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.

Kök Hücre kaynaklı RPE 1. Yönetmen Farklılaşma Not: Tüm bekletme işlemleri,% 5 CO2 içinde 37 ° C sıcaklıkta gerçekleştirilmektedir <ol style="margin-left: 40px;"><li> Bakım medya (Tablo 1 MM) de embriyonik kök veya HIPS hatları (günlük yem) koruyun. Hat kalite kontrol yeterli standartlarını yakalamış sonra, fare embriyonik besleyici bir hücre tabakası bunları muhafaza (MEF&#39;ler) 250,000 olan bir yoğunluk / gözenek bir altı yuvalı plaka olarak tohumlanır. Xeno içermeyen kültürler de Tucker ve ark. 40 tarafından oluşturulan protokol takip edilerek üretilebilir. </li><li> Kök hücre kolonileri confluency ulaşmak için bekleyin. </li><li> Nikotinamid (Tablo 1 DM / NIC) ile farklılaşma medya geçin; Günlük yem. </li><li> RPE farklılaşmasını geliştirmek, kültür üç hafta sonra, nikotinamid ve ya Aktivin-A veya IDE-1 (Tablo 1 DM / NIC / AA veya DM / NIC / ide1) ile farklılaşma medya geçmek. </li&gt; <li> Kültür beş hafta sonra, DM / NIC geri dönün. Günlük beslenmeye artık beslendikten sonra sarı bir gün dönüm medya durağı pH göstergelerinin kez gereklidir. </li><li> (Şekil 2D F ve 3A bakınız) kadar kesilmesi ve alınması için büyük görünmesi pigmentli hücre adacıkları bekleyin. </li></ol> 2. Yalıtımlı Pigmentli Islets <ol style="margin-left: 40px;"><li> Coat doğal hücre ortamı (xeno-ücretsiz tercih edilir) taklit eden bir matris ile 24 plaka kuyu. NOT: Kornea bıçaklar ve keskin, ince uçlu forseps seçmek için çok yararlıdır. toplama sırasında farklılaşmış hücrelerin tüm levha ayırabilirsiniz. Dikkatli çalışıyor ve başlangıçta yemeğin ortasında koloniler seçerek bu kaçının. </li><li> (Tablo 1 DM) gerektiği gibi farklılaşma ortam maddesi ile 24 oyuklu plaka gibi çok sayıda çukur doldurun. Pigmentli koloni sayısına bağlı olacaktır Bu kararToplanan. </li><li> Diseksiyon kapsamı ile bir hücre kültürü kaputu, el pigmentli adacıklar kesip. Yaklaşık 2-6 adet bir neşter kullanılarak orijinal plaka altındaki her adacık kıymak ve keskin forseps ile pigmentli parçaları kapmak. </li><li> Her 24 kuyunun içine 1-2 adet pigmentli aktarın. </li><li> Hücreler uygun kadar 3-5 gün süreyle ortamı değiştirmek etmeyin, daha sonra Ortam haftada üç kez değişen başlar. (Bu saatten sonra uygun değilse, olabilirlik onlar asla o iyidir). </li><li> (Tablo 1 DM) - tipik kültür bir görüntü Şekil 3B&#39;de gösterilmiştir farklılaşma ortamda 3-4 hafta boyunca hücrelerin aç. Hücreler hala tüm kuyu doldurmak olmayabilir ama uzun bekleme yardım görünmüyor. Haftada hücreler üç kez besleyin. </li><li> Yaklaşık 3 hafta sonra, elle keskin forseps ile gerekirse kümeleri beyaz hücrelerin kümeleri kaldırın. Gerekli olmadıkça bu adımı yapmayın - bu kirletici tanıtmak için kolaydır. Bu ideal birAdım 1.6 orijinal pigmentli koloniler gelen RPE saf popülasyonlarının edinmeye çalışın. </li></ol> 3. Pasajlanması Kök Hücre türetilen RPE <ol style="margin-left: 40px;"><li> Bir hücre ayrışma çözeltisi ile 200 ul hücreleri ayırmak 37 ° C&#39;de 5-8 dakika boyunca (tercihen seyreltme tercih edilir ile inaktive edilebilir reaktif-a, tripsin olan), ve DM ile sulandırarak bu etkisiz hale getirirler. Tüm hücreleri yıkayın ve (iki 24-kuyulardan hücreleri havuzu düşünün, sadece bir kaç hücreleri içeren iyi seçtiyseniz) onları tekrar süspansiyon 200 ul pipet kullanın. </li><li> Santrifüj (5 dakika için 800 xg), 3 ml DM medya süpernatant ve tekrar süspansiyon atın. </li><li> Yeniden plaka hücreleri bir (veya iki) oyuk başına DM 3 ml bir matris kaplı 6-kuyuya 24 kuyu ile ilgili (1: 5 genişleme). </li><li> Hücreler ~ 90% konfluent kadar 1-2 hafta genişlet; tam olarak farklılaşmış saf kültürünün bir görüntü Şekil 3C &#39;de gösterilmiştir. </li><li> 1 ml hücre d hücreleri ayırmak37 ° C&#39;de yaklaşık 5-8 dakika boyunca issociation Çözelti, DM ile seyreltme ile inaktive tüm hücreleri yıkamak için 1000 ul pipet kullanın, ve bunları yeniden süspanse edin. </li><li> (5 dk için 800 xg) Spin 18 ml DM medyada süpernatant ve tekrar süspansiyon atın. </li><li> (: 6 genişletme 1) ya da 75 cm 2 balon içinde bir kaplanmış (1: 7.5 genişleme) altı matris içine bir 6-yuvalı her birinden yeniden plaka hücreleri, oyuk başına DM 3 ml 6-kuyu kaplama. </li><li> Yeterli hücre elde edilinceye kadar tekrarlayın 3.4-3.8 olarak gerekli adımları. NOT: yerine her geçiş RPE dediferansiyonunu indükler beri Pasajlanması aracılığıyla kültürleri yükseltmek için planlama daha genişleme için mümkün olduğunca çok koloniler toplamaya çalışın. </li></ol>

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None of the authors have any commercial disclosures to declare.

Bu yazıda verimli saf iPS-RPE kültürlerin çok sayıda oluşturmak için adımları özetlemektedir. Biz şiddetle transkriptomik, proteomik, metabolomik ve işlevselliğine dayalı hRPE benzer iPS-RPE üretebilir Aktivin A ile yönlendirilmiş farklılaşma protokolü kullanarak önceden göstermiştir, ve RCS sıçanlarda 12,31,32 implante zaman retina dejenerasyonu geciktirir . iPS-RPE üretme süreci, zaman alıcıdır, ancak zahmetli (Şekil 1). IPS kültürleri konfluense ulaştıkla...

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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			<td style="width:145px;">Life Technologies</td>
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			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">15140-148</td>
			<td>20 mL x 1</td>
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			<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>
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			<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>
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			<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>
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			<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>
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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.

Şekil 1 &#39;de gösterildiği gibi, bu metinde belirtilen adımlar, daha önce rapor edildiği gibi 10,12 kolayca kök hücreler RPE oluşturmak için de kullanılabilir. Birkaç hafta iPS hatları sağlandıktan sonra, pigmentli koloniler 5-7 hafta (7 haftalık kültürler Şekil 2A-C gösterilmiştir) sonra kolonilerde görünmeye başlar. Bu koloniler, kültür muhafaza edildiği gibidir hafta boyunca büyümeye devam edebilirler. Şekil 3A&#39;da göste...

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.

Kök Hücreleri Retina pigment epiteli hücrelerinin Verimli türetilmesi

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

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Developmental Biology

9.1K Görüntüleme.  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.