高效的小鼠胚胎干细胞分化成运动神经元
Summary
我们开发了一个新的协议，以提高效率，在体外分化成运动神经元的小鼠胚胎干细胞。分化的胚胎干细胞获得运动神经元功能的神经元和运动神经元采用免疫组化技术标记表达证明。
Abstract
功能运动神经元分化成胚胎干细胞（ES细胞）的直接代表一个有前途的资源，研究疾病机理，筛选新的药物化合物，并制定，如脊肌萎缩症（SMA）和肌萎缩性侧索硬化症的运动神经元疾病的新疗法（ ALS）的。目前许多协议使用维甲酸（RA）和刺猬（SHH）的组合分化成运动神经元1-4小鼠胚胎干细胞（MES）。然而，mES细胞分化成运动神经元的效率只达到中等成功。我们已经开发了一个两步分化协议5，显着提高目前建立的协议相比，分化效率。第一步是Noggin与成纤维细胞生长因子（FGFs）的加入，以加强neuralization过程。 Noggin与骨形态发生蛋白（BMP）受体拮抗剂和被牵连在神经诱导1ccording神经发生和结果形成前神经图案6默认模型。 FGF信号协同诱导神经组织的形成，促进后神经身份7-9 Noggin的行为。在这一步，mES细胞被引两天，以促进对神经谱系分化与Noggin的，碱性成纤维细胞生长因子，FGF-8。第二步是诱发运动神经元的规范。 Noggin与/ FGFs暴露mES细胞培养与RA和Shh的激动剂，理顺激动剂（凹陷），再过5天，以促进运动神经元生成。为了监测乱七八糟的分化成运动神经元，我们使用胚胎干细胞系，从转基因小鼠表达EGFP派生控制下运动神经元特异性启动子HB9 1。使用这个强大的协议，我们实现了51±0.8％的分化效率（N = 3，P <0.01，学生t-检验）5。从免疫的结果染色表明，绿色荧光蛋白+细胞表达运动神经元的特异性标志物，胰岛-1和胆碱乙酰转移酶（CHAT）。我们两步分化协议mES细胞分化成脊髓运动神经元提供了一条有效途径。
Protocol
Discussion
mES细胞的质量是最关键的参数为运动神经元的高效发电。 mES细胞必须培养，对PMEF防止自发分化。此外LIF的帮助mES细胞保持在未分化状态。由于MES细胞分裂每12至15小时，培养基变得枯竭迅速，必须每天更换。
高效活化Shh的途径是一个关键的参数，需要引起运动神经元的规范。 Shh蛋白（研发体系），Shh信号激动剂，如HH激动剂HhAg1.3（Curis公司），purmorphamine（Calbiochem公司），?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这手稿是致力于到文澜王博士的记忆2011年05月26日，通过了。我们感谢道格拉斯A·克尔博士慷慨提供的HBG3 MES在这项研究中使用的细胞。这项工作是由内穆尔，赠款（2 RR016472-10）根据国家研究资源中心（NCRR），和1 COBRE补助金从NCRR（5 P20的RR020173-05），以支持中心的INBRE方案儿童，美国特拉华州威尔明顿，在阿尔弗雷德一，杜邦医院的儿科研究。
Materials
| Name of the reagent | Company | Catalogue number | Final concentration |
| PMEF | Millipore | PMEF-H | N.A. |
| PMEF medium | |||
| DMEM | Invitrogen | 11965-118 | N.A. |
| FBS | Invitrogen | 16140-071 | 10% |
| L-glutamine | Invitrogen | 25030-081 | 1% |
| Penicillin/streptomycin | Invitrogen | 15240-063 | 1% |
| mES medium | |||
| DMEM | StemCell Technologies | 36250 | N.A. |
| ES Cell-Qualified FBS | Invitrogen | 16141-079 | 15% |
| GlutaMax-I | Invitrogen | 35050-061 | 1% |
| Non-essential amino acids | Invitrogen | 11140-050 | 1% |
| Nucleosides | Millipore | ES-008-D | 1% |
| β-mercaptoethanol | Millipore | ES-007-E | 0.1 mM |
| Penicillin/streptomycin | Millipore | TMS-AB2-C | 1% |
| LIF | Millipore | LIF2010 | 10 ng/ml |
| Neural Differentiation medium | |||
| DMEM | StemCell Technologies | 36250 | N.A. |
| ES Cult FBS | StemCell Technologies | 06905 | 15% |
| Non-essential amino acids | Invitrogen | 11140-050 | 1% |
| Mono-thio glycerol | Sigma-Aldrich | M-6145 | 1mM |
| Noggin | Invitrogen | PHC1506 | 50 ng/ml |
| FGF-8 | Invitrogen | PHG0274 | 20 ng/ml |
| bFGF | Invitrogen | PHG0024 | 20 ng/ml |
| MND medium (differentiation) | |||
| ES-Cult Basal Medium-A | StemCell Technologies | 5801 | N.A. |
| Knockout serum replacement | Invitrogen | 10828-028 | 10% |
| N-2 supplement | Invitrogen | 17502-048 | 1% |
| ITS Supplement-B | StemCell Technologies | 07155 | 1% |
| Ascorbic acid | StemCell Technologies | 07157 | 1% |
| Penicillin/streptomycin | Millipore | TMS-AB2-C | 1% |
| GlutaMax-I | Invitrogen | 35050-061 | 1% |
| D-glucose | Sigma-Aldrich | G-8270 | 0.15% in d2H2O |
| Fibronectin | StemCell Technologies | 07159 | 5 μg/ml |
| Heparin | Sigma-Aldrich | H3149 | 20 μg/ml in d2H2O |
| β-mercaptoethanol | Millipore | ES-007-E | 0.1 mM |
| Retinoic Acid | Sigma-Aldrich | R-2625 | 1 μM |
| SAG | EMD Chemicals | 566660 | 1 μM |
| MND medium (Motor Neuron culture) | |||
| ES-Cult Basal Medium-A | StemCell Technologies | 5801 | N.A. |
| Knockout serum replacement | Invitrogen | 10828-028 | 10% |
| N-2 supplement | Invitrogen | 17502-048 | 1% |
| ITS Supplement-B | StemCell Technologies | 07155 | 1% |
| Ascorbic acid | StemCell Technologies | 07157 | 1% |
| Penicillin/streptomycin | Millipore | TMS-AB2-C | 1% |
| GlutaMax-I | Invitrogen | 35050-061 | 1% |
| D-glucose | Sigma-Aldrich | G-8270 | 0.15% in d2H2O |
| Fibronectin | StemCell Technologies | 07159 | 5 μg/ml |
| Heparin | Sigma-Aldrich | H3149 | 20 μg/ml in d2H2O |
| β-mercaptoethanol | Millipore | ES-007-E | 0.1 mM |
| BDNF | R&D Systems | 248-BD-005/CF | 10 ng/ml |
| CNTF | R&D Systems | 257-NY-010/CF | 10 ng/ml |
| GDNF | R&D Systems | 212-GD-010/CF | 10 ng/ml |
| NT-3 | R&D Systems | 267-N3-005/CF | 10 ng/ml |
N.A. = Non-applicable.
Table 1. PMEF and Media for mES cell culture or differentiation.
| Name of the reagent | Company | Catalogue number | Final Concentration |
| 0.1% Gelatin | StemCell Technologies | 07903 | N.A. |
| Poly-DL-ornithine | Sigma-Aldrich | P0421 | 0.1 mg/ml in d2H2O |
| Mouse laminin | Millipore | CC095 | 2 μg/ml in PBS |
| 0.25% Trypsin/EDTA | StemCell Technologies | 07901 | N.A. |
| Accumax | Millipore | SCR006 | N.A. |
N.A. = Non-applicable.
Table 2. Reagents for coating and dissociation.
Riferimenti
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