从诱导多能干得出视网膜色素上皮（RPE）（IPS），由胚体大小不同的细胞
Summary
此报告的目的是描述协议来导出从诱导的多能干（iPS细胞）使用不同尺寸的胚状体的视网膜色素上皮细胞（RPE）。
Abstract
Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.
Introduction
诱导多能干细胞（iPS细胞）是由重编程体细胞与外在因素1派生的类型的多能干细胞。与此相反，胚胎干细胞（ESCs），另一种类型的多能干细胞，从胚泡2-3的内细胞团生成。尽管他们的不同的起源，iPS细胞和胚胎干细胞是可比在其无限的容量在体外和在其分化成任何细胞类型的能力4-5进行复制。 iPS细胞的这些特性使其成为理想的候选人在个性化的再生医学应用。最近的研究工作集中在开发健壮的分化协议用 于生产特殊成年细胞包括视网膜色素上皮（RPE）6-11。
于iPS来源的细胞潜在的临床应用中，定向分化为特定的细胞类型是至关重要的。有多种方法公布为胚胎干细胞和iPS细胞定向分化为RPE的良莠不齐的工作效率6-7，12-16。我们仍然不知道有多少，发展和分化过程中执政细胞/组织命运的分子事件。在最近几年，已经作出努力来开发该分化方案，可以模仿的胚胎发育尽可能。在胚泡阶段，干细胞的未提交的人口都一起在三维微环境。因此，采用了不同的策略，使ESC / iPS细胞聚集在一起，在三个维度成长他们。这些干细胞聚集体被称为胚体（EB）。有研究表明，干细胞的分化的EB模仿胚胎发育的早期阶段，并且可以自发地产生原始内胚层在其外表面上。后来，随着EB开发的进展，所有三个胚谱系分化细胞的表型出现17-18。日erefore，胚基分化协议已经吸引了大量的关注在体外分化ESC / iPS细胞，是一个很好的候选人RPE代从多能干细胞13。
胚可以使用多种方法从ESC / iPS细胞进行。最初，胚被刮去附着的菌落，并保持它们在非粘附悬浮培养制成。然而，这种方法产生的EB，导致低再现性的异质群体。悬滴细胞培养和微孔基EBS形成其他流行的技术胚形成而产生定义尺寸的高度重复性的同质胚。另外，微孔的技术可以产生大量的聚集体以较少的努力。
内的EB细胞分化是通过从细胞外和细胞内的微形态线索多路复用调节。与此相反，以分化的纹olayer格式的EB为细胞的复杂的组装和细胞间信号发生17的平台。有趣的是，观察到用来制作个别的EB多能干细胞的数量来影响细胞的命运。例如，在人类胚胎干细胞的造血分化的研究中，观察到500个细胞EB对促进髓系分化，而1000细胞EB推向红系20。在另一项研究中，较小的EB青睐定形内胚层分化而促进向神经外胚层分化11,17更大的EB。
这些过去的研究有力地表明，用于制造单个的EB ESC / iPS细胞的数量的影响根据分化成任何细胞类型的胚。然而，据我们所知，有一些已经阐明其倾向胚大小的影响，区分对RPE目前没有研究。这项研究的目的是表征的影响EB大小对诱导多能干细胞（iPS细胞） – 视网膜色素上皮细胞（IPS-RPE）的分化，并确定最佳的细胞数量，使胚的对RPE细胞系定向分化。
Protocol
Representative Results
Discussion
为了实现多能干细胞用于细胞疗法的充分承诺，有必要调节其分化以一致和可再现的方式。这份报告描述了协议，形成利用微孔板技术尺寸控制EBS，开始分化为RPE，并确定RPE的蛋白质和基因标记。同步在体外分化，胚的大小均匀已知数字的iPS细胞在微孔板强迫聚集离心的形成。免疫细胞化学和逆转录聚合酶链反应（RT-PCR），用于监视所述表达式的RPE的蛋白质和基因说明。最后，分化的不同?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. This research was performed while the authors Alberto Muñiz, Ramesh R Kaini, Whitney A Greene and Jae-Hyek Choi held a National Research Council Postdoctoral Research Associateship at the USAISR. This work was supported by U.S. Army Clinical Rehabilitative Medicine Research Program (CRMRP) and Military Operational Medicine Research Program (MOMRP).
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| mTeSR1 media + 5X supplement | Stem Cell Technologies | 5850 | |
| Y-27632 (Rock Inhibitor) | Stem Cell Technologies | 72304 | |
| DMEM/F12 | Life Technologies | 11330-032 | |
| 2-Mercaptoethanol | Sigma | M-7154 | |
| Non essential amino acids | Hyclone(Fisher) | SH30853.01 | |
| Knockout serum replacement | Life Technologies | 10828-028 | |
| Gentamicin | Life Technologies | 15750-060 | |
| L-Glutamine | Life Technologies | 25030-081 | |
| MEM media | Life Technologies | 10370-021 | |
| N1 supplement | Sigma | N-6530-5ML | |
| Taurine | Sigma | T-8691-25G | |
| Hydrocortisone | Sigma | H0888-1G | |
| Fetal bovine serum | Hyclone(Fisher) | SH3008803HI | |
| Triiodo-l-thyronine sodium salt | Sigma | T6397 | |
| Sodium hydroxide | Sigma | S5881 | |
| Dispase | Life Technologies | 17105-041 | |
| Matrigel | BD Biosciences | 354277 | |
| Phosphate buffered saline | Hyclone(Fisher) | 10010-023 | |
| Aggrewell 400 plate | Stem Cell Technologies | 27940 | |
| AggreWell medium | Stem Cell Technologies | 5893 | |
| Accutase | Stem Cell Technologies | 7920 | |
| BD Cytofix/Cytoperm Fixation/Permeabilization Kit | BD Biosciences | 554714 | |
| Mouse Anti-PAX6 antibody | Developmental Studies Hybridoma Bank | ||
| Rabbit Anti- RX antibody | Abcam | Ab23340 | |
| Mouse Anti-MITF antibody | Thermo Scientific | MS-772-P | |
| Rabbit Anti-ZO-1 antibody | Invitrogen | 40-2200 | |
| RNeasy plus mini kit | Qiagen | 74134 | |
| PCR master mix | promega | M7502 | |
| High capacity RNA to c DNA kit | Life Technologies | 4387406 | |
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