We wish to thank the following individuals: Drs. Tim Krohne and Eyal Banin (along with Dr. Mandy Lehmann and David Friedlander) for generous help developing the differentiation protocols. Dr. Felicitas Bucher provided assistance differentiating the RPE cells used in this study. We also acknowledge the National Eye Institute (NEI grants EY11254 and EY021416), California Institute for Regenerative Medicine (CIRM grant TR1-01219), and the Lowy Medical Research Institute (LMRI) for very generous funding for this project.

干细胞衍生的RPE 1.定向分化注：所有的孵育步骤都在37℃下在5％CO 2 <ol style="margin-left: 40px;"><li>维护保养媒体（MM; 表1）胚胎或臀部线条（每日喂）。一旦线已达到质量控制的足够标准，维持它们在层上的小鼠胚胎饲养细胞（MEFs细胞）接种在250,000 /孔的密度的6孔板中。无异物培养，也可以产生以下由Tucker 等。40建立的协议。 </li><li>允许干细胞集落达到汇合。 </li><li>切换到分化培养基与烟酰胺（DM / NIC; 表1）;每天喂。 </li><li>三周培养后，切换到分化培养基与烟酰胺，要么激活素A或IDE-1（DM / NIC / AA或DM / NIC / IDE1; 表1）加强RPE分化。 </l我&gt; <li> 5星期培养后，切换回的DM /网卡。每日喂食不再需要一次在媒体停止喂养后变黄天pH指示剂。 </li><li>等待色素细胞的胰岛显示是足够大的要被切割和除去（参见图2D-F和3A）。 </li></ol> 2.隔离色素胰岛<ol style="margin-left: 40px;"><li>外套24孔板用基质模仿天然细胞环境（无异物优选）的孔中。 注：角膜刀和锋利，细尖的镊子对采摘非常有用的。分化细胞的整个工作表可以分离而采摘。通过仔细的工作，最初在盘中央采摘殖民地避免这种情况。 </li><li>补的24孔板用分化培养基的许多孔（DM; 表1），为需要的。这个决定将取决于着色的菌落数收集。 </li><li>在细胞培养罩与解剖范围，手动切出含颜料的胰岛。砍每个胰岛在约2-6个使用手术刀原板的底部，并抓住着色零件用锋利的钳子。 </li><li>转1-2色素块放入每24孔。 </li><li>不要为3-5天更换介质，直到细胞粘附，然后开始改变媒体每周三次。 （如果他们在此时间之后不坚持，可能是很好的，他们永远不会）。 </li><li>扩大细胞3-4周在分化培养基（DM; 表1） -的典型培养物的图像示于图3B。细胞可能仍然无法填满整个很好，但等待的时间似乎并没有帮助。每周喂细胞三次。 </li><li>约3周后，手动删除在必要时用尖锐的镊子团块簇的白细胞。不要做这一步，除非它是必要的 - 这是容易引入污染物。它是理想的尝试获得RPE的最纯净的人口从原来的色素殖民地步骤1.6。 </li></ol> 3.传代干细胞衍生的RPE <ol style="margin-left: 40px;"><li>分离的细胞用200μl用细胞解离溶液（优选不胰一个可灭活通过稀释是优选的试剂）为5-8分钟，在37℃下，并通过与DM稀释灭活它。用200μl的移液管，以洗掉所有的细胞，并重新悬浮它们（如果​​所选井只包含几个细胞，考虑汇集的细胞从两个24孔）。 </li><li>离心机（800 XG为5分钟），弃上清，重悬在3毫升DM媒体。 </li><li>重板的细胞从一个（或两个）24孔到一个基质涂敷的6孔中每孔3毫升的DM（1:5展开）。 </li><li>扩大为1-2周，直至细胞为〜90％汇合;完全分化的纯培养物的图像示于图3C。 </li><li>分离的细胞用1毫升单元d在37℃进行约5-8分钟issociation溶液，失活其稀释用的DM，使用1000微升吸管洗掉所有的细胞，并重新悬浮它们。 </li><li>降速（800 XG为5分钟），弃去上清液，并重新悬浮于18ml DM媒体。 </li><li>重板的细胞从一个6孔各为六个矩阵涂覆6-孔在3毫升的DM每孔的（1:6展开）或1被覆75厘米2烧瓶（1：7.5的扩展）。 </li><li>直到有足够的细胞获得3.4-3.8根据需要重复步骤。 注：尽量收集尽可能多的殖民地尽可能扩张，而不是通过规划传代，因为每个通过诱导分化RPE扩增培养。 </li></ol>

<ol>
	<li>Strauss, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+pigment+epithelium+in+visual+function.">The retinal pigment epithelium in visual function.</a> Physiol Rev. 85 (3), 845-881 (2005).</li><li>Congdon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+prevalence+of+visual+impairment+among+adults+in+the+United+States.">Causes and prevalence of visual impairment among adults in the United States.</a> Arch Ophthalmol. 122 (4), 477-485 (2004).</li><li>Friedman, D. S. <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+age-related+macular+degeneration+in+the+United+States.">Prevalence of age-related macular degeneration in the United States.</a> Arch Ophthalmol. 122 (4), 564-572 (2004).</li><li>Resnikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+data+on+visual+impairment+in+the+year.">Global data on visual impairment in the year.</a> Bull World Health Organ. 82 (11), 844-851 (2002).</li><li>Bird, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+targets+in+age-related+macular+disease.">Therapeutic targets in age-related macular disease.</a> The Journal of Clinical Investigation. 120 (9), 3033-3041 (2010).</li><li>Jong, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration.">Age-related macular degeneration.</a> N Engl J Med. 355 (14), 1474-1485 (2006).</li><li>Binder, S., Stanzel, B. V., Krebs, I., Glittenberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+In+Retinal+And+Eye+Research.">Progress In Retinal And Eye Research.</a> Transplantation of the RPE in AMD. 26 (5), 516-554 (2007).</li><li>Cruz, L., Chen, F. K., Ahmado, A., Greenwood, J., Coffey, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPE+transplantation+and+its+role+in+retinal+disease.">RPE transplantation and its role in retinal disease.</a> Progress In Retinal And Eye Research. 26 (6), 598-635 (2007).</li><li>Carr, A. J. <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+human+iPS-derived+retinal+pigment+epithelium+cell+transplantation+in+the+retinal+dystrophic+rat.">Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat.</a> PLoS One. 4 (12), e8152 (2009).</li><li>Idelson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+embryonic+stem+cells+into+functional+retinal+pigment+epithelium+cells.">Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells.</a> Cell Stem Cell. 5 (4), 396-408 (2009).</li><li>Klimanskaya, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+comparative+assessment+of+retinal+pigment+epithelium+from+human+embryonic+stem+cells+using+transcriptomics.">Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics.</a> Cloning Stem Cells. 6 (3), 217-245 (2004).</li><li>Krohne, T. <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+pigment+epithelial+cells+from+small+molecules+and+OCT4-reprogrammed+human+induced+pluripotent+stem+cells.">Generation of retinal pigment epithelial cells from small molecules and OCT4-reprogrammed human induced pluripotent stem cells.</a> Stem Cells Translational Medicine. 1 (2), 96-109 (2012).</li><li>Lund, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+cells+rescue+visual+function+in+dystrophic+RCS+rats.">Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.</a> Cloning Stem Cells. 8, 189-199 (2006).</li><li>Vugler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+the+phenomenon+of+HESC-derived+RPE:+anatomy+of+cell+genesis,+expansion+and+retinal+transplantation.">Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation.</a> Exp Neurol. 214 (2), 347-361 (2008).</li><li>Algvere, P. V., Berglin, L., Gouras, P., Sheng, Y. <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+fetal+retinal+pigment+epithelium+in+age-related+macular+degeneration+with+subfoveal+neovascularization.">Transplantation of fetal retinal pigment epithelium in age-related macular degeneration with subfoveal neovascularization.</a> Graefes Arch Clin Exp Ophthalmol. 232 (12 ), 707-716 (1994).</li><li>Binder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outcome+of+transplantation+of+autologous+retinal+pigment+epithelium+in+age-related+macular+degeneration:+a+prospective+trial.">Outcome of transplantation of autologous retinal pigment epithelium in age-related macular degeneration: a prospective trial.</a> Invest Ophthalmol Vis Sci. 45 (11), 4151-4160 (2004).</li><li>Binder, S. <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+autologous+retinal+pigment+epithelium+in+eyes+with+foveal+neovascularization+resulting+from+age-related+macular+degeneration:+a+pilot+study.">Transplantation of autologous retinal pigment epithelium in eyes with foveal neovascularization resulting from age-related macular degeneration: a pilot study.</a> Am J Ophthalmol. 133 (2), 215-225 (2002).</li><li>Falkner-Radler, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+retinal+pigment+epithelium+(RPE)+transplantation:+outcome+after+autologous+RPE-choroid+sheet+and+RPE+cell-suspension+in+a+randomised+clinical+study.">Human retinal pigment epithelium (RPE) transplantation: outcome after autologous RPE-choroid sheet and RPE cell-suspension in a randomised clinical study.</a> British Journal of Ophthalmology. 95 (3), 370-375 (2011).</li><li>Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+safety+and+efficacy+of+human-induced+pluripotent+stem+cell+(iPS)+grafts+in+a+preclinical+model+of+retinitis+pigmentosa.">Long-term safety and efficacy of human-induced pluripotent stem cell (iPS) grafts in a preclinical model of retinitis pigmentosa.</a> Molecular Medicine (Cambridge, Mass). 18, 1312-1319 (2012).</li><li>Lu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Safety+and+Function+of+RPE+from+Human+Embryonic+Stem+Cells+in+Preclinical+Models+of+Macular+Degeneration).">Long-Term Safety and Function of RPE from Human Embryonic Stem Cells in Preclinical Models of Macular Degeneration).</a> Stem Cells. 27 (9), 2126-2135 (2009).</li><li>Peyman, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+retinal+pigment+epithelium+transplantation+for+age-related+macular+degeneration+secondary+to+extensive+subfoveal+scarring.">A technique for retinal pigment epithelium transplantation for age-related macular degeneration secondary to extensive subfoveal scarring.</a> Ophthalmic Surgery. 22 (2), 102-108 (1991).</li><li>Schwartz, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+trials+for+macular+degeneration:+a+preliminary+report.">Embryonic stem cell trials for macular degeneration: a preliminary report.</a> The Lancet. 379 (9817), 713-720 (2012).</li><li>Wang, N. K. <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+reprogrammed+embryonic+stem+cells+improves+visual+function+in+a+mouse+model+for+retinitis+pigmentosa.">Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for retinitis pigmentosa.</a> Transplantation. 89 (8), 911-919 (2010).</li><li>Cruz, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+the+receptor+tyrosine+kinase+gene+Mertk+in+the+retinal+dystrophic+RCS+rat.">Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat.</a> Human Molecular Genetics. 9 (4), 645-651 (2000).</li><li>Buchholz, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+functional+retinal+pigmented+epithelium+from+induced+pluripotent+stem+cells.">Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells.</a> Stem Cells. 27 (10), 2427-2434 (2009).</li><li>Hirami, Y. <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+cells+from+mouse+and+human+induced+pluripotent+stem+cells.">Generation of retinal cells from mouse and human induced pluripotent stem cells.</a> Neurosci Lett. 458 (3), 126-131 (2009).</li><li>Meyer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+early+retinal+development+with+human+embryonic+and+induced+pluripotent+stem+cells.">Modeling early retinal development with human embryonic and induced pluripotent stem cells.</a> Proceedings of the National Academy of Sciences. 106 (39), 16698-16703 (2009).</li><li>Osakada, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+retinal+cells+from+human+pluripotent+stem+cells+by+small-molecule+induction.">In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction.</a> J Cell Sci. 122 (17), 3169-3179 (2009).</li><li>Carr, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+and+functional+analysis+of+phagocytosis+by+human+embryonic+stem+cell-derived+RPE+cells+using+a+novel+human+retinal+assay.">Molecular characterization and functional analysis of phagocytosis by human embryonic stem cell-derived RPE cells using a novel human retinal assay.</a> Mol Vis. 15, 283-295 (2009).</li><li>Kokkinaki, M., Sahibzada, N., Golestaneh, N. <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-Derived+Retinal+Pigment+Epithelium+(RPE)+Cells+Exhibit+Ion+Transport,+Membrane+Potential,+Polarized+Vascular+Endothelial+Growth+Factor+Secretion,+and+Gene+Expression+Pattern+Similar+to+Native+RPE.">Human Induced Pluripotent Stem-Derived Retinal Pigment Epithelium (RPE) Cells Exhibit Ion Transport, Membrane Potential, Polarized Vascular Endothelial Growth Factor Secretion, and Gene Expression Pattern Similar to Native RPE.</a> Stem Cells. 29 (5), 825-835 (2011).</li><li>Westenskow, P., Friedlander, M., Werne, J. S., Chalupa, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+111.">Ch. 111.</a> The New Visual Neurosciences. , 1611-1626 (2013).</li><li>Westenskow, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+flow+cytometry+to+compare+the+dynamics+of+photoreceptor+outer+segment+phagocytosis+in+iPS-derived+RPE+cells.">Using flow cytometry to compare the dynamics of photoreceptor outer segment phagocytosis in iPS-derived RPE cells.</a> Invest Ophthalmol Vis Sci. 53 (10), 6282-6290 (2012).</li><li>Algvere, P. V., Gouras, P., Dafgard Kopp, ., E, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+outcome+of+RPE+allografts+in+non-immunosuppressed+patients+with+AMD.">Long-term outcome of RPE allografts in non-immunosuppressed patients with AMD.</a> Eur J Ophthalmol. 9 (3), 217-230 (1999).</li><li>Zhang, X., Bok, D. <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+retinal+pigment+epithelial+cells+and+immune+response+in+the+subretinal+space.">Transplantation of retinal pigment epithelial cells and immune response in the subretinal space.</a> Invest Ophthalmol Vis Sci. 39 (6), 1021-1027 (1998).</li><li>Gamm, D. M., Phillips, M. J., Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+retinal+degenerative+diseases+with+human+iPS-derived+cells:+current+status+and+future+implications.">Modeling retinal degenerative diseases with human iPS-derived cells: current status and future implications.</a> Expert Review Of Ophthalmology. 8, 213-216 (2013).</li><li>Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPS+cell+modeling+of+Best+disease:+insights+into+the+pathophysiology+of+an+inherited+macular+degeneration.">iPS cell modeling of Best disease: insights into the pathophysiology of an inherited macular degeneration.</a> Hum Mol Genet. 22 (3), 593-607 (2013).</li><li>Buchholz, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+directed+differentiation+of+human+pluripotent+stem+cells+into+retinal+pigmented+epithelium.">Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium.</a> Stem Cells Transl Med. 2 (5), 384-393 (2013).</li><li>Fuhrmann, S., Levine, E. M., 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=Extraocular+mesenchyme+patterns+the+optic+vesicle+during+early+eye+development+in+the+embryonic+chick.">Extraocular mesenchyme patterns the optic vesicle during early eye development in the embryonic chick.</a> Development (Cambridge, England). 127 (21), 4599-4609 (2000).</li><li>Borowiak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecules+efficiently+direct+endodermal+differentiation+of+mouse+and+human+embryonic+stem+cells.">Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells.</a> Cell Stem Cell. 4 (4), 348-358 (2009).</li><li>Tucker, B. A., Anfinson, K. R., Mullins, R. F., Stone, E. M., Young, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+synthetic+xeno-free+culture+substrate+for+induced+pluripotent+stem+cell+induction+and+retinal+differentiation.">Use of a synthetic xeno-free culture substrate for induced pluripotent stem cell induction and retinal differentiation.</a> Stem Cells Transl Med. 2 (1), 16-24 (2013).</li><li>Ramsden, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+in+retinal+regeneration:+past,+present+and+future.">Stem cells in retinal regeneration: past, present and future.</a> Development (Cambridge, England). 140 (12), 2576-2585 (2013).</li><li>Zhao, T., Zhang, Z. -. N., Rong, Z., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+of+induced+pluripotent+stem+cells.">Immunogenicity of induced pluripotent stem cells.</a> Nature. 474 (7350), 212-215 (2011).</li><li>Zhang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+conversion+of+human+fibroblasts+into+retinal+pigment+epithelium-like+cells+by+defined+factors.">Direct conversion of human fibroblasts into retinal pigment epithelium-like cells by defined factors.</a> Protei., &amp; Cell. 5 (1), 48-58 (2013).</li></ol>

None of the authors have any commercial disclosures to declare.

在这份手稿，我们概述的步骤有效地产生大量的纯的iPS-RPE文化。我们已经使用这种定向分化协议与激活素A，我们可以生成的iPS-RPE强烈类似于视网膜色素上皮基于转录组学，蛋白质组学，代谢组学和功能前所示，他们延缓视网膜变性时，在RCS大鼠植入12,31,32 。产生的iPS-RPE的过程是耗时的，但不费力（ 图1）。一旦培养的iPS达到汇合它们必须与分化培养基，每天供给。我们既用...

The various cell types that occupy the retina are organized in a functional architecture. The photoreceptors in the back of the retina are responsible for converting light into electrical impulses through phototransduction. However, phototransduction cannot occur without the coordinated efforts of other neighboring cell types including Mueller glia and retinal pigment epithelium (RPE) cells. A monolayer of RPE cells partitions the sensory retina from the choriocapillaris, the primary blood supply for photoreceptors, and are ideally situated to control multiple functions important for photoreceptor homeostasis. In fact, the RPE and photoreceptors are so co-dependent they are widely considered to be one single functional unit. (For a review of all the diverse functions of the RPE see Strauss, 20051.) Death or dysfunction of retinal pigment epithelium cells can induce age-related macular degeneration (AMD), the leading cause of permanent vision loss in industrialized countries2-4. 
AMD is a multifactorial disease of RPE, photoreceptors, and the choroidal vasculature; risk factors are diverse and include combinations of environmental and genetic influences5,6. Treatments for AMD are very limited, but one promising potential therapy is RPE cell replacement7,8. While the outcomes have been mixed, the transplantation of RPE cells in AMD patients (and in other patients with retinal degeneration) and also in rodent models of retinal degeneration, has the potential to transiently prevent significant photoreceptor atrophy9-23. (The animal model commonly used for these studies are Royal College of Surgeons (RCS) rats, which harbor a mutation in the MerTK gene. This mutation renders RPE cells incapable of phagocytosing photoreceptor outer segments and promotes retinal degeneration24.) While the reported survival rates of implanted RPE in the subretinal space of RCS rats and mice vary greatly, there is potential for them to survive for several months or years9,10,12,20. 
RPE cells can be obtained in sufficient numbers for transplantation by deriving them from pluripotent stem cells9-14,25-28. Several independent groups have demonstrated that these cells function in similar ways to their somatic counterparts, and long term studies suggest that they are safe upon implantation in rat and mouse disease models9,10,12,14,19,20,25,29-32. The use of induced pluripotent stem cells instead of embryonic stem cells may be advantageous since ethical issues and immunological challenges associated with allogeneic RPE may be obviated33,34. Another exciting application for iPS technology is disease modeling35. The ability to interrogate large numbers of RPE cells derived from patients with RPE diseases could be invaluable for understanding their molecular bases. This type of study has been performed recently with Best disease patient RPE and could pave the way for similar studies of inherited maculopathies36. 
The derivation of RPE from stem cells is a relatively simple process and can be done entirely in xeno-free conditions. The simplest strategy is to derive monolayers of RPE cells spontaneously, however, the yield can be significantly improved using directed differentiation techniques. But these techniques involve the use of exogenous protein factors that can be expensive and often generated in bacteria or other non-human sources10,12,37. In our studies we followed an established protocol10 that utilizes nicotinamide and Activin A, a signaling factor that has been shown to be sufficient for RPE specification38. Here we will demonstrate that the small molecule IDE-1 can adequately replace Activin A, thus reducing costs and alleviating concerns associated with the use of recombinant proteins39. Additionally, we utilize xeno-free serum replacement, and we culture the differentiating RPE cells on a synthetic xeno-free substrate. RPE cells have been shown previously to differentiate very effectively using this approach40.
When differentiated as a monolayer, we visualize pigmented colonies containing RPE cells after as early as five weeks12. Once they reach sufficient size, they can be manually excised and transferred to another dish for expansion. RPE cells are notorious for dedifferentiating with each passage, and the use of anything older than five passages should be avoided (we find that sufficient numbers of cells for characterization and transplantation in animal models can easily be generated after two or three passages). Once fully differentiated, we employ multiple techniques to characterize the cells anatomically and functionally to ensure that they will serve as adequate replacements for diseased RPE. The description of these techniques, and protocol for implanting the iPS-RPE in the subretinal space of rodents, are beyond the scope of this methods paper and have been previously published12,32,41. 
While developing standardized protocols for effective derivation of iPS-RPE is clearly important for the clinics, there is also significant preclinical work to still be done in animal models. There are concerns regarding immunogenicity of iPS-derived cells, and multiple different implantation techniques, including implanting cells on artificial substrates, are being explored42,43. For these reasons, we feel that the publication of standardized protocols is beneficial to facilitate both clinical and preclinical studies. Especially if direct comparisons will be done of iPS-RPE cells derived in different labs by different research groups.

<table border="0" cellpadding="0" cellspacing="0" width="849">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Name of Material/ Equipment (A-Z)</td>
			<td style="width:145px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Corneal knife&#160;</td>
			<td style="width:145px;">Surgipro</td>
			<td style="width:119px;">SPOI-070</td>
			<td>knife x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">DMEM/F-12, HEPES</td>
			<td style="width:145px;">Life Technologies</td>
			<td>11330-032</td>
			<td>500 mL x 4&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate-Buffered Saline, 1X w/out Ca or Mg</td>
			<td style="width:145px;">VWR</td>
			<td style="width:119px;">45000-434</td>
			<td>500 mL x 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Fetal Bovine Serum, Regular (Heat Inactivated)</td>
			<td style="width:145px;">VWR</td>
			<td style="width:119px;">45000-736</td>
			<td>500 mL x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">FGF-Basic (AA 10-155) Recombinant Human Protein</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">PHG0021</td>
			<td>100 ug x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">IDE-1</td>
			<td style="width:145px;">Stemgent</td>
			<td style="width:119px;">04-0026</td>
			<td>2 mg x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Knockout DMEM</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">10829-018</td>
			<td>500 mL x 1&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">KnockOut Serum Replacement</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td>500 mL x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">L-Glutamine 200 mM&#160;</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td>100 mL x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution 100X&#160;</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td>100 mL x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Nicotinamide</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:119px;">N0636-100G</td>
			<td>100 g x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">15140-148</td>
			<td>20 mL x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Recombinant Human/Murine/Rat Activin A&#160;</td>
			<td style="width:145px;">PeproTech</td>
			<td style="width:119px;">120-14E</td>
			<td>10 ug x 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Synthemax-T Surface 6 Well Plates</td>
			<td style="width:145px;">Corning</td>
			<td style="width:119px;">3877</td>
			<td>Case(12) x 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">TrypLE-Express Enzyme (1X), no phenol red&#160;</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">12604-021</td>
			<td>500 mL x 1&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Vacuum Filter/Storage Bottle System, 0.1&#181;m pore, 500mL&#160;</td>
			<td style="width:145px;">Corning</td>
			<td style="width:119px;">431475</td>
			<td>Case(12) x 1&#160;</td>
		</tr>
	</tbody>
</table>

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.

在此概述的手稿，如在图1中所描绘的步骤，可用于容易地产生从干细胞的RPE如先前报道10,12。维持数周的iPS线后，色素的菌落开始5-7周（7周龄培养物示于图2A-C）的后出现在菌落。这些殖民地可以继续增长数周的文化得以维持。一旦达到足够的尺寸， 如图2D-F（8周龄培养物），它们可以被手动地切除， 如图3A所示 。小心切除，以避免污染...

Watch this Scientific Journal Video about Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells at JoVE.com

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

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.

视网膜色素上皮细胞的干细胞高效的推导

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 ...

efficient-derivation-retinal-pigment-epithelium-cells-from-stem

Research

JoVE Journal

Developmental Biology

9.1K 次观看.  The Scripps Research Institute. 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.