小鼠胚胎干细胞向皮质 Interneuron 前体细胞的分化
概要
本议定书描述了一种方法, 产生皮层 interneuron 祖细胞和后有丝分裂 interneuron 前体从小鼠胚胎干细胞使用改良的胚体-单层法。这些祖/前体可用于体外或荧光分类和移植到新生儿大脑皮层研究命运确定, 或用于治疗应用。
Abstract
gaba 皮质神经元是一种异构的细胞群体, 在调节兴奋性锥体神经元的输出以及同步锥体神经元集合的输出方面起着关键作用。interneuron 功能的缺陷已经牵连到各种神经精神疾病, 包括精神分裂症, 自闭症和癫痫。从胚胎干细胞衍生的皮质神经元不仅允许研究其发展和功能, 但提供了深入的分子机制的基础上, 皮层 interneuron 相关疾病的发病机理。神经元也有非凡的能力, 生存, 迁移, 并集成到宿主皮质电路后移植, 使他们成为理想的候选细胞的治疗方法。在这里, 我们提出了一个可伸缩, 高效, 改良的胚体的单层方法, 以 Nkx2.1 表达 interneuron 祖细胞和他们的后代从小鼠胚胎干细胞 (mESCs)。使用 Nkx2.1:: mCherry: Lhx6:: GFP 双报告制线、Nkx2.1 祖细胞或其 Lhx6-expressing 后有丝分裂后代, 可通过荧光活化单元分类 (资产管制系统) 隔离, 随后用于许多下游应用。我们还为发育 (PV) 或生长抑素 (SST) interneuron 亚群提供了丰富的方法, 这可能有助于研究命运的确定, 或用于治疗应用, 将受益于 interneuron 亚组丰富移植.
Introduction
在两个小鼠和人类中, 大约一半的皮质抑制神经元 (CIns) 起源于一个短暂的皮层下结构, 称为内侧节隆起 (MGE), 上皮祖和其他神经元和神经胶质子组表示转录因子 Nkx2.11,2。通过相交形态学、神经、电生理学和连通性特征3,4来定义这些子类型。MGE 衍生的 CIns 可以分为大多数非重叠子群的基础上, 其表达的 PV 或 SST, 其表达与特定的电生理和连接倾向5。神经元, 特别是 PV 亚群的功能障碍, 已牵连多神经精神疾病和疾病6,7。该方法的总目标是产生干细胞衍生的有丝分裂祖细胞和迁移前体, 丰富的 PV 或 SST 的命运, 研究皮质 interneuron 生物学和用于细胞基础治疗。
我们已经开发了一个可伸缩的, 高效的方法来推导 Nkx2.1 表达 interneuron 祖及其后代从 mESCs。使用 Nkx2.1:: mCherry: Lhx6:: GFP 双报告制线8, Nkx2.1 祖细胞或其 Lhx6-expressing 后有丝分裂后代可通过外地资产管制系统隔离, 随后用于若干下游应用。通过操纵一些信号通路、培养时间和神经再生模式, 我们可以获得数百万个荧光标记的 interneuron 前体, 适合于下游应用的宿主。
虽然存在一些其他方法来生成 MGE 样的祖 mESCs9,10,11,12,13,14, 我们的方法, 这依赖于 Wnt拮抗剂 XAV-939, 是特别有效的产生 Foxg1/Nkx2.1 co 表达 telencephalic 祖细胞。此外, 通过我们的双报告系统选择 interneuron 祖细胞或其后有丝分裂 Lhx6-expressing 后代的能力, 极大地提高了产生不同祖和后代的能力。
Protocol
注: 本协议中描述的双记者制线可根据要求提供 (sande@mail.med.upenn.edu sande@mail 大学)。 1. 媒体准备 注意: 在细胞培养使用前, 请将所有介质预热到37° c。 小鼠胚胎成纤维细胞 (MEF) 培养基 (准备500毫升) 添加50毫升胎牛血清 (FBS) 到449毫升 Dulbecco 氏改良鹰的培养基 (DMEM), 并通过500毫升0.22 µm 孔过滤器过滤器。 在过滤后加入1?…
Representative Results
本文所描述的协议是我们已发布协议的修改版本15,16,17 , 并已针对我们的 Nkx2.1 进行了优化:: mCherry: Lhx6:: GFP 双报告制线。通过添加 Wnt 抑制剂 XAV-939 从 DD0-5, 结合重新电镀在 DD8, 我们实现了鲁棒 Nkx2.1 诱导, 其中50% 以上的 DAPI + 核在文化中也 Nkx2.1 表达 (图 1A, B)。免疫组…
Discussion
虽然这种方法是非常有效的模式 J1-derived mESCs (SCRC-1010), 我们已经经历了可变的成功与其他制线和克隆分离株。例如, Foxg1::venus mESCs (EB3-derived;Danjo et al.13) 对此协议的响应很差, DD12 的 Foxg1 归纳通常按1-2% 的顺序进行。出于我们不完全理解的原因, 另一个 Nkx2.1:: mCherry: Lhx6:: GFP 双报告克隆 (称为 JQ59), 同时被隔离为该协议中描述的线 (称为 JQ27), 生长时产生少于 1% Nkx2.1 表达的细…
開示
The authors have nothing to disclose.
Acknowledgements
我们感谢 Nkx2.1 的发展:: mCherry: Lhx6: GFP 双重记者制线以及詹妮弗. 泰森、Maroof 和蒂姆. 彼得罗斯米为帮助开发这一协议而进行的早期工作。我们还感谢印章流式细胞仪的核心技术援助。这项工作得到了 NIH R01 MH066912 (SA) 和 F30 MH105045-02 (DT) 的支持。
Materials
| Bottle-top vacuum filter system | Corning | CLS430769 | |
| Test Tube with Cell Strainer Snap Cap | ThermoFisher | Corning 352235 | |
| Mouse embryonic fibroblasts (CF-1 MEF IRR 7M) | MTI-Globalstem | GSC-6101G | 1 vial of 7M MEFs is sufficient for four 10-cm TC plates. References: 29,35 |
| FBS | Atlanta Biologicals | S11150H | |
| Primocin | Invivogen | Ant-pm-2 | Also known as antimicrobial agent. Do not filter with base media — add after filtration. References: 9,11,36,37 |
| N2 supplement-B | Stemcell Technologies | 7156 | Do not filter with base media — add after filtration |
| Glutamax (100x) | ThermoFisher | 35050061 | Also known as L-alanine-L-glutamine. References: 9,11,38,39 |
| KnockOut Serum Replacement (KSR) | ThermoFisher | 10828028 | Also known as serum-free medium supplement. References: 9,11 |
| L-glutamine (100x) | ThermoFisher | 25030081 | |
| MEM-NEAA (100x) | ThermoFisher | 11140050 | |
| 2-Mercaptoethanol | ThermoFisher | 21985023 | |
| KnockOut DMEM | ThermoFisher | 10829018 | Also known as non-glutamine containing DMEM. References: 9,11 |
| Hyclone FBS | VWR | 82013-578 | Also known as stem cell grade FBS. References: 9,11 |
| Tissue culture treated dish (10cm) | BD Falcon | 353003 | |
| Non-adherent sterile petri dish (10cm) | VWR | 25384-342 | |
| Leukemia inhibitory factor (mLIF) | Chemicon | ESG1107 | Do not freeze, store at 4'C. References: 9,11 |
| DMEM/F12 | ThermoFisher | 11330032 | |
| 0.1% Gelatin Solution | ATCC | ATCC PCS-999-027 | |
| Laminin | Sigma | L2020 | |
| Poly-L-lysine | Sigma | P6282 | |
| Trypsin-EDTA (0.05%) | ThermoFisher | 25300054 | |
| Accutase | ThermoFisher | A1110501 | Also known as non-trypsin containing cell dissociation reagent. References: 9,11 |
| RQ1 RNase-Free DNase | Promega | M610A | |
| LDN-193189 | Stemgent | 04-0074 | Resuspend in DMSO and store at -80'C in single use aliquots |
| XAV939 | Stemgent | 04-0046 | Resuspend in DMSO and store at -80'C in single use aliquots |
| rhFGF-2 | R&D Systems | 233-FB | Resuspend in PBS with 0.1% BSA and store at -80'C in single use aliquots |
| rhIGF-2 | R&D Systems | 291-G1 | Resuspend in PBS with 0.1% BSA and store at -80'C in single use aliquots |
| ROCK inhibitor (Y-27632) | Tocris | 1254 | Resuspend in DMSO and store at -80'C in single use aliquots |
| Smoothened agonist (SAG) | Millipore | 566660-1MG | Resuspend in H20 and store at -80'C in single use aliquots |
| rm Sonic Hedgehog/SHH | R&D Systems | 464-SH-025 | Resuspend in PBS with 0.1% BSA and store at -80'C in single use aliquots |
| PKCζ Pseudosubstrate Inhibitor, Myristoylated | EMD Millipore | 539624 | Resuspend in H20 and store at -80'C in single use aliquots |
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