Summary

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

Published: June 14, 2017
doi:

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

所有涉及动物的手续均严格按照NIH“实验动物护理和使用指南”进行,并经香港大学李嘉诚医学院动物实验动物教学与研究委员会批准。获得知情同意后,从健康供体的髂嵴获得人骨髓样品。香港大学机构审查委员会批准议案。 1.大鼠MSC培养物的制备 从股骨收获MSC 在使用前,将所有的夹层工具( 即精细剪切剪刀,钝头切割剪刀和齿形钳子)…

Representative Results

我们的协议关键阶段的概述如图1所示 。总之,通过依靠组织培养塑料选择大鼠和人类MSC。扩张的MSC被缺氧预处理,然后经历神经球形成条件。电镀神经球,使其分化成SCLC。 SCLC与纯化的DRG神经元共培养以产生命运赋予的施旺细胞。 培养的大鼠和人类MSC的形态如图2所示 。显示出与?…

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…

Divulgazioni

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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Citazione di questo articolo
Tsui, Y., Mung, A. K., Chan, Y., Shum, D. K., Shea, G. K. Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells. J. Vis. Exp. (124), e55794, doi:10.3791/55794 (2017).

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