视网膜色素上皮细胞的干细胞高效的推导

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Westenskow, P., Sedillo, Z., Barnett, A., Friedlander, M. Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells. J. Vis. Exp. (97), e52214, doi:10.3791/52214 (2015).

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Abstract

Protocol

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 。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。
  5. 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-F3A)。

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。

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Representative Results

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
图1:示意图描绘的iPS-RPE推导 点击此处查看该图的放大版本。

图2
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)。(BC)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
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

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Discussion

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,RAXCRX 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。

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Materials

Name Company Catalog Number Comments
Corneal knife  Surgipro SPOI-070 knife x 1
DMEM/F-12, HEPES Life Technologies 11330-032 500 ml x 4 
Dulbecco's Phosphate-Buffered Saline, 1X w/out Ca or Mg VWR 45000-434 500 ml x 6
Fetal Bovine Serum, Regular (Heat Inactivated) VWR 45000-736 500 ml x 1
FGF-Basic (AA 10-155) Recombinant Human Protein Life Technologies PHG0021 100 µg x 1
IDE-1 Stemgent 04-0026 2 mg x 1
Knockout DMEM Life Technologies 10829-018 500 ml x 1 
KnockOut Serum Replacement Life Technologies 10828-028 500 ml x 1
L-Glutamine 200 mM  Life Technologies 25030-081 100 ml x 1
MEM Non-Essential Amino Acids Solution 100X  Life Technologies 11140-050 100 ml x 1
Nicotinamide Sigma-Aldrich N0636-100G 100 g x 1
Penicillin-Streptomycin (10,000 U/ml) Life Technologies 15140-148 20 ml x 1
Recombinant Human/Murine/Rat Activin A  PeproTech 120-14E 10 µg x 2
Synthemax-T Surface 6 Well Plates Corning 3877 Case(12) x 1
TrypLE-Express Enzyme (1X), no phenol red  Life Technologies 12604-021 500 ml x 1 
Vacuum Filter/Storage Bottle System, 0.1µm pore, 500ml  Corning 431475 Case(12) x 1 

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