视网膜器官诱导系统，用于从人类多能干细胞中提取3D视网膜组织
Summary
在这里，我们描述了一个优化的视网膜器官诱导系统，它适用于各种人类多能干细胞系，以产生具有高可重复性和效率的视网膜组织。
Abstract
视网膜退行性疾病是无法逆转的失明的主要原因，没有有效的治疗。多能干细胞有可能分化成所有类型的视网膜细胞，甚至小视网膜组织，为这些疾病患者带来巨大的希望，在疾病建模和药物筛查方面也有很多机会。然而，从 hPSC 到视网膜细胞的诱导过程是复杂而耗时的。在这里，我们描述了一个优化的视网膜诱导协议，以产生具有高可重复性和效率的视网膜组织，适合各种人类多能干细胞。此协议执行时不添加视网膜酸，这有利于圆锥体光感受器的浓缩。本协议的优点是量化EB尺寸和电镀密度，显著提高视网膜诱导的效率和可重复性。使用这种方法，所有主要视网膜细胞依次出现并重新概括视网膜发育的主要步骤。它将促进下游应用，如疾病建模和细胞治疗。
Introduction
视网膜退行性疾病 （RD）， 如与年龄相关的黄斑变性 （AMD） 和视网膜炎色素 （RP）， 其特点是光感受细胞功能障碍和死亡， 通常导致不可逆转的视力丧失， 没有有效的方法来治愈1.这些疾病背后的机制在很大程度上是未知的，部分原因是缺乏人类疾病模型2。在过去的几十年里，通过干细胞技术在再生医学方面取得了重大进展。许多研究人员，包括我们自己，已经表明，人类多能干细胞（hPSCs），包括人类胚胎干细胞（hESCs）和人类诱导的多能干细胞（hiPSCs），可以通过各种分化方法3，4，5，6，7，8，9，10，分化成所有类型的视网膜细胞，甚至小视网膜组织，11，在疾病建模和细胞治疗方面提供巨大的潜力12，13，14。
然而，从 hPSC 到视网膜细胞的诱导过程非常复杂且耗时，可重复性低，这需要具有丰富经验和高技能的研究人员。在复杂和动态的诱导过程中，许多因素将影响视网膜组织15，16，17的产量。此外，不同的诱导方法往往在视网膜标记的正时和强健的表达上有很大的不同，这可能会混淆样本收集和数据解释3。因此，需要一个直接的视网膜分化协议，从hPSC与分步指导。
在这里，根据我们发表的研究18，19，20，21，一个优化的视网膜诱导协议，以产生视网膜器官（ROs）与丰富的锥形光感受器从hPSCs描述，这不需要视网膜酸（RA）的补充。此协议侧重于生成神经视网膜和 RPE 的多步骤方法的描述。EB 形成是早期诱导阶段的重要组成部分。BB的大小和电镀密度都经过定量优化，科学地提高了视网膜组织的产量，提高了可重复性。在感应的第二部分，光学囊泡（OVs）在依从文化和ROs形式中自我组织在悬浮文化中：这部分的时间课程和效率在不同的 hPSC 行中差异很大。ROs中视网膜细胞的成熟和规范主要发生在诱导的中后期。如果不添加 RA，可以生产具有丰富圆锥体和棒子的成熟感光器。
此协议的目的是定量描述和详细说明每个步骤，让缺乏经验的研究人员重复。通过此协议，各种 hPSC 线已成功诱导到 ROs 中，具有丰富的锥体视网膜组织的丰产和高可重复性。使用此协议的 HPSC 衍生 ROs 可以回顾 体内视网膜发育的主要步骤，并长期存活，从而促进下游应用，如疾病建模、药物筛选和细胞治疗。
Protocol
1. hPSC的文化和扩展 HPSC 文化 将两口 6 井板的油井涂上细胞外基质（ECM、hESC 合格矩阵）。在杜尔贝科的改良鹰介质 （DMEM） 中准备 50 mL 的 ECM 解决方案，其中包含 8-12 μg/mL 的 ECM。在 49 mL 的 DMEM 中，加入 1 mL 解冻的 ECM 库存解决方案 （50 倍）。在 6 井板的每个井中加入 1 mL 的 ECM 解决方案。在 37 °C 和 5% CO2的孵化器中孵育 1 小时。 根据制造商的说明准?…
Representative Results
本协议中的视网膜诱导过程模拟了人类胎儿视网膜的发展。为了启动视网膜分化，hPSC 被分离成小团块，并在悬浮中培养，以诱导 BB 的形成。在 D1 上，均匀的细胞聚合或 EB 形成 （图 1C）。文化媒介逐渐向NIM过渡。在 D5 上，EB 被镀在 ECM 涂层的文化菜肴上。细胞逐渐从EB中迁移出来（图1D）。从 D10 开始，眼场在粘附 BB 的外围区域自行组织。在 D16 上，?…
Discussion
在这个多步骤视网膜诱导协议中，hPSC 被引导一步一步地获得视网膜的命运，并自行组织成包含层压 NR 和 RPE 的视网膜器官。在分化过程中，hPSC回顾了人类视网膜 发育在体内的所有主要步骤，从EF、OV和RPE，到视网膜层压，生成视网膜细胞的所有亚型，包括视网膜结节细胞、单体细胞、双极细胞、棒状和锥形光感受器，以及空间和时间顺序的摩尔胶质细胞。视网膜发育的回顾将有利于下游?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究得到了中国国家重点研发计划（2016YFC110103，2017YFA0104101）、广州科技项目基金（201803010078）、广东省科技项目（2017B020230003）、中国自然科学基金（NSF）（81570874） 81970842）、中山大学百人计划（PT1001010）和国家眼科重点实验室基础研究基金。
Materials
| (−)-Blebbistatin | Sigma | B0560-5mg | ROCK-inhibitor |
| 1 ml tips | Kirgen | KG1313 | 1 ml |
| 10 ml pipette | Sorfa | 3141001 | Pipette |
| 100 mm Tissue culture | BIOFIL | TCD000100 | 100 mm Petri dish |
| 100 mm Tissue culture | Falcon | 353003 | 100 mm Petri dish |
| 15 ml Centrifuge tubes | BIOFIL | CFT011150 | Centrifuge tubes |
| 35 mm Tissue culture dishes | Falcon | 353001 | 35 mm Petri dish |
| 5 ml pipette | Sorfa | 313000 | Pipette |
| 50 ml Centrifuge tubes | BIOFIL | CFT011500 | Centrifuge tubes |
| 6 wells tissue culture plates | Costar | 3516 | Culture plates |
| Anti-AP2α Antibody | DSHB | 3b5 | Primary antibody |
| ANTIBIOTIC ANTIMYCOTIC 100X | Gibco | 15240062 | Antibiotic-Antimycotic |
| Anti-ISL1 Antibody | Boster | BM4446 | Primary antibody |
| Anti-Ki67 Antibody | Abcam | ab15580 | Primary antibody |
| Anti-L/M opsin Antibody | gift from Dr. jeremy | / | Primary antibody |
| Anti-PAX6 Antibody | DSHB | pax6 | Primary antibody |
| Anti-rabbit 555 | Invitrogen | A31572 | Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 |
| Anti-Recoverin Antibody | Millipore | ab5585 | Primary antibody |
| Anti-Rhodopsin Antibody | Abcam | ab5417 | Primary antibody |
| Anti-sheep 555 | Invitrogen | A21436 | Donkey anti-Sheep IgG (H+L) Secondary Antibody, Alexa Fluor 555 |
| Anti-SOX9 Antibody | Abclonal | A19710 | Primary antibody |
| Anti-VSX2 Antibody | Millipore | ab9016 | Primary antibody |
| B-27 supplement W/O VIT A (50X) | Gibco | 12587010 | Supplement |
| Cryotube vial | Thermo scientific-NUNC | 375418 | 1.8 ml |
| DAPI | DOJINDO | D532 | 4',6-Diamidino-2-phenylindole dihydrochloride; multiple suppliers |
| Dimethyl sulphoxide(DMSO) Hybri-max | Sigma | D2650-100ML | Multiple suppliers |
| DMEM | Gibco | C11995500BT | Medium |
| DMEM /F12 | Gibco | C11330500BT | Medium |
| EDTA | Invitrogen | 15575-020 | 0.5 M PH 8.0 |
| FBS | NATOCOR | SFBE | Serum |
| Filter | Millipore | SLGP033RB | 0.22μm, sterile Millex filter |
| GlutaMax, 100X | Gibco | 35050061 | L-alanyl-L-glutamine |
| Heparin | Sigma | H3149 | 2 mg/ml in PBS to use |
| Matrigel, 100x | Corning | 354277 | Extracellular matrix (ECM) |
| MEM Non-Essential Amino Acids Solution (100X) | Gibco | 11140050 | MEM NEAA |
| mTeSR1 | STEM CELL | 85850 | hPSCs maintenance medium (MM) |
| N2 supplement | Gibco | 17502048 | Supplement |
| Phosphate-buffered saline (PBS) buffer | GNM | GNM10010 | Without Ca+,Mg+,PH7.2±0.1 0.1M |
| Taurine | Sigma | T0625 | Supplement |
| Ultra-low attachment culture dishes 100mm petri dish, low-attachment | Corning | CLS3262-20EA | Petri dish |
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Cite This Article
Guan, Y., Xie, B., Zhong, X. Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells. J. Vis. Exp. (170), e62435, doi:10.3791/62435 (2021).