the laboratory.Begin by preparing 50 mL of ECM solution by adding 1 mL of the third 50 times stock solution to 50 mL of DMEM. Then, add 1 mL of this prepared ECM solution to each well of a six-well plate and incubate it for one hour at 37 degrees Celsius and 5%carbon dioxide. Prepare hPSC maintenance medium according to the manufacturer's instruction and prewarm it to room temperature for 30 minutes.Pour a cryogenic vial of hPSCs from a liquid nitrogen tank by incubating it in a water bath at 37 degrees Celsius for 30 seconds. Carefully disinfect it using a 75%alcohol spray and put it in a bio-safety cabinet. Transfer the cell suspension from the vial to a 15 millimeter tube.Then, add 5 mL of pre-warmed maintenance medium drop-by-drop to the tube using a 5 mL pipette, while gently shaking the tube to blend the hPSCs. Centrifuge the tube at 170 times G for five minutes. Carefully remove most of the supernatant using a 1 mL pipette, leaving behind approximately 50 microliters of supernatant to avoid losing the cells.Re-suspend the pellet with 1 mL of maintenance medium by pipetting up and down. Remove ECM from the pre-coated wells and add 1.5 milliliters of maintenance medium to each well. Then distribute 0.5 milliliters of cell suspension into each well.Gently shake the plate to distribute the hPSCs uniformly. Put the plate in an incubator at 37 degrees Celsius and 5%carbon dioxide for at least 24 hours to promote cell adherence. Change medium every day and passage hPSCs at 80%confluence.On Day-0, initiate the differentiation by removing the 80%confluence cells from one well of the six-well plate. Collect the cells with EDTA dissociation solution as described in the text manuscript. Remove the EDTA solution and add 1 mL of maintenance medium containing 10 micromolar fluvastatin to stop cell dissociation.Collect the cells with a 1 mL pipette. Transfer the cell suspension to a 100 millimeter ultra-low attached Petri dish and add 9 mL of maintenance medium containing 10 micromolar fluvastatin to the dish. Gently shake the dish twice to distribute the cells uniformly.Then, put it in the incubator at 37 degrees Celsius and 5%carbon dioxide. After the cells are cultured for at least 24 hours, observe them under the microscope. Add 9 mL of maintenance medium and 3 mL of NIM to a 15 mL tube.Transfer the cell cultures to a 15 mL centrifuge tube and add 10 mL of the pre-warmed NIM mixture to the dish. Centrifuge the tube at 60 times G for 3 minutes to collect the aggregates. Then remove the supernatant using a 5 mL pipette, leaving behind approximately 500 microliters to avoid losing cells.Add 2 mL of the mixture to the tube and transfer the suspension back to the same Petri dish. Gently shake the dish to uniformly distribute the cell aggregates. Then, put the dish back in the incubator.On Day-5, remove ECM from the pre-coated dishes and add 10 mL pre-warmed NIM to each dish. Take out the dish containing Ebs and check the quality of EBs under the microscope, making sure that they are bright and round. Collect all EBs in a 15 mL tube and allow them to settle for 5 minutes.Then, remove most of the supernatant, leaving behind about 2 mL of medium. After counting the EBs, seed them drop-by-drop at a density of approximately 2-3 EBs per square centimeter into coated dishes containing 10 mL of NIM. Gently shake the dishes to distribute the EBs uniformly and put them in the incubator for at least 24 hours.Use a tungsten needle or a needle with a 1 mL syringe to mechanically detach the morphologically identifiable optic vesicles, along with the adjacent retinal pigment epithelium on days 28 to 35. Culture them in suspension. Put 50-60 optic vesicles into each 100 millimeter low attachment culture dish, containing 15 mL of RDM for retinal organoid formation.Change the medium every 2 to 3 days until Day-42. To initiate the retinal differentiation, hPSCs were dissociated into small clumps and cultured in suspension, which formed EBs since Day-1. On Day-5 EBs were plated onto the ECM-coated culture dishes and the cells gradually migrated out of the EBs.On Day-16, the induction medium was replaced by RDM, causing the neural retina domains to form and gradually protrude from the dish, as well as, cell forming optic vesicle-like structures surrounded by RPE cells. During Days-28 to 35, retinal organoids consisting of the neural retina, attached to an RPE sphere. At one side, cells formed.As retinal differentiation and specification progressed, hPSCs produced subtypes of neural retina that gradually lined up in layers, mimicking the architectural features of a native human retina. Retinal ganglion cells were first generated from retinal progenitors and accumulated in the basal side of the neuro retina. With this protocol, retinal organoids developed into highly mature photoreceptors with both, rich rods and cones.Photoreceptor cells were located in the apical side, while amacrine cells, horizontal cells, bipolar cells and muller glial cells were all located in the intermediate layer of the neural retina. The key points of this protocol are creating high quality of Ebs and seeding them at the right density.

Research

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