小鼠胚胎干细胞中神经祖细胞和神经元的分化与表征
Summary
我们描述了使用悬挂滴法将小鼠胚胎干细胞体外分化为神经元细胞的过程。此外，我们通过RT-qPCR，免疫荧光，RNA-seq和流式细胞术进行全面的表型分析。
Abstract
我们描述了将小鼠胚胎干细胞培养和分化为神经元谱系的分步程序，然后进行一系列测定以表征分化细胞。利用E14小鼠胚胎干细胞通过悬挂滴法形成胚体，然后通过视黄酸诱导分化为神经祖细胞，最后分化成神经元。定量逆转录聚合酶链反应（RT-qPCR）和免疫荧光实验表明，神经祖细胞和神经元分别在分化后第8天和第12天表现出相应的标志物（神经祖的巢巢蛋白和神经元的神经丝）。在表达Sox1启动子驱动的GFP报告基因的 E14 系上进行流式细胞术实验表明，第8天约60%的细胞为GFP阳性，表明该阶段神经祖细胞成功分化。最后，使用RNA-seq分析来分析全局转录组学变化。这些方法可用于分析特定基因和途径在神经元分化过程中调节细胞身份转变的参与。
Introduction
自从它们第一次从发育中的小鼠囊胚1、2的内细胞质量中衍生出来以来，小鼠胚胎干细胞（mESC）已被用作研究干细胞自我更新和分化的强大工具3。此外，研究mESC分化导致对分子机制的深刻理解，这些机制可以提高基于干细胞的治疗治疗神经退行性疾病等疾病的效率和安全性4。与动物模型相比，这种体外系统具有许多优点，包括实践和评估的简单性，与动物相比，维持细胞系的成本较低，以及遗传操作的相对容易性。然而，分化细胞类型的效率和质量往往受到不同的mESCs系以及分化方法5，6的影响。此外，评估分化效率的传统检测依赖于对所选标记基因的定性检查，这些标记基因缺乏稳健性，因此无法掌握基因表达的全局变化。
在这里，我们的目标是使用一系列测定来系统地评估神经元分化。使用对所选标记物和RNA-seq的传统体外分析，我们建立了一个平台来测量分化效率以及在此过程中的转录组学变化。基于先前建立的方案7，我们通过悬挂滴技术产生类胚体（EB），然后使用超生理量的视黄酸（RA）诱导产生神经祖细胞（NPC），随后用神经诱导培养基分化为神经元。为了检查分化的效率，除了传统的RT-qPCR和免疫荧光（IF）测定外，我们还进行了RNA-seq和流式细胞术。这些分析提供了对阶段特异性分化进展的全面测量。
Protocol
1. 微微微网络培养 用0.1%明胶涂覆10厘米组织培养处理的平板，让明胶凝固至少15-30分钟，然后再吸出。 在预热的mESC培养基（Dulbecco的改性鹰培养基（DMEM）中培养15%胎牛血清（FBS），非必需氨基酸，β巯基乙醇，L-谷氨酰胺，青霉素/链霉素，丙酮酸钠，LIF，PD0325901（PD）和Chir99021（CH））中培养mESCs之前一天，种子γ辐照小鼠胚胎成纤维细胞（MEFs）。 在培养E14细胞之前，允许γ…
Representative Results
作为我们方法的代表，我们在E14细胞上进行了EB，NPC和神经元分化实验。E14细胞在γ辐照的MEF上培养（图1A），直到γ辐照的MEF群体被稀释。我们通过对Nanog和Oct4标记物进行碱性磷酸酶（AP）染色（图1B）和后来的RT-qPCR（见下文）证实了E14细胞的多能性。然后使用图2A中概述的方案诱导γ照射的不含MEF的E14细胞进行分化?…
Discussion
小鼠胚胎干细胞的神经分化方法已经建立了几十年，研究人员继续修改以前的方案或为各种目的创建新的方案7，10，11。我们利用一系列检测方法，综合分析了mESCs对神经元分化阶段的效率和进展，可用于分析小鼠或人类ESC的其他谱系分化。此外，我们的方法已被证明是评估特定基因或途径对体外神经元分化的影响的有用工具<s…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH（1R35GM133496-01）对Z. Gao的资助。我们要感谢Ryan Hobbs博士在切片方面的帮助。我们感谢宾夕法尼亚州立大学医学院的核心设施，包括基因组科学和生物信息学，高级光学显微镜成像和流式细胞术。我们还感谢 Yuka Imamura 博士在 RNA-seq 分析方面的帮助。
Materials
| 0.05% Trypsin + 0.53mM EDTA 1X | Corning | 25-052-CV | |
| 0.1% Gelatin | Sigma | G1890-100G | Prepared in de-ionized water |
| 16% Paraformaldehyde | Thermo Scientific | 28908 | Diluted in 1X PBS |
| 40-μm cell strainer | Falcon | 352340 | |
| Albumax | Thermo Fisher Scientific | 11020021 | |
| AlexaFluor 488 goat anti-mouse IgG (H+L) | Invitrogen | A11001 | Antibody was diluted at 1:500 for IF |
| Alkaline Phosphatase Staining Kit II | Stemgent | 00-0055 | |
| AzuraQuant Green Fast qPCR Mix LoRox | Azura Genomics | AZ-2105 | |
| B27 supplement | Thermo Fisher Scientific | 17504044 | |
| BD FACSCanto | BD | 657338 | |
| bFGF | Sigma | 11123149001 | |
| BioAnalyzer High Sensitivity DNA Kit | Agilent | 5067-4626 | |
| Chir99021 | Cayman Chemicals | 13122 | |
| Chloroform | C298-500 | Fisher Chemical | |
| DAPI | Invitrogen | R37606 | |
| DMEM | Corning | 10-017-CM | |
| DMEM/F12 medium | Thermo Fisher Scientific | 11320033 | |
| EB buffer | Qiagen | 19086 | |
| Ethanol | 111000200 | Pharmco | Diluted in de-ionized water |
| Fetal bovine serum | Atlanta Biologicals | S10250 | |
| Fisherbrand Superfrost Plus Microscope Slides | Fisher Scientific | 12-550-15 | |
| HiSeq 2500 Sequencing System | Illumina | SY-401-2501 | |
| Isopropanol | BDH1133-4LG | BDH VWR Analytical | Diluted in de-ionized water |
| L-glutamine | Thermo Fisher Scientific | 25030024 | |
| LIF | N/A | N/A | Collected from MEF supernatant |
| m18srRNA primers | IDTDNA | N/A | 5'-GCAATTATTCCCCATGAACG-3' 5'-GGCCTCACTAAACCATCCAA-3' |
| MEM Non-essential amino acids | Corning | 25-025-Cl | |
| mNanog primers | IDTDNA | N/A | 5'-AGGCTTTGGAGACAGTGAGGTG-3' 5'-TGGGTAAGGGTGTTCAAGCACT-3' |
| mNes primers | IDTDNA | N/A | 5'-AGTGCCCAGTTCTAGTGGTGTCC-3' 5'-CCTCTAAAATAGAGTGGTGAGGGTTG-3' |
| mNeuroD1 primers | IDTDNA | N/A | 5'-CGAGTCATGAGTGCCCAGCTTA-3' 5'-CCGGGAATAGTGAAACTGACGTG-3' |
| mOct4 primers | IDTDNA | N/A | 5'-AGATCACTCACATCGCCAATCA-3' 5'-CGCCGGTTACAGAACCATACTC-3' |
| mPax6 primers | IDTDNA | N/A | 5'-CTTGGGAAATCCGAGACAGA-3' 5'-CTAGCCAGGTTGCGAAGAAC-3' |
| N2 supplement | Thermo Fisher Scientific | 17502048 | |
| Nestin primary antibody | Millipore | MAB5326 | Antibody was diluted at 1:200 for IF |
| Neural basal | Thermo Fisher Scientific | 21103049 | |
| Neurofilament primary antibody | DSHB | 2H3 | |
| NEXTflex Illumina Rapid Directional RNA-Seq Library Prep Kit | BioO Scientific | NOVA-5138-07 | |
| PD0325901 | Cayman Chemicals | 13034 | |
| Penicillin/streptomycin | Corning | 30-002-Cl | |
| Phosphate-buffered saline (PBS) | N/A | N/A | Prepared in de-ionized water |
| – Potassium chloride | P217-500G | VWR | |
| – Potassium phosphate monobasic anhydrous | 0781-500G | VWR | |
| – Sodium chloride | BP358-10 | Fisher Bioreagents | |
| – Sodium phosphate, dibasic, heptahydrate | SX0715-1 | Milipore | |
| Random hexamer primer | Thermo Scientific | SO142 | |
| Retinoic acid | Sigma | R2625 | Prepared in DMSO |
| Sodium pyruvate | Corning | 25-000-Cl | |
| Sucrose | Sigma | 84097 | Diluted in 1X PBS |
| SuperScript III Reverse Transcriptase | Invitrogen | 18064022 | |
| Tissue-Tek O.C.T. compound | Sakura | 4583 | |
| TriPure Isolation Reagent | Sigma-Aldrich | 11667165001 | |
| TruSeq Rapid | Illumina | 20020616 | |
| β-mercaptoethanol | Fisher BioReagents | BP176-100 |
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Cite This Article
Hanafiah, A., Geng, Z., Wang, Q., Gao, Z. Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells. J. Vis. Exp. (159), e61446, doi:10.3791/61446 (2020).