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.

줄기 세포 유래 망막 색소 상피 1. 감독 차별화 주 : 모든 인큐베이션 단계는 5 % CO 2에서 37 ℃에서 수행 <ol style="margin-left: 40px;"><li> 유지 보수 미디어 (; 표 1 MM)의 배아 줄기 또는 엉덩이 라인 (매일 공급)를 유지한다. 라인이 품질 관리 기준을 충분히 도달했으면, 마우스 배아 피더 세포의 층에이를 유지 보수 (MEFs에)은 250,000의 밀도 / 웰의 여섯 웰 플레이트에 시딩. 제노없는 배양 또한 터커 외. (40)에 의해 확립 된 프로토콜은 다음 생성 될 수있다. </li><li> 줄기 세포 식민지 포화 상태에 도달 할 수 있습니다. </li><li> 니코틴 아미드 (; 표 1 DM / NIC)와 차별화 미디어로 전환; 매일 공급. </li><li> RPE 차별화를 강화하기 위해, 문화 삼주 후, 니코틴 및 중 하나를 Activin-A 또는 IDE-1 (표 1 DM / NIC / AA 또는 DM / NIC / IDE1)와 차별화 미디어로 전환합니다. </lI&gt; <li> 문화 오주 후, DM / NIC로 전환. 매일 먹이를 더 이상 공급 후 노란색 하루를 돌려 미디어 정지의 pH를 표시 한 번 필요하지 않습니다. </li><li> (그림 2D-F와 3A 참조)만큼 잘라 제거 할 큰이 나타나는 색소 세포의 섬 기다립니다. </li></ol> 2. 분리 안료 독도 <ol style="margin-left: 40px;"><li> 외투 세포 자연 환경 (이종이없는 것이 바람직하다) 본뜬 매트릭스 24- 웰 플레이트의 웰. 참고 : 각막 블레이드 날카로운, 뾰족한 집게 따기위한 매우 유용합니다. 따기 동안 분화 된 세포의 전체 시트를 분리 할 수​​ 있습니다. 신중하게 작업하고 처음에 접시의 중앙에 식민지를 선택하여이 피하십시오. </li><li> (; 표 1 DM) 필요에 따라 차별화 미디어를 24 웰 플레이트의 많은 우물을 입력합니다. 착색 된 콜로니의 수에 의존 할 것이다 이러한 결정수집. </li><li> 해부와 세포 배양 후드에서 수동으로 착색 된 섬을 잘라. 약 2-6 조각으로 메스를 사용하여 원래 판의 맨 아래에 각각의 섬을 잘라 날카로운 집게로 착색 된 부분을 잡아. </li><li> 각 24 웰에 1-2 착색 된 조각을 전송합니다. </li><li> 세포가 준수 할 때까지 3~5일을 위해 미디어를 변경하지 마십시오, 다음 미디어에게 일주일에 세 번 변경 시작합니다. (그들은이 시간 이후 준수하지 않는 경우, 가능성은 결코 좋은입니다). </li><li> (; 표 1 DM) - 전형적인 문화의 이미지가 그림 3b에 도시되어 차별화 미디어 3-4 주 동안 세포를 확장합니다. 세포는 여전히 전체를 잘 채울 수 있지만 더 이상 기다리는 것은 해결 될 수 없다. 일주일에 세포를 세 번 피드. </li><li> 약 3 주 후, 수동 날카로운 집게로 필요한 경우 대단히 짧은 흰색 세포의 클러스터를 제거합니다. 필요한 경우가 아니면이 단계를 수행하지 마십시오 - 그것은 오염 물질을 소개하기 쉽습니다. 그것은에 이상적입니다단계 1.6 원래 색소 식민지에서 RPE의 순수한 인구를 얻으려고. </li></ol> 3.과 Passaging 줄기 세포 유래 망막 색소 상피 <ol style="margin-left: 40px;"><li> 세포 해리 용액 200 μL를 가진 세포를 분리하여 37 ℃에서 5-8 분 동안 (바람직 희석 바람직하다 통하여 불 활성화 될 수있는 시약의 트립신 생략), 및 DM 희석하여 비활성화. 모든 세포를 씻어하고 (두 24 우물에서 세포를 풀링을 고려, 몇 세포가 포함되어 잘 선택한 경우)을 재현 탁하기 위해 200 μL 피펫을 사용합니다. </li><li> 원심 분리기 (5 분 800 XG)는, 3 ㎖의 DM 미디어에서 뜨는 및 재현 탁를 폐기합니다. </li><li> 다시 플레이트 세포 하나 (또는​​ 2) 잘 당 DM의 3 ㎖ 한 매트릭스 코팅 6 자에 24 우물에서 (1 : 5 확장). </li><li> 세포가 ~ 90 % 컨 플루 언트 될 때까지 1~2주 대한 확대; 완전히 차별화 된 순수 문화의 이미지는 그림 3C에 표시됩니다. </li><li> 1 ml의 세포 D와 세포를 분리37 ° C에서 약 5-8 분 issociation 솔루션은, DM 희석하여 비활성화 모든 세포를 씻어 1,000 μL 피펫을 사용하고이를 재현 탁. </li><li> (5 분 800 XG) 스핀 다운 18 ML의 DM 미디어에서 뜨는 및 재현 탁를 폐기합니다. </li><li> (6 팽창 1) 또는 (2) 75cm 플라스크 하나가 코팅 된 (1 : 7.5 확장) 여섯 매트릭스로 한 각 6- 웰 플레이트에서 다시 셀 웰 당 3 ㎖ DM 6- 웰 코팅. </li><li> 충분한 세포를 얻을 때까지 반복 3.4-3.8 필요한 단계를 반복합니다. 참고 : 오히려 각 통로 RPE의 탈분화를 유도하기 때문에 계대를 통해 문화를 증폭 계획보다 확장 가능한 한 많은 식민지를 수집하려고합니다. </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.

이 논문에서 우리는 효율적으로 순수 만능 줄기-RPE 문화의 큰 숫자를 생성하는 단계를 설명합니다. 우리는 우리가 강하게 transcriptomics, 단백질 체학, 대사 체학 및 기능에 따라 hRPE 유사 만능 줄기-RPE를 생성 할 수 있습니다를 Activin이 감독 분화 프로토콜을 사용하여 이전에 보여 주었다 및 RCS 쥐 12,31,32를 이식 할 때 그들은 망막 변성을 지연하는 것이 . 만능-RPE를 생성하는 과정은 시간이 ?...

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.

도 1에 도시 된 바와 같이,이 논문에서 설명하는 단계는, 이전에보고 된 바와 같이 (10, 12)를 용이하게 줄기 세포에서 RPE를 생성하는데 사용될 수있다. 몇 주 동안 만능 줄기 라인을 유지 한 후, 색소 식민지 5-7주 (7 주 된 문화가 그림 2A-C에 나와 있습니다) 후 식민지에서 나타나기 시작. 이러한 식민지 문화가 유지 될 때 주 동안 계속 증가 할 수 있습니다. 도 3...

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.

줄기 세포에서 망막 색소 상피 세포의 효율적인 유도

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 조회수.  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.