体外重建人视网膜母细胞瘤
Summary
我们描述了一种通过在人胚胎干细胞（hESC）中引入双等位基因RB1突变来产生人视网膜母细胞瘤（RB）的方法。RB细胞系也可以在培养皿中使用分离的RB成功培养。
Abstract
人类RB是小儿癌症，如果不进行治疗，它是致命的。由于RB起源于锥形前体，在啮齿动物模型中比较少见，同时关于人类与啮齿动物的种间差异，源自人类的疾病模型更有利于揭示人类RB的机制，寻找治疗目标。在此，该协议描述了两个基因编辑的hESC系的产生，分别具有双等位基因RB1点突变（RB1 Mut / Mut）和RB1敲除突变（RB1–/-）。在视网膜发育过程中，观察到RB的形成。RB细胞系也是通过从RB类器官中分离而建立的。总之，通过使用2D和3D组合分化方案将基因编辑的hESC系分化为视网膜类器官，我们已经成功地在培养皿中重建了人类RB并确定了其锥体前体起源。它将为观察视网膜母细胞瘤的发生、增殖和生长以及进一步开发新型治疗剂提供有用的疾病模型。
Introduction
人视网膜母细胞瘤（RB）是一种罕见的致命肿瘤，来源于视网膜视锥细胞前体1，2，3，是儿童期最常见的眼内恶性肿瘤类型4。RB1 基因的纯合失活是 RB5 的起始遗传病变。然而，具有RB1突变的小鼠无法形成视网膜肿瘤2。尽管小鼠肿瘤可以通过Rb1突变和其他基因修饰的组合产生，但它们仍然缺乏人类RB6的特征。由于视网膜类器官分化的发展，可以获得hESC衍生的RB，显示人类RB1的特征。
在过去的十年中，已经建立了许多用于视网膜类器官分化的方案，包括2D7，3D8以及2D和3D9的组合。这里用于生成人类RB的方法是粘附培养物和浮动培养物的合并9。通过将 RB1 突变的hESC分化为视网膜类器官，在第45天左右检测到RB的形成，然后在第60天左右迅速增殖。在第90天，可以分离RBs并生成RB细胞系;此外，RB在第120天包围几乎所有视网膜类器官。
hESC 衍生的 RB 是一种探索 RB 起源、肿瘤发生和治疗的创新模型。在该协议中，详细描述了基因编辑hESC的产生，RB的分化和RB的表征。
Protocol
Representative Results
Discussion
人视网膜母细胞瘤（RB）是由 RB1 失活和Rb蛋白功能障碍引起的。在该协议中， RB1-KO hESC是在培养皿中产生RB的关键步骤。虽然即使使用 RB1-/- hESC，但由于视网膜类器官分化的方法，也有可能没有RB形成10。在该协议中，从贴壁培养到漂浮培养的转移在分化过程中至关重要。囊肿的密度、多能干细胞的类型和增殖速率都是影响脱离时间的变量。当细胞扩?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢502团队的所有帮助。这项工作得到了北京市自然科学基金（Z200014）和国家重点研发计划（2017YFA0105300）的部分支持。
Materials
| 2-mercaptoethanol | Life Technologies | 21985-023 | |
| Anti-ARR3 | Sigma | HPA063129 | Antibody |
| Anti-CRX (M02) | Abnove | ABN-H00001406-M02 | Antibody |
| Anti-Ki67 | Abcam | ab15580 | Antibody |
| Anti-Syk (D3Z1E) | Cell Signaling Technology | 13198 | Antibody |
| BbsI | NEB | R3539S | Restriction enzymes |
| Dispase (1U/mL) | Stemcell Technologies | 7923 | |
| DMEM basic | Gibco | 10566-016 | |
| DMEM/F-12-GlutaMAX | Gibco | 10565-042 | |
| DMSO | Sigma | D2650 | |
| DPBS | Gibco | C141905005BT | |
| EDTA | Thermo | 15575020 | |
| Fetal Bovine Serum (FBS), Qualified for Human Embryonic Stem Cells | Biological Industry | 04-002-1A | |
| Glutamine | Gibco | 35050-061 | |
| Ham's F-12 Nutrient Mix (Hams F12) | Gibco | 11765-054 | |
| MEM Non-essential Amino Acid Solution (100X) | Sigma | M7145 | |
| Neurobasal Medium | Gibco | 21103-049 | |
| P3 Primary Cell 4D-Nucleofector X Kit S | Lonza | V4XP-3032 | Nucleofection kit |
| Pen Strep | Gibco | 15140-122 | |
| Puromycin | Gene Operation | ISY1130- 0025MG | |
| QIAquick PCR Purification Kit | QIAGEN | 28104 | |
| ncEpic-hiPSC/hESC culture medium | Nuwacell | RP01001 | ncEpic-hiPSC/hESC culture medium in 1.2.1 |
| Growth factor reduced basement membrane matrix | BD | 356231 | Matrigel in 1.2.1 |
| Cell dissociation enzyme | Gibco | 12563-011 | TrypLE Express in 1.2.8 |
| RNeasy Midi Kit | QIAGEN | 75144 | |
| RNeasy Mini Kit | QIAGEN | 74104 | |
| Supplement A | Life Technologies | 17502-048 | N-2 Supplement (100X), liquid, supplemet in medum I |
| Supplement B | Life Technologies | 17105-041 | B-27 Supplement (50X),liquid, supplemet in medum I,II,III |
| T4 Polynucleotide Kinase | Life Technologies | EK0032 | |
| Taurine | Sigma | T-8691-25G | |
| Y-27632 2HCl | Selleck | S1049 | |
| pX330-U6- Chimeric BB-CBh-hSpCas9-2A-Puro | Addgene | 42230 | |
| Nucleofector 4D | Lonza | ||
| RPMI | Sigma | R0883-500ML |
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