视网膜色素上皮细胞的干细胞高效的推导

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.

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.

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 countries^2-4.

AMD is a multifactorial disease of RPE, photoreceptors, and the choroidal vasculature; risk factors are diverse and include combinations of environmental and genetic influences^5,6. Treatments for AMD are very limited, but one promising potential therapy is RPE cell replacement^7,8. 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 atrophy^9-23. (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 years^9,10,12,20.

RPE cells can be obtained in sufficient numbers for transplantation by deriving them from pluripotent stem cells^9-14,25-28. 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 models^{9,10,12,14,19,20,25,29-32}. 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 obviated^33,34. 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 sources^10,12,37. 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 published^12,32,41.

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 explored^42,43. 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.

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

1. 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。
2. 允许干细胞集落达到汇合。
3. 切换到分化培养基与烟酰胺（DM / NIC; 表1）;每天喂。
4. 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。
5. 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。
6. 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）。

2.隔离色素胰岛

1. 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。
2. 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。
3. 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。
4. 转1-2色素块放入每24孔。
5. 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）。
6. 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。
7. 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。

3.传代干细胞衍生的RPE

1. 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）。
2. 离心机（800 XG为5分钟），弃上清，重悬在3毫升DM媒体。
3. 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）。
4. 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。
5. 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。
6. 降速（800 XG为5分钟），弃去上清液，并重新悬浮于18ml DM媒体。
7. 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）。
8. 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。

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，以避免污染与非RPE细胞将大大促进足够纯的RPE培养物（ 图3B-C）的生成。

图1:示意图描绘的iPS-RPE推导 。幼稚干细胞是在维持培养基中培养直至达到融合。在每天0分化培养基（DM）缺乏的bFGF但含有烟酰胺加（DM / NIC）。将细胞每日供给与此媒体三个星期。在3周结束时，DM培养基补充有重组激活素A（DM / NIC / AA）或IDE-1（DM / NIC / IDE1）增强RPE说明书和细胞饲喂该介质两周。在这种治疗色素殖民地开始出现，这些扩大在未来数周，8周，可以手动删除，转移到新的板块的扩张DM。 （见表1特定媒体组件） 点击此处查看该图的放大版本。

图2:激活素A的ðIDE-1增强的iPS-RPE的产率。（A）的小色素菌落开始即粘附到6孔板的底部7周在培养自发非RPE细胞中的单个片之间后出现（一些标有箭头）。（B和C）的补充有IDE-1或活化素A导致在更色素菌落的外观（一些标有交流箭头）表明补充或者激活素A或IDE-1增强的RPE分化。（DF）8周补充有IDE-1或活化素A的影响后更加显着。既着色胰岛的数目及尺寸定向分化后大。比例尺=5毫米

图3:扩展和终末分化色素的iPS细胞集落RPE是足够大的消费色素的iPS-RPE。（A）代表的形象。非RPE细胞包围菌落在一个6孔板的底部。蓝色轮廓标记，将被切除的区域。后汇合未成熟的iPS-RPE细胞在培养的第一个膨胀步骤之后的（B）中的图像。（℃）两月龄终末分化的iPS-RPE细胞。注意明显小区边界和色素沉着的同质水平展示先进分化的存在。比例尺= 100微米

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。我们既用激活素A或更便宜的小分子IDE-1补充媒体，并继续喂养两周同时监测文化的色素的iPS-RPE菌落一般出现5-7周（ 图2A-C）之后。一旦达到足够的大小（ 图2D-F和图3A）中，科洛尼ES手动移除，并转移到新的板进行扩展。发生这种情况大致8周后，是整个协议的最费力的步骤。

最具挑战性的方面是由偶然收集无用的相邻小区中或周围色素菌落避免污染与非RPE细胞中的扩大培养。根据污染程度，非视网膜色素上皮细胞的小岛，可以手动膨胀期间除去，虽然每次培养物的处理引入细菌或真菌污染物增加的额外风险。值得注意的是，在我们的经验了IPS-RPE实际上可以在传代步骤"outcompete"少数污染的细胞。但是，我们强烈建议您采摘殖民地的时候，以确保它们尽可能的纯净，以避免诉诸这些步骤采取非常谨慎。由第二或第三通道的的iPS-RPE培养是足够纯和suffi可用于表征和植入细胞cient号码。

这里报道的技术当然不是用于导出干细胞衍生的RPE的唯一方法。事实上，它是既不容易也不是最快的。最简单和最广泛的方法是使用自发分化。 （但是，补充的IDE-1两周便宜且大大增加了的iPS-RPE的产率。）的iPS-RPE已经于分化成偏振RPE细胞的球体，可以迫使粘附表面胚体也产生培养板并展开为单层。 RPE细胞生成使用这种方法也已经很大力特点和强烈类似于视网膜色素上皮^27。 RPE可以极其从干细胞通过补充媒体与烟酰胺分化迅速（在仅14天），IGF1，头蛋白，的Dkk1，和bFGF将它们转换为神经视网膜祖命运，后来加入的亲RPE因子烟酰胺一第二激活素A ^37。 RPE也可以由转导的成纤维细胞与一组转录因子包括最小cMYC的，MITF，OTX2，RAX和CRX ⁴⁴从成纤维细胞产生更迅速地直接在大约一个月。然而，虽然这些结果是非常令人鼓舞的RPE生成这最后两种技术尚未严格特征在于植入他们体内 。因此，我们建议读者考虑所有RPE推导仔细选择决定哪些在他们的研究中使用的时候。

利用这里所概述的协议的优点是它的简单性和非常高品质的RPE ³¹的一致的高产量。补充的IDE-1，而不是激活素A大大降低了整体成本，并降低了涉及使用重组蛋白的风险。因为它是目前尚不清楚是否选择使用不同的分化方法将有对最终产品的影响，它可能是有利的，利用标准化的协议，特别是如果在不同的实验室中产生的RPE之间的直接比较，将是必要的（也许特别是在疾病模型的情况下）。一个简单的协议，像这样的一个需要很少的专业知识和试剂，并产生高收益的iPS-RPE的，可能是理想的这些情况。

None of the authors have any commercial disclosures to declare.

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.