骨髓衍生祖细胞的低氧预处理作为成熟雪旺细胞产生的来源
Summary
具有神经电位的骨髓基质细胞（MSC）存在于骨髓内。我们的方案通过缺氧预处理丰富了这种细胞群，之后引导他们成为成熟的雪旺氏细胞。
Abstract
该手稿描述了一种从骨髓基质细胞（MSC）群体中丰富神经祖细胞并随后将其引导至成熟的施万细胞命运的手段。我们将大鼠和人类MSC接受短暂的缺氧条件（1％氧气16小时），随后在含有表皮生长因子（EGF）/碱性成纤维细胞生长因子（bFGF）补充的低附着底物上扩增为神经球。将神经球接种到聚-D-赖氨酸/层粘连蛋白包被的组织培养塑料上，并在含有β-Heregulin，bFGF和血小板衍生生长因子（PDGF）的胶质细胞混合物中培养以产生施旺细胞样细胞（SCLC）。 SCLC通过共同培养2周，以E14-15怀孕的Sprague Dawley大鼠获得的纯化的背根神经节（DRG）神经元为指导。成熟的雪旺氏细胞表现出S100β/ p75表达的持续性，并可形成髓鞘细胞。以这种方式生成的细胞具有潜力脊髓损伤后自体细胞移植中的应用，以及疾病建模。
Introduction
神经祖细胞及其衍生物的移植显示出创伤神经损伤1,2和神经变性3,4之后的治疗策略的希望。在临床应用之前，必须确保：i）获取和扩大自体来源的干/祖细胞的方法，以及ii）将其引导到相关的成熟细胞类型的手段3 。我们对细胞治疗脊髓损伤的兴趣导致我们从成人组织中寻找一个健壮的自体细胞来源的神经祖细胞。
MSCs的亚群源自神经嵴，并且容易从骨髓腔进入。这些细胞是可以产生神经元和神经胶质的神经祖细胞5 。脑缺血的动物模型表明，缺氧促进了唾液脑内神经祖细胞的多发性和多能性6 。这是利用缺氧预处理作为扩展骨髓来源的神经祖细胞的手段的基础。
将施万细胞移植到损伤的脊髓中促进再生2 。 SCLC可以通过补充Gliogenic因子（ 即， β-Heregulin，bFGF和PDGF-AA）从MSC产生，但表现出表型不稳定性。退出生长因子后，它们恢复成为成纤维细胞样表型7 。由于异常分化和致癌的风险，细胞移植中的表型不稳定性是不合需要的。由于施旺细胞前体与胚胎周围神经8内的轴突束相关，我们被引导与纯化的胚胎DRG神经元共培养SCLC 7 ，ass =“xref”> 9。结果成熟的雪旺氏细胞是命运决定的，并在体外证明功能7,9和体内 10 。
我们用于富集MSC的神经祖细胞的方案是简单和有效的，导致后续测定的细胞数量增加。通过共培养平台推导命运致命的雪旺氏细胞允许研究胶质细胞分化和产生用于潜在临床应用的稳定和功能的施万细胞。
Protocol
Representative Results
Discussion
在通过缺氧预处理和神经球培养富集神经祖细胞之前，必须保持MSC的“干性”。根据我们的经验，可以通过其细长的成纤维细胞样形态可靠地鉴定多能MSC。相比之下，采用更扁平，四边形形态，具有突出的细胞骨架应力纤维的MSC不容易采用神经细胞命运，应该被丢弃。一般来说，我们不使用通道数大于8的MSC。为了保持它们的干性，在达到100％汇合之前及时通过MSC是至关重要的。相反，保持MSC在一…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者衷心感谢王培acknowledge博士提供的缺氧室仪器，以及Alice Lui女士的技术支持。
Materials
| αMEM | Sigmaaldrich | M4526 | |
| DMEM/F12 | Thermofisher scientific | 12400-024 | |
| Neurobasal medium | Thermofisher scientific | 21103-049 | |
| FBS | Biosera | FB-1280/500 | |
| B27 | Thermofisher scientific | 17504-001 | |
| Epidermal growth factor (EGF) | Thermofisher scientific | PHG0313 | |
| Basic fibroblast growth factor (bFGF) | Peprotech | 100-18B/100UG | |
| Nerve growth factor (NGF) | Millipore | NC011 | |
| Platelet-derived growth factor-AA (PDGF-AA) | Peprotech | 100-13A | |
| Heregulin beta-3, EGF domain (β-Her) | Millipore | 01-201 | |
| Uridine | Sigmaaldrich | U3003 | |
| 5-Fluro-2' – deoxyuridine (FDU) | Sigmaaldrich | F0503 | |
| Poly-D-lysine (PDL) | Sigmaaldrich | P7886-1G | |
| Laminin | Thermofisher scientific | 23017015 | |
| GlutaMAX | Thermofisher scientific | 35050061 | |
| Penicillin / streptomycin (P/S) | Thermofisher Scientific | 15140-122 | |
| TrypLE Express | Thermofisher Scientific | 12604-013 | |
| 10 cm plate for adherent culture | TPP | 93100 | Used for selection of MSCs by tissue culture adherence |
| 6-well plate for adherent culture | TPP | 92006 | Used for expansion of MSCs following passaging |
| UltraLow 6-well plate for non-adherent culture | Corning | 3471 | Used for neural progenitor enrichment |
| anti-human CD90(Thy-1) | BD Biosciences | 555593 | |
| anti-human CD73 | BD Biosciences | 550256 | |
| anti-human/rat STRO-1 | R&D Systems | MAB1038 | |
| anti-human nestin | R&D Systems | MAB1259 | |
| anti-human CD45 | BD Biosciences | 555480 | |
| anti-rat CD90(Thy-1) | BD Biosciences | 554895 | |
| anti-rat CD73 | BD Biosciences | 551123 | |
| anti-rat nestin | BD Biosciences | MAB1259 | |
| anti-rat CD45 | BD Biosciences | 554875 | |
| Anti-S100β | Dako | Z031101 | |
| Anti-p75 | Millipore | MAB5386 | |
| Anti-GFAP | Sigmaaldrich | G3893 | |
| Anti-Class III-beta tubulin (Tuj-1) | Covance | MMS-435P | |
| Anti-Human nuclei | Millipore | MAB1281 | |
| Hypoxia chamber | Billups-Rothenberg | MIC-101 | |
| HEPES buffer | Sigmaaldrich | H4034-100G |
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