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Neuroscience

小鼠初级少突胶质的快速特异磁分离

Published: May 21, 2018
doi:
10.3791/57543
, ,
1Program in Molecular and Cellular Biology,State University of New York Downstate Medical Center, 2Department of Neurology and Rehabilitation,University of Illinois at Chicago

Summary

我们描述了主鼠标少突胶质的磁隔离, 它允许对体外区域性的细胞进行快速和特定的隔离。

Abstract

高效、健壮的分离和培养原少突胶质 (生命线)是体外研究 oligodendroglia 的重要工具, 也是脱髓鞘疾病 (如多发性硬化) 的生物学和Pelizaeus Merzbacher 样疾病 (PMLD)。在这里, 我们提出了一个简单而有效的选择方法, 磁隔离三期 O4+ preoligodendrocytes 细胞从新生小鼠幼崽。由于未成熟的 OL 在产后 7 (P7) 的啮齿动物脑白质中占80% 以上, 这种隔离方法不仅保证了高细胞的产量, 而且还对已经致力于 oligodendroglial 谱系的生命线的具体隔离, 减少了可能隔离污染的细胞, 如星形胶质细胞和其他电池从老鼠的大脑。这种方法是对以前报告的技术的修改, 并提供少突胶质的纯度在80% 以上约4小时。

Introduction

少突胶质 (生命线) 是中枢神经系统 (CNS)1 的 myelinating 细胞。在严格调节的环境中, 主少突胶质的分离和培养是体外研究 oligodendroglia 的重要工具, 也是脱髓鞘疾病 (如多发性硬化症) 的生物学基础2.这需要一个高效且健壮的少突胶质隔离和区域性方法3。在本研究中, 我们利用了一个独特的少突胶质细胞表面标记的表达, 以实现一个快速和具体的改进的隔离技术。

已经确定了四个不同的少突胶质成熟阶段, 每一个发育阶段都表现出独特的细胞表面标记(图 1)。这些单元格表面标记可以通过特定的抗体4、5识别, 并可用于在特定阶段隔离生命线。在第一阶段, 少突胶质前体细胞 (OPCs) 有能力增殖, 迁移, 并明确表达血小板衍生生长因子受体 (血小板 Rα)6, 苷 A2B5, 蛋白多糖NG27, 8, 聚唾液酸-神经细胞黏附分子9和脂肪酸结合蛋白 7 (FABP7) 10. OPCs 具有双极性形态学, 细胞体的对立两极产生的短过程很少, 这是神经前体细胞11的特征。

Figure 1
图 1: 在鼠标少突胶质开发过程中单元格表面标记的表达式。A2B5、GalC (O1)、NG2、O4 和血小板因子 Rα等生命线细胞表面标记物可用于在特定发育阶段用特定抗体分离少突胶质。  请单击此处查看此图的更大版本.

在第二阶段, OPCs 产生 preoligodendrocytes 和表达的细胞膜不仅 OPC 标记, 而且 sulfatide (硫酸 galactolipid) 承认 O4 抗体12,13, GPR17 蛋白14,一直持续到未成熟的少突胶质 (OL) 阶段。在这个阶段, preoligodendrocytes 扩展多极短过程。Preoligodendrocytes 是大鼠和鼠脑白质中 2 (P2) 的主要 OL 阶段, 其小种群为未成熟的生命线15。

在第三阶段, 未成熟的生命线继续表达 O4, 失去 A2B5 和 NG2 标记的表达, 并开始表达 galactocerebroside C16。在这个阶段, 苏丹生命线行动致力于 oligodendroglial 谱系, 成为后有丝分裂细胞与长分支分支17,18。未成熟的 OL 构成80% 以上的啮齿动物白色物质在 P7 和此时第一个 MBP+单元格被观察15,19,20,21。因此, 在 P7 中分离生命线能保证细胞高产。

在 OL 发育的最后和第四阶段, 成熟的 myelinating 蛋白 (髓鞘碱性蛋白 (MBP), proteolipid 蛋白 (), 髓鞘相关糖蛋白 (MAG) 和髓鞘少突胶质糖蛋白 (MOG) 22, 23 ,24,25,26。在这个阶段, 成熟的生命线延伸膜, 形成紧凑的包裹鞘围绕轴突和能够 myelinate。这与观察老鼠和老鼠大脑的情况一致, 在 P1419,20,21中, MBP+细胞变得越来越丰富。

自从 Fewster 和同事在1967年首次隔离少突胶质27 以来, 已实施了几种方法, 用于从啮齿动物中枢神经系统中分离生命线, 其中包括immunopanning28、29、30、荧光活化细胞分选 (资产管制署) 利用细胞表面特异抗原28,31, 差分梯度离心32,33,34,35和基于不同中枢神经胶质细胞差异粘附的震动方法36,37。然而, 现有的文化方法有局限性, 特别是在纯度、产量和执行程序38所需的时间方面。因此, 需要更有效的少突胶质隔离方法。

本文提出了一种简便、有效的选择方法, 用于磁三 O4+ preoligodendrocytes 细胞与新生小鼠幼崽的分离。此方法是对金刚砂et所报告的技术的修改。39和 Dincman et 。40 , 并提供在大约4小时以上80% 的少突胶质制备纯度。

Protocol

本研究中使用的小鼠根据纽约州立大学南部医学中心实验室动物资源 (DLAR) 协议15-10492 号的指导方针予以照顾。 注: 原少突胶质与新生儿 (P5-P7 野生型 C57Bl/6N) 小鼠分离。在这一阶段, 不成熟的生命线构成了80% 以上的啮齿动物白质, 确保高细胞产量。所有缓冲和试剂组合物都可在材料表的末尾提供。 1. 盖玻片准备 注: 聚 d-赖?…

Representative Results

本研究的目的是为 O4+主鼠标少突胶质建立一个改进的隔离方法, 要求对目标单元格进行尽可能少的操作。整个过程从幼崽的安乐死到电镀细胞在盖玻片需要大约 4 h, 并且这里显示的数据代表三个独立实验。组织离解后, 每项独立实验的平均 4.3 0.46 x 107细胞被隔离, 其生存能力为 91% @ 5.6%。在使用 anti-O4 标记磁性微珠磁隔离之后, 平均获得了6.9 个 0.38 x 106</…

Discussion

本文提出了一种高效分离高纯度未成熟小鼠少突胶质培养的方法。与以前发布的协议39、40相比, 此方法产生了更高的纯度, 具有低得多的 GFAP 阳性星形胶质细胞和其他非特征细胞的极低百分比。重要的是要指出, 这些是未成熟的生命线行动已经致力于 oligodendroglial 血统。因此, 这些细胞对于研究分化的早期阶段是不有用的。

Dincman 协?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究得到了国家多发性硬化协会 (RG4591A1/2) 和国立卫生研究院 (R03NS06740402) 的赠款的支持。作者感谢 Ivan 博士及其实验室成员提供实验室空间、设备和建议。

Materials

10ml serological pipets Fisher Scientific 13-676-10J
10ml syringe Luer-Loc tip BD, Becton Dickinson 309604
15ml conical tubes Falcon 352097
24-well tissue culture plates Falcon 353935
40µm cell strainer Fisher Scientific 22368547
50ml conical tubes Falcon 352098
5ml serological pipets Fisher Scientific 13-676-10H
60mm tissue culture plates Falcon 353002
70µm cell strainer Fisher Scientific 22363548
Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody Invitrogen A11001
Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody Invitrogen A21042
Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody Invitrogen A11008
Alexa Fluor 594 goat anti-chicken IgG (H+L) secondary antibody Invitrogen A11042
Anti-O4 beads- Anti-O4MicroBeads Miltenyi Biotec 130-094-543
Apo-Transferrin human Sigma T1147
Autofil complete bottle top filter assembly, 0.22um filter, 250ml USA Scientific 6032-1101
Autofil complete bottle top filter assembly, 0.22um filter, 250ml USA Scientific 6032-1102
B27 Supplement Invitrogen 17504-044
Boric acid Sigma B7660
Bovine Growth Serum (BGS) GE Healthcare Life Sciences SH30541.03
BSA Fisher Scientific BP-1600-100
CNTF Peprotech 450-50
d-Biotin Sigma B4639
Desoxyribonuclease I (DNAse I) Worthington LS002007
EDTA Fisher Scientific S311
Epifluorescence microscope with an Olympus DP70 camera Olympus Bx51
Feather disposable scalpels Andwin Scientific EF7281C
Forskolin Sigma F6886
German glass coverslips, #1 thickness, 12mm diameter round NeuVitro GG-12-oz
GFAP antibody Aves GFAP
Glucose Fisher Scientific D16-1
GlutaMAX Invitrogen 35050-61
Insulin Invitrogen 12585-014
Magnetic separator stand – MACS multistand Miltenyi Biotec 130-042-303
Magnetic separator-MiniMACS separator Miltenyi Biotec 130-042-302
Millex PES 0.22µm filter unit Millipore SLG033RS
Mounting media- Prolong Gold with DAPI Thermo Fisher P36930
N-acetyl-cysteine (NAC) Sigma A8199
Natural mouse laminin Invitrogen 23017-015
Neurobasal Medium A Invitrogen 10888-022
Neurotrophin-3 (NT-3) Peprotech 450-03
NG2 antibody Millipore AB5320
Papain Worthington LS003126
PBS without Ca2+ and Mg2+ Sigma D5652
PDGF Peprotech 100-13A
Petri dishes Falcon 351029
Poly-D-Lysine Sigma P6407
Primocin Invivogen ant-pm-2
Progesterone Sigma P8783
Putrescine Sigma P5780
Selection column-LS columns Miltenyi Biotec 130-042-401
Sodium Selenite Sigma S5261
Trace elements B Corning 25-000-CI
Triiodothyronine (T3) Sigma T6397
Triton-X Sigma T8787
Trypan Blue Solution Corning 25-900-CI
Tween 20 Sigma P1379
B27NBMA 487.75 mL Neurobasal Medium A; 10 mL B27 Supplement; 1 mL Primocin; 1.25 mL Glutamax; Filter sterilize and store at 4 °C until use.
B27NBMA + 10% BGS 27 mL B27NBMA; 3 mL Bovine growth serum
CNTF solution stock (10 µg/ml; 1000X) Order from Peprotech (450-50). Make up at 0.1 to 1 mg/ml according to Manufacturer’s instruction (may vary from lot to lot) in buffer (e.g. DPBS + 0.2% BSA). Store at -80 °C.
Working solution (10 µg/ml, 1000X)
1. Make on 0.2% BSA (Fisher scientific BP-1600-100) in DPBS solution and filter sterilize.
2. Dilute master stock aliquot to 10µg/ml in sterile, chilled 0.2% BSA/DPBS.
3. Aliquot (20µl/tube) and snap freeze in liquid nitrogen.
4. Store aliquots at -80 °C.
d-Biotin stock solution (50 µg/ml; 5000X) Resuspend d-Biotin (Sigma-B4639) in double-distilled H2O at 50 µg/ml (e.g. 2.5 mg in 50 ml of ddH2O). Resuspension might take fair amount of agitation/vortexing, or mild warming briefly at 37°C. If the d-Biotin still will not solubilize, it is fine to make up a less concentrated (e.g. 10µg/ml), and to add a higher volume to the B27NBMA (1/1000), instead of 1/5000). Store at 4°C.
DNase I stock solution 1. Dissolve at 12,500 U Deoxyribonuclease I / ml in HBSS chilled on ice.
2. Filter sterilize on ice
3. Aliquot at 200 µl and freeze overnight at -20°C.
4. Store aliquots at -20 to -30°C.
Dulbecco’s Phosphate Buffered Saline (w/o Ca2+ and Mg2+) Dissolve pouch in 1 Liter of water to yield 1 liter of medium at 9.6 grams of powder per liter of medium. Store at 2-8 °C.
Forskolin stock solution (4.2 mg/ml; 1000X) Add 1 ml of sterile DMSO to 50 mg Forskolin in bottle (Sigma-F6886) and pipette until resuspended. Transfer to a 15 ml centrifuge tube and add 11 ml of sterile DMSO to bring to 4.2 mg/ml. Aliquot (e.g. 20 µl) and store at -20°C.
Hank’s balanced salts (HBSS) (Sigma 1. Measure 900 ml of water (temperature 15-20 °C) in a cylinder and stir gently.
2. Add the power and stir until dissolved.
3. Rinse original package with a small amount of water to remove all traces of the powder.
4. Add to the solution in step 2.
5. Add 0.35 gr of sodium bicarbonate (7.5% w/v) for each liter of final volume.
6. Keep stirring until dissolved.
7. Adjust the pH of the buffer while stirring to 0.1-0.3 units below pH= 7.4 since it may rise during filtration. The use of 1N HCl or 1N NaOH is recommended to adjust the pH.
8. Add additional water to bring the final volume to 1L.
9. Sterilize by filtration using a membrane with a porosity of 0.22 microns.
10. Store at 2-8 °C.
Insulin stock solution (4000 µg/ml) Thaw the bottle and aliquot 25 µl per microcentrifuge tube and store at -20°C.
Laminin solution Slowly thaw laminin in the cold (2°C to 8°C) to avoid gel formation. Then, aliquot into polypropylene tubes. Store at 5° C to -20° C in aliquots (e.g. 20 µl) and do not freeze/thaw repeatedly. Laminin may be stored at these temperatures for up to six months.
Magnetic Cell Sorting (MCS) Buffer Prepare the solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA), 0.5 mM EDTA, 5µg/ml Insulin, 1 g/L Glucose. Sterilize and degas by filtration the buffer by passing it through a 0.22 µm Millex filter. Store the buffer at 4°C until use
N-Acetyl-L-cysteine (NAC) stock solution (5mg/ml; 1000X) Dissolve N-Acetyl-L-cysteine (Sigma-A8199) at 5 mg/ml in DMEM (e.g. 50 mg NAC in 10 ml B27NBMA). Filter sterilize and aliquot (e.g. 20 µl). Store at -20°C.
NT3 stock solution (1 µg/ml; 1000X) Master stock:
Order from Peprotech (450-03). Make up at 0.1 to 1 mg/ml according to manufacturer’s instructions (may vary from lot to lot), in buffer (e.g. DPBS + 0.2% BSA). Store at -80°C.

Working stock (1µg/ml; 1000X):
1. Make on 0.2% BSA in DPBS solution and filter sterilize.
2. Dilute master stock aliquot to 1 µg/ml in sterile, chilled 0.2% BSA/DPBS.
3. Aliquot (e.g. 20µl/tube) and snap freeze in liquid nitrogen.
4. Store aliquots at -80°C.
PDGF stock solution (10 µg/ml; 1000X) Master stock:
Order from Peprotech (100-13A). Make up at 0.1 to 1 mg/ml according to manufacturer’s instructions (may vary from lot to lot) in buffer (e.g. DPBS) + 0.2% BSA). Store at -80°C.

Working stock (1µg/ml; 1000X):
1. Make on 0.2% BSA in DPBS solution and filter sterilize.
2. Dilute master stock aliquot to 1µg/ml in sterile, chilled 0.2% BSA/DPBS.
3. Aliquot (e.g. 20µl/tube) and snap freeze in liquid nitrogen.
4. Store aliquots at -80°C.
Poly-D-lysine (1mg/ml; 100X) Resuspend poly-D-lysine, molecular weight 70-150 kD (Sigma P6407) at 0.5mg/ml in 0.15M boric acid pH 8.4 (e.g. 50mg in 50ml borate buffer). Filter sterilize and aliquot (e.g. 100µl/tube). Store at -20°C. Prior to use, dilute the 100X stock (1mg/ml) to 50 µg/ml in sterile water.
Oligodendrocyte proliferation media see Supplementary Table 1
Oligodendrocyte differentiation media see Supplementary Table 1
Sato supplement (100X) see Supplementary Table 1
References: the list of reagents and recipes were adopted from the protocols previously described by Emery et. al. 2013 (Emery, B. & Dugas, J. C. Purification of oligodendrocyte lineage cells from mouse cortices by immunopanning. Cold Spring Harb Protoc. 2013 (9), 854-868, doi:10.1101/pdb.prot073973, (2013)) and Dincman et. al. (Dincman, T. A., Beare, J. E., Ohri, S. S. & Whittemore, S. R. Isolation of cortical mouse oligodendrocyte precursor cells. J Neurosci Methods. 209 (1), 219-226, doi:10.1016/j.jneumeth.2012.06.017, (2012))
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Immunomagnetic IsolationMouse Primary OligodendrocytesMyelinMyelinationNeural ScienceCell CultureDissociationNeurobasal A MediumBovine Growth SerumDNase IPipetting

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Flores-Obando, R. E., Freidin, M. M., Abrams, C. K. Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes. J. Vis. Exp. (135), e57543, doi:10.3791/57543 (2018).
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