Name: retinal organoid induction system for derivation of 3d retinal tissues from human pluripotent stem cells
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本研究得到了中国国家重点研发计划（2016YFC110103，2017YFA0104101）、广州科技项目基金（201803010078）、广东省科技项目（2017B020230003）、中国自然科学基金（NSF）（81570874） 81970842）、中山大学百人计划（PT1001010）和国家眼科重点实验室基础研究基金。

1. hPSC的文化和扩展
<ol><li>HPSC 文化
	<ol>
<li>将两口 6 井板的油井涂上细胞外基质（ECM、hESC 合格矩阵）。在杜尔贝科的改良鹰介质 （DMEM） 中准备 50 mL 的 ECM 解决方案，其中包含 8-12 μg/mL 的 ECM。在 49 mL 的 DMEM 中，加入 1 mL 解冻的 ECM 库存解决方案 （50 倍）。在 6 井板的每个井中加入 1 mL 的 ECM 解决方案。在 37 °C 和 5% CO2的孵化器中孵育 1 小时。</li>
		<li>根据制造商的说明准?...

<ol>
	<li>Flaxman, S. 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=Global+causes+of+blindness+and+distance+vision+impairment+1990-2020:+a+systematic+review+and+meta-analysis.">Global causes of blindness and distance vision impairment 1990-2020: a systematic review and meta-analysis.</a> The Lancet Global Health. 5 (12), 1221-1234 (2017).</li><li>Sayed, N., Liu, C., 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=Translation+of+human-induced+pluripotent+stem+cells:+From+clinical+trial+in+a+dish+to+precision+medicine.">Translation of human-induced pluripotent stem cells: From clinical trial in a dish to precision medicine.</a> Journal of American College of Cardiology. 67 (18), 2161-2176 (2016).</li><li>Capowski, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+and+staging+of+3D+human+retinal+organoids+across+multiple+pluripotent+stem+cell+lines.">Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.</a> Development. 146 (1), (2019).</li><li>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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+cell+fate+specification+in+vertebrates.">Photoreceptor cell fate specification in vertebrates.</a> Development. 142 (19), 3263-3273 (2015).</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=Generation,+transcriptome+profiling,+and+functional+validation+of+cone-rich+human+retinal+organoids.">Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids.</a> Proceedings of the National Academy of Science U. S. A. 116 (22), 10824-10833 (2019).</li><li>Lowe, A., Harris, R., Bhansali, P., Cvekl, A., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+adhesion-dependent+cell+survival+and+ROCK-regulated+actomyosin-driven+forces+mediate+self-formation+of+a+retinal+organoid.">Intercellular adhesion-dependent cell survival and ROCK-regulated actomyosin-driven forces mediate self-formation of a retinal organoid.</a> Stem Cell Reports. 6 (5), 743-756 (2016).</li><li>Carcamo-Orive, I., 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=Analysis+of+transcriptional+variability+in+a+large+human+ipsc+library+reveals+genetic+and+non-genetic+determinants+of+heterogeneity.">Analysis of transcriptional variability in a large human ipsc library reveals genetic and non-genetic determinants of heterogeneity.</a> Cell Stem Cell. 20 (4), 518-532 (2017).</li><li>DeBoever, 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=Large-scale+profiling+reveals+the+influence+of+genetic+variation+on+gene+expression+in+human+induced+pluripotent+stem+cells.">Large-scale profiling reveals the influence of genetic variation on gene expression in human induced pluripotent stem cells.</a> Cell Stem Cell. 20 (4), 533-546 (2017).</li></ol>

在这个多步骤视网膜诱导协议中，hPSC 被引导一步一步地获得视网膜的命运，并自行组织成包含层压 NR 和 RPE 的视网膜器官。在分化过程中，hPSC回顾了人类视网膜 发育在体内的所有主要步骤，从EF、OV和RPE，到视网膜层压，生成视网膜细胞的所有亚型，包括视网膜结节细胞、单体细胞、双极细胞、棒状和锥形光感受器，以及空间和时间顺序的摩尔胶质细胞。视网膜发育的回顾将有利于下游?...

视网膜退行性疾病 （RD）， 如与年龄相关的黄斑变性 （AMD） 和视网膜炎色素 （RP）， 其特点是光感受细胞功能障碍和死亡， 通常导致不可逆转的视力丧失， 没有有效的方法来治愈1.这些疾病背后的机制在很大程度上是未知的，部分原因是缺乏人类疾病模型2。在过去的几十年里，通过干细胞技术在再生医学方面取得了重大进展。许多研究人员，包括我们自己，已经表明，人类多能干细胞（hPSCs），包括人类胚胎干细胞（hESCs）和人类诱导的多能干细胞（hiPSCs），可以通过各种分化方法3，4，5，6，7，8，9，10，分化成所有类型的视网膜细胞，甚至小视网膜组织，11，在疾病建模和细胞治疗方面提供巨大的潜力12，13，14。
然而，从 hPSC 到视网膜细胞的诱导过程非常复杂且耗时，可重复性低，这需要具有丰富经验和高技能的研究人员。在复杂和动态的诱导过程中，许多因素将影响视网膜组织15，16，17的产量。此外，不同的诱导方法往往在视网膜标记的正时和强健的表达上有很大的不同，这可能会混淆样本收集和数据解释3。因此，需要一个直接的视网膜分化协议，从hPSC与分步指导。
在这里，根据我们发表的研究18，19，20，21，一个优化的视网膜诱导协议，以产生视网膜器官（ROs）与丰富的锥形光感受器从hPSCs描述，这不需要视网膜酸（RA）的补充。此协议侧重于生成神经视网膜和 RPE 的多步骤方法的描述。EB 形成是早期诱导阶段的重要组成部分。BB的大小和电镀密度都经过定量优化，科学地提高了视网膜组织的产量，提高了可重复性。在感应的第二部分，光学囊泡（OVs）在依从文化和ROs形式中自我组织在悬浮文化中：这部分的时间课程和效率在不同的 hPSC 行中差异很大。ROs中视网膜细胞的成熟和规范主要发生在诱导的中后期。如果不添加 RA，可以生产具有丰富圆锥体和棒子的成熟感光器。
此协议的目的是定量描述和详细说明每个步骤，让缺乏经验的研究人员重复。通过此协议，各种 hPSC 线已成功诱导到 ROs 中，具有丰富的锥体视网膜组织的丰产和高可重复性。使用此协议的 HPSC 衍生 ROs 可以回顾 体内视网膜发育的主要步骤，并长期存活，从而促进下游应用，如疾病建模、药物筛选和细胞治疗。

<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

视网膜退行性疾病是无法逆转的失明的主要原因，没有有效的治疗。多能干细胞有可能分化成所有类型的视网膜细胞，甚至小视网膜组织，为这些疾病患者带来巨大的希望，在疾病建模和药物筛查方面也有很多机会。然而，从 hPSC 到视网膜细胞的诱导过程是复杂而耗时的。在这里，我们描述了一个优化的视网膜诱导协议，以产生具有高可重复性和效率的视网膜组织，适合各种人类多能干细胞。此协议执行时不添加视网膜酸，这有利于圆锥体光感受器的浓缩。本协议的优点是量化EB尺寸和电镀密度，显著提高视网膜诱导的效率和可重复性。使用这种方法，所有主要视网膜细胞依次出现并重新概括视网膜发育的主要步骤。它将促进下游应用，如疾病建模和细胞治疗。

本协议中的视网膜诱导过程模拟了人类胎儿视网膜的发展。为了启动视网膜分化，hPSC 被分离成小团块，并在悬浮中培养，以诱导 BB 的形成。在 D1 上，均匀的细胞聚合或 EB 形成 （图 1C）。文化媒介逐渐向NIM过渡。在 D5 上，EB 被镀在 ECM 涂层的文化菜肴上。细胞逐渐从EB中迁移出来（图1D）。从 D10 开始，眼场在粘附 BB 的外围区域自行组织。在 D16 上，?...

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

在这里，我们描述了一个优化的视网膜器官诱导系统，它适用于各种人类多能干细胞系，以产生具有高可重复性和效率的视网膜组织。

视网膜器官诱导系统，用于从人类多能干细胞中提取3D视网膜组织

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

从 hPSC 到视网膜细胞的诱导过程既复杂又耗时。此优化协议可以生成视网膜组织，具有高可重复性和无成本。本协议的优点是量化EB尺寸和电镀密度，显著提高hPSC视网膜诱导的效率和可重复性。使用这种方法，所有主要视网膜细胞依次出现并重新概括视网膜发育的主要步骤。它将促进视网膜退行性疾病的疾病建模和细胞治疗。演示程序，我们与袁元冠，博士生，和谢冰冰，从实验室的技术员。首先准备 50 mL 的 ECM 解决方案，将第三个 50 倍库存溶液的 1 mL 添加到 50 mL 的 DMEM 中。然后，在六井板的每口井中加入 1 mL 的预制 ECM 溶液，并在 37 摄氏度和 5% 的二氧化碳下孵育一小时。根据制造商的指示准备 hPSC 维护介质，并预热至室温 30 分钟。将一小瓶低温的 hPSC 从液氮罐中倒入 37 摄氏度的水浴中，浸泡 30 秒。使用 75% 酒精喷雾剂仔细消毒，并将其放入生物安全柜中。将细胞悬架从小瓶转移到 15 毫米管。然后，使用 5 mL 移液器向管中加入 5 mL 预热维护介质滴管，同时轻轻摇动管子以混合 hPSC。以 170 倍 G 离心管，时间为 5 分钟。使用 1 mL 移液器小心地取出大部分超高分子，留下大约 50 微升的超高纳坦，以避免失去细胞。通过上下管道，用 1 mL 的维护介质重新悬挂颗粒。从预涂层的油井中取出 ECM，并在每口油井中加入 1.5 毫升的维护介质。然后向每口井中分配 0.5 毫升的细胞悬浮。轻轻摇动盘子，均匀分配 hPSC。将板放在37摄氏度和5%二氧化碳的孵化器中至少24小时，以促进细胞的依从性。每天更改介质，通过 hPSC 的交汇度为 80%。第 0 天，从六井板的一口井中取出 80% 的汇合细胞，从而启动分化。收集带有 EDTA 分离解决方案的细胞，如文本手稿中所述。取出EDTA溶液，加入含有10微摩尔氟化物的1mL维护介质，以阻止细胞分离。用 1 mL 移液器收集细胞。将细胞悬浮转移到 100 毫米超低附加的 Petri 盘中，并在盘中加入含有 10 微摩尔氟伐他汀的 9 毫升维护介质。轻轻摇动两次盘，均匀地分配细胞。然后，在37摄氏度和5%的二氧化碳的孵化器中放入。细胞培养至少24小时后，在显微镜下观察它们。在 15 mL 管中加入 9 mL 的维护介质和 3 mL 的 NIM。将细胞培养物转移到 15 mL 离心机管中，并将 10 mL 预加热的 NIM 混合物添加到盘中。以 60 倍 G 离心管 3 分钟收集聚合物。然后使用 5 mL 移液器取出超高分子，留下大约 500 微升，以避免丢失细胞。将混合物的 2 mL 加入管中，并将悬架移回同一个培养皿。轻轻摇动盘，均匀地分配细胞聚集物。然后，将盘子放回孵化器中。第 5 天，从预涂层的菜肴中取出 ECM，并在每道菜中加入 10 mL 预热 NIM。拿出含有 Ebs 的菜肴，在显微镜下检查 EBS 的质量，确保它们明亮而圆润。在 15 mL 管中收集所有 BB，并允许它们沉淀 5 分钟。然后，去除大部分超高纳特，留下约2兆L的介质。在计算了 BB 后，以每平方厘米约 2-3 EB 的密度逐滴播种到含有 10 mL NIM 的涂层盘子中。轻轻摇动盘子，均匀地分发EBs，并把它们放在孵化器中至少24小时。使用钨针或带 1 mL 注射器的针头，在 28 至 35 日机械地分离形态上可识别的光学囊泡，以及相邻的视网膜色素上皮。文化他们在暂停。将50-60个光学囊泡放入每个100毫米的低附件培养皿中，含有15毫升的RDM用于视网膜器官形成。每 2 到 3 天更改一次介质，直到第 42 天。为了启动视网膜分化，hPSC 被分离成小团块，并在悬浮中培养，从第 1 天起就形成了 EBS。第5天，BB被镀在ECM涂层的培养皿上，细胞逐渐从EB中迁移出来。在第16天，感应介质被RDM取代，导致神经视网膜域形成并逐渐从盘中突出，以及由RPE细胞包围的细胞形成视囊状结构。在28至35天期间，视网膜器官由神经视网膜组成，附着在RPE球体上。在一边，细胞形成。随着视网膜分化和规范的进展，hPSC 产生了神经视网膜的亚型，逐渐成层排列，模仿本地人视网膜的建筑特征。视网膜结节细胞最初由视网膜祖体生成，并积聚在神经视网膜的基底侧。有了这个协议，视网膜器官发展成高度成熟的光感受器，既丰富又具有棒和圆锥体。光受体细胞位于 apal 侧，而单细胞、水平细胞、双极细胞和摩尔胶质细胞都位于神经视网膜的中间层。此协议的要点是创建高质量的 Ebs，并以正确的密度播种。

Research

JoVE Journal

Medicine

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

简化技术<em&gt;原位</em&gt;切除角膜和视网膜组织剜人眼地球

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

科研

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

基因表达谱分析人视网膜手术标本杂志

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

定量自体荧光眼底视网膜疾病的评价

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

毛喉素诱导在肠组织体溶胀：一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Using Retinal Imaging to Study Dementia

利用视网膜成像研究痴呆

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

睡眠美人转座转染人视网膜色素上皮细胞氧化应激的诱导与分析

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...