人类神经干细胞从外周血造血祖细胞直接诱导
Summary
的方法的开发是为了直接从外周血细胞富集的造血祖细胞衍生的人神经干细胞。
Abstract
人类疾病的特定的神经元的文化是产生的体外模型人类神经系统疾病至关重要。然而，由于缺乏获得初级成人神经文化提出了独特的挑战。在诱导多能干细胞的最新发展（IPSC）提供了另一种方法，通过患者特定的iPSC以导出从皮肤成纤维细胞的神经培养物，但是这个过程是劳动密集的，需要特殊的专业知识和大量资源，并且可能需要数月。这可以防止该技术的广泛应用，以神经系统疾病的研究。为了克服其中的一些问题，我们已经开发出一种方法，可直接从人成人外周血衍生的神经干细胞，从而绕过的iPSC推导过程。从成人外周血富集造血祖细胞在体外培养，并用含有转录因子Sox2的，十月仙台病毒载体3/4，Klf4和c-Myc的。转染的结果，其中通过使用人神经祖介质含有碱性成纤维细胞生长因子（bFGF）和血管内皮生长因子（VEGF）进一步选择在细胞形态学变化。将得到的细胞的特征在于为神经干细胞标记物，如巢蛋白和Sox2的表达。这些神经干细胞可进一步分化为神经元，星形胶质细胞和在指定的分化培养基少突胶质细胞。使用便利的人外周血样品中，该方法可以被用来推导神经干细胞进一步分化为神经细胞的体外模型神经障碍并可以前进与这些疾病的发病机制和治疗的研究。
Introduction
在体外的神经元培养物已被用作用于神经性疾病的研究中的基本工具。主动物（大多数啮齿类）神经培养1,2和从胶质瘤或其他肿瘤衍生的人神经细胞系是最常用的此类研究。但是，人们已经认识到，有啮齿动物和人类细胞间显著差异。基于啮齿动物的许多研究结果不能被转换为人类。此外，在分析海量基因组信息和比较容易的基因编辑和全基因组测序的迅速发展，这种趋势越来越侧重发现疾病容易发生基因和划分职能作用的特定疾病，这使得一些人的神经细胞系只有有限的用法。从理论上说，从病人的神经系统的样品得出人类的主要神经文化是最好的选择，但他们都无法获得;因此替代甲基ODS是必要的。近年来，一些方法已被追求的，有两个是最区分。下列产生诱导性多能干细胞（IPSC），使用小鼠和人类体细胞的技术的发展3，4，神经细胞可进一步分化，从他们5-7。然而，产生和表征IPSC的要求密集型劳动，技术和时间的输入，有时甚至令人望而却步。不久之后，另一种方法是开发可直接由体细胞8,9变换神经元细胞。作为所得到的神经元都是非增殖性，它限制了它在深入细致的研究和药物筛选，这需要大量的细胞的应用。取这两种技术，神经干的直接推导的优点/由体细胞祖细胞已探索由几组10-12，它绕过的iPSC产生和表征，但仍PROVI的繁琐过程DES神经干细胞后神经分化的一个体面的数量。我们以前曾表明，在采用新的山中转录因子的成造血祖细胞，神经干细胞可以直接使用神经祖细胞在选择培养基中13产生。这里，我们报告了详细的方法。
Protocol
Representative Results
Discussion
提供了一种详细的协议，以直接产生从外周造血祖细胞的神经干细胞。
相比成纤维细胞，人外周血是更容易获得。使用所提出的协议，大于1×10 5个造血祖细胞，或CD34阳性细胞中，可以在10毫升的全血被富集。虽然污染血小板通常不是可预防的，它可以容易地通过低速离心降低，它不与神经干细胞的产生干扰。然而，CD34 +细胞的质量和数量将决定神经干细胞的最终生?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors have no conflicts of interest to disclose.
Materials
| Material List | |||
| Name | Company | Catalog Number | Comments |
| Lymphocyte Separation Medium | Lonza | 17-829E | 1 X |
| SepMate | Stemcell Technologies | 15415 | 15 mL |
| Human Serum | Invitrogen | 34005-100 | |
| Antibiotics | Gibco | 15240-062 | 1% |
| CD34 MultiSort Kit | Miltenyi Biotec | 130-056-701 | |
| EDTA | Cellgro | 46-034-Cl | 2mM |
| MACS LS Column | Miltenyi Biotec | 130-042-401 | |
| StemSpan SFEM Medium | Stemcell Technologies | 9650 | 1X |
| IMDM | Quality Biologicals | 112-035-101 | 1X |
| TPO | Peprotech | 300-18 | 100 ng/mL |
| Flt-3 | Peprotech | 300-19 | 100 ng/mL |
| SCF | Peprotech | 300-07 | 100 ng/mL |
| IL-6 | Peprotech | 200-06 | 20 ng/mL |
| IL-7 | Peprotech | 200-07 | 20 ng/mL |
| IPS Sendai Reprogramming Kit | Life technologies | A1378001 | |
| Cell scraper | Sarstedt | 83.183 | |
| DMSO | Sigma | D2650 | |
| CryoTube vials | Thermo | 368632 | |
| Mr. Frosty container | Thermo | 5100-0001 | |
| DMEM/F12 | Life technologies | 12400-024 | 1X |
| N2 supplement | Life technologies | 17502-048 | 1X |
| Bovine serum albumin | Sigma | A2934 | 0.1% (w/v) |
| bFGF | Peprotech | 100-18B | 20 ng/ml |
| EGF | Peprotech | AF-100-15 | |
| B27 supplement | Life technologies | 17504-044 | 1X |
| NSC serum free medium | Life Technologies | A1050901 | 1X |
| Poly-D-lysine/laminin coated cover slips | BD Bioscences | 354087 | |
| cAMP | Sigma | A9501 | 300 ng/ml |
| Vitamin C | Sigma | A0278 | 0.2 mM |
| BDNF | Peprotech | 450-02 | 10 ng/ml |
| GDNF | Peprotech | 450-10 | 10 ng/ml |
| Poly-L-Ornithine | Sigma | P4957 | 1X |
| PDGF-AA | Peprotech | 100-13A | 10 ng/ml |
| NT-3 | Peprotech | 450-03 | 2 ng/ml |
| Shh | Peprotech | 1314-SH/CF | 2 ng/ml |
| T3 | Sigma | T6397 | 3 nM |
| PFA | Sigma | P6148 | 4% |
| PBS | Quality Biological | 119-069-101 | 1X |
| Goat serum | Sigma | G9023 | 4% |
| TritonX-100 | Sigma | T9284 | |
| Mouse monoclonal anti-Nestin | Millipore | AB5922 | 1:1000 dilution |
| Anti-SOX2 antibody | Applied Stemcell | ASA0120 | Ready to use |
| Mouse anti-βIII-tubulin antibody | Promega | G712A | 1:1000 dilution |
| Rabbit anti-GFAP antibody | Sigma | G4546 | 1:100 dilution |
| Anti-O4 antibody | R&D Systems | MAB1326 | IgM; 1 ng/ml |
| Alexa Fluor 594 goat anti-rabbit antibody | Life techniologies | A11012 | 1:400 dilution |
| Alexa Fluor 488 goat anti-mouse antibody | Life techniologies | A11001 | 1:400 dilution |
| Alexa Fluor 488 goat anti-mouse IgM antibody | Life techniologies | A21042 | 1:250 dilution |
| DAPI | Sigma | D9542 | 1 ug/ml |
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