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.

1. diferenciación dirigida de Stem Cell-RPE derivados NOTA: Todos los pasos de la incubación se realizó a 37 ° C en el 5% de CO 2 <ol style="margin-left: 40px;"><li> Mantener el SUH o ​​caderas líneas (alimentar diariamente) en medio de mantenimiento (MM, Tabla 1). Una vez que las líneas han llegado a las normas suficientes de control de calidad, mantenerlos en una capa de células alimentadoras de ratón embrionario (MEFs) sembradas a una densidad de 250.000 / pocillo de una placa de seis pocillos. Culturas-Xeno libre también pueden ser generados siguiendo el protocolo establecido por Tucker et al. 40. </li><li> Permita que las colonias de células madre para llegar a la confluencia. </li><li> Cambie a medio de diferenciación con nicotinamida (DM / NIC, Tabla 1); alimentar a diario. </li><li> Después de tres semanas en cultivo, cambiar a medio de diferenciación con nicotinamida y, o bien activina-A o IDE-1 (DM / NIC / AA o DM / NIC / IDE1, Tabla 1) para mejorar la diferenciación RPE. </li&gt; <li> Después de cinco semanas en cultivo, volver a DM / NIC. Alimentación diaria ya no son necesarios una vez que los indicadores de pH en la parada de los medios de comunicación se tornan amarillas el día después de la alimentación. </li><li> Espere a islotes de células pigmentadas en aparecer que son lo suficientemente grandes como para ser corta y se retira (Ver Figuras 2D-F y 3A). </li></ol> 2. Aislamiento pigmentadas Islotes <ol style="margin-left: 40px;"><li> Cubra los pocillos de una placa de 24 pocillos con una matriz que imita el entorno natural de la célula (es preferible-xeno gratis). NOTA: cuchillas de córnea y afilados, pinzas de punta fina son muy útiles para el picking. Toda la hoja de células diferenciadas puede separar al recoger. Evitar esto trabajando cuidadosamente y recogiendo inicialmente colonias en el centro del plato. </li><li> Rellene tantos pocillos de la placa de 24 pocillos con medio de diferenciación (DM; Tabla 1), según sea necesario. Esta decisión dependerá de la cantidad de colonias pigmentadasrecogido. </li><li> En una campana de cultivo de células con un microscopio de disección, corte manualmente islotes pigmentadas. Picar cada islote en la parte inferior de la placa original utilizando un bisturí en unos 2-6 piezas y agarrar las partes pigmentadas con pinzas cortantes. </li><li> Transferencia 1-2 piezas pigmentadas en cada uno de 24 pocillos. </li><li> No cambie los medios de comunicación durante 3-5 días hasta que las células se adhieran, a continuación, empezar a cambiar los medios de comunicación, tres veces por semana. (Si no se adhieren después de este tiempo, la probabilidad es bueno que ellos nunca lo hará). </li><li> Expandir las células durante 3-4 semanas en medio de diferenciación (DM; Tabla 1) - Una imagen de un cultivo típico se muestra en la Figura 3B. Las células todavía no pueden llenar todo el bien pero espera más largo no parece ayudar. Alimentar a las células tres veces por semana. </li><li> Después de 3 semanas, retire manualmente grupos de células blancas en grupos si es necesario con pinzas cortantes. No hacer este paso a menos que sea necesario - es fácil de introducir contaminantes. Es ideal paratratar de obtener poblaciones puras de RPE de las colonias pigmentadas originales en el paso 1.6. </li></ol> 3. pases de células madre derivadas de RPE <ol style="margin-left: 40px;"><li> Separar las células con 200 l con una solución de disociación celular (preferiblemente no tripsina-un reactivo que puede ser inactivado mediante dilución es preferible) durante 5-8 min a 37 ° C, e inactivar por dilución con DM. Usar una pipeta de 200 l para lavar todas las células y resuspender ellos (Si el pozo seleccionado contiene sólo unas pocas células, considere la agrupación de las células a partir de dos 24-pozos). </li><li> Se centrifuga (800 xg durante 5 min), desechar el sobrenadante y resuspender en 3 ml de medio DM. </li><li> Células Re-placa de uno (o dos) 24-pozos en una sola matriz recubierta de 6 pocillos en 3 ml de DM por pocillo (1: 5 de expansión). </li><li> Expandir durante 1-2 semanas hasta que las células son ~ 90% de confluencia; una imagen de cultivo puro totalmente diferenciada se muestra en la Figura 3C. </li><li> Separe las células con 1 ml de células dissociation solución durante aproximadamente 5-8 min a 37 ° C, inactivar por dilución con DM, utilice 1.000 l pipeta para lavar todas las células, y resuspender ellos. </li><li> Centrifugar (800 xg durante 5 min), desechar el sobrenadante y resuspender en 18 ml de medio DM. </li><li> Re células de placa de un 6 pocillos cada uno en seis matriz recubierta 6 pocillos en 3 ml de DM por pocillo (1: 6 de expansión) o uno-recubierto 75cm 2 matraz (1: 7,5 de expansión). </li><li> Repita los pasos 3.4 a 3.8, según sea necesario hasta que se obtengan suficientes células. NOTA: Trata de recoger la mayor cantidad de colonias como sea posible para la expansión en lugar de planificar para amplificar las culturas a través de pases ya que cada paso induce RPE desdiferenciación. </li></ol>

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None of the authors have any commercial disclosures to declare.

En este manuscrito resumimos los pasos para generar eficientemente un gran número de culturas iPS-EPR puros. Hemos demostrado anteriormente utilizando este protocolo diferenciación dirigida con activina A que podemos generar iPS-EPR que se asemejan fuertemente hRPE basado en la transcriptómica, proteómica, metabolómica y funcionalidad, y que retardar la degeneración de la retina cuando se implanta en ratas RCS 12,31,32 . El proceso de generación de iPS-RPE es mucho tiempo, pero no laboriosa (Fi...

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.

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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.

Los pasos descritos en este manuscrito, como se representa en la Figura 1, pueden ser utilizados para generar fácilmente RPE partir de células madre como se informó anteriormente 10,12. Después de mantener las líneas iPS para varias semanas, las colonias pigmentadas comienzan a aparecer en las colonias después de 5-7 semanas (7 semanas viejas culturas se muestran en la Figura 2A-C). Estas colonias pueden continuar creciendo durante semanas mientras se mantienen las cult...

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.

Derivación eficiente de células de la retina epitelio pigmentario de Células Madre

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 ...

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9.1K vistas.  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.