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Neural Stem Cell Differentiation and Prion Infection
神经干细胞分化和朊病毒感染   

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Abstract

Prion diseases are transmissible, fatal, neurodegenerative diseases in human and animals. The molecular basis of neurodegeneration in prion diseases is largely unclear. Developing a cellular model capable of monitoring prion-induced cytotoxicity would be a promising approach for better understanding the prion pathogenesis. One candidate cellular assay is a model based on neurospheres, which contains neural stem cells (NSCs). Both undifferentiated and differentiated NSCs have been demonstrated to be permissive to prion infection, and prion-induced cytopathic changes in differentiated neruosphere cultures were reported (Iwamaru et al., 2013). This protocol describes the procedure to induce differentiation of NSCs from transgenic mice overexpressing prion protein (tga20 mice) into cultures susceptible for prion infection.

Keywords: Neural stem cell(神经干细胞), Differentiation culture(分化培养), Prion infection(朊病毒感染), Cellular pathogenesis(细胞病变作用)

Materials and Reagents

  1. Mouse-adapted prion infected mice brains (terminally ill CD-1 female mice intracerebrally inoculated with mouse adapted scrapie, at ca. 6 months of age)
  2. Ethanol (99.5% EtOH) (Nacalai Tesque, catalog number: 14713-95 )
  3. Dulbecco's phosphate buffered saline without Ca and Mg (D-PBS) (Nacalai Tesque, catalog number: 14249-95 )
  4. Hank's balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H8264 )
  5. Dulbecco's modified Eagle's medium/nutrient F-12 Ham (DMEM/F12 Ham) (Sigma-Aldrich, catalog number: D8437 )
  6. N-2 supplement (Life Technologies, catalog number: 17502-048 )
  7. Bovine serum albumin solution (30% BSA in D-PBS) (Sigma-Aldrich, catalog number: A9576 )
  8. Accutase (Innovative Cell Technologies, catalog number: AT-104 )
  9. 99% Acetone (Nacalai Tesque, catalog number: 00310-95 )
  10. Water for embryo transfer (sterile-filtered) (Sigma-Aldrich, catalog number: W1503 )
  11. 35% hydrochlolic acid (HCl) ( Nacalai Tesque, catalog number: 18321-05 )
  12. Penicillin-Streptomycin (Sigma-Aldrich, catalog number: P0781 )
  13. Epidermal growth factor human (EGF) (Sigma-Aldrich, catalog number: E9644 ) (see Recipes)
  14. Fibroblast growth factor-basic human (bFGF) (Sigma-Aldrich, catalog number: F0291 ) (see Recipes)
  15. Neurosphere basal medium (see Recipes)
  16. Fibronectin (Sigma-Aldrich, catalog number: F4759 ) (see Recipes)
  17. Glass wash solution (see Recipes)
  18. Differentiation-inducing medium (see Recipes)

Equipment

  1. Falcon 15 ml conical centrifuge tubes (Corning, catalog number: 352096 )
  2. Falcon 50 ml conical centrifuge tubes (Corning, catalog number: 352070 )
  3. Corning primaria 6 well clear multiwell plate (Corning, catalog number: 353846 )
  4. Nunc 60mm dish with HydroCell surface (Thermo Fisher Scientific, catalog number: 174912 )
  5. Glass coverslips (22 x 22 mm) (Asahi Techno Glass Corporation, catalog number: 2918 )
  6. ART 1000 REACH tips (Thermo Fisher Scientific, catalog number: 2079 )
  7. Multi-beads shocker cell disruptor (Yasui Kikai Corporation)
  8. 2 ml screw-cap micro centrifuge tubes-freestanding (Sarstedt K.K., catalog number: 72.664.002 )
  9. Zirconia ball (1 mm diameter) (As One YTZ1)
  10. Plastic square dishes (Eiken Chemical, model: AW2000 )
  11. 1.5 ml screw-cap micro centrifuge tubes (Sarstedt K.K., catalog number: 72.687 .028J)
  12. Sealed sonicator (Biorupter®, model: UCD250 )
  13. Centrifuge (Kubota Corporation, model: 5220 )
  14. CO2 incubator (set at 37 °C and 5% CO2-95% air)
  15. Reciprocal shaker (Taitec, model: NR-1 )
  16. Bio-safety cabinet (ORIENTAL GIKEN, model: LAD-1300XB3 )
  17. Deep freezer (set at -80 °C)
  18. Fine forceps (NAPOX A-5, 110 mm; NAPOX MA-45, 110 mm)
  19. C-Chip disposable hemocytometer (iN CYTO, DHC-N01 Neubauer Improved)
  20. Electric balance (Mettler Toledo, model: AB104-S )

Procedure

  1. Preparation of glass coverslips
    1. Immerse 24 glass coverslips in a plastic square dish containing glass wash solution (25 -30 ml) for 12-16 h at 25 °C with gentle rocking using a reciprocal shaker at 50 rpm (Figure 1).


      Figure 1. Washing process of glass coverslips in a plastic square dish on a reciprocal shaker

    2. Place the coverslips in 50 ml conical tube and wash the coverslips with 35 ml acetone for 1 min three times with inversion of the tube.
    3. In a bio-safety cabinet, after air-dry for 30 sec, pass an individual coverslip over a flame briefly and place one cover slip on the bottom of each well of 6 well plate.
    4. In a bio-safety cabinet, coat coverslips with 1.5 ml of fibronectin solution and incubate at 37 °C for 12-16 h. After aspiration of fibronectin solution, air-dry coverslips laid on the bottom of the well for 30 min (Figure 2).


      Figure 2. Air-drying of fibronectin-coated glass coverslips laid in 6 well plate in a bio-safety cabinet

  2. Preparation of prion-infected brain homogenate
    Prions, the infectious agents of fatal neurological diseases in humans and animals, are known to be highly resistant to conventional physical and chemical procedures for inactivating or eliminating microbes. To minimize the bio-hazard risk, before you handle prion-infected tissues, you have to contact with biosafety officers in your department and comply with the biosafety regulations. Required personal protection equipments, decontamination methods, waste management and procedures should be referred to each institutional bio-safety regulations. For example, please see the guidelines for prion experiments published elsewhere (Biosafety in Microbiological and Biomedical Laboratories 5th Edition: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf).
    1. In a bio-safety cabinet, weigh a brain with an electric balance, cut it sagitally along the midline and place each hemisphere containing brain stem and cerebellum in 2 ml screw-capped micro centrifuge tube containing zirconia balls (around 800 mg), as shown in Figure 3.


      Figure 3. Dissection of prion-infected brain tissue and loading into a screw-capped micro centrifuge tube containing zirconia balls

    2. Add 1 ml of sterile D-PBS at 4 °C and homogenize it by Multi beads shocker at 3,000 rpm for 1 min at 25 °C. Repeat this homogenization process once more.
    3. Place brain homogenate into 15 ml conical tube and add appropriate volume of sterile D-PBS at 4 °C to adjust at 10% weight per volume.
    4. Aliquot them (250 μl) in 1.5 ml screw-cap micro centrifuge tubes and stored at -80 °C until use. After thawing at 25 °C, sonicate the content for 30 sec using a sealed sonicator before use.

  3. Preparation of NSC cultures
    1. Centrifuge neurosphere suspensions (ca.100-150 μm diameter) isolated from transgenic mice overexpressing murine prion protein (tga20 mice) at 200 x g for 5 min at room temperature (Iwamaru et al., 2013; Iwamaru et al., 2014). Discard the supernatant, resuspend in 2 ml of Accutase, and incubate at 37 °C for 10 min.
    2. Gently pipet up and down using ART 1000 Reach tip to dissociate neurospheres into single cells. Add 12 ml of HBSS and count the cell density of cell suspension (10 μl) by using C-Chip disposable hemocytometer. Centrifuge at 200 x g for 5 min at 25 °C and discard the supernatant. Resuspend the pellet at the concentration of 100,000 cells/ml in the Neurosphere basal medium. Dispense 2 ml of the cell suspension into a well of 6 well plates containing a glass coverslip which is prepared in step A4.
    3. Add 1 ml of Neurosphere basal medium to the cultures after two days of incubation at 37 °C in CO2 incubator.
    4. Incubate for additional one to two days until the cultures reach at 90% of confluency.


      Figure 4. Differential interference contrast micrograph of undifferentiated neural stem cell culture at 90% of confluency. (Scale bar = 100 μm)

    5. Wash the cultures with 2 ml of HBSS at 25 °C, gently hold the edge of the glass coverslips with fine forceps and transfer them into new 6 well plates which contain 2 ml of Neurosphere basal medium without bFGF (differentiation-inducing medium) per well.
    6. Change 2 ml of the differentiation-inducing medium every 2 days.
    7. After 6 days of differentiation, replace the culture medium with prion-infected brain homogenate diluted at 1:1,000 in 2 ml of the differentiation-inducing medium.
    8. After two days of infection, replace 2 ml of the culture medium with freshly prepared prion-infected brain homogenate at the same dilution, and continue to incubate for another two days.
    9. Change the differentiation-inducing medium every 2 days, until biochemical and infectivity assays of prion are performed.

Recipes

  1. Epidermal growth factor human (EGF)
    Dissolved at 50 μg /ml in sterile DMEM/F12 Ham containing 1% BSA
    Aliquoted (100 μl) and stored at -20 °C
    Use within 6 months
  2. Fibroblast growth factor-basic human (bFGF)
    Dissolved at 50 μg /ml in sterile DMEM/F12 Ham containing 1% BSA
    Aliquoted (50 μl) and stored at -20 °C
    Use within 6 months
  3. Neurosphere basal medium
    Reagents
    Volume
    Final conc.
    DMEM/F12 Ham
    487.33 ml

    EGF
    500 μl
    50 ng/ml
    bFGF
    500 μl
    50 ng/ml
    BSA
    1.67 ml
    0.1%
    N2 supplement
    5 ml
    1%
    Penicillin-streptomycin
    5 ml
    100 IU/ml and 100 μg/ml, respectively
  4. Fibronectin
    Dissolve 1 mg of fibronectin in 1 ml of sterile water for a stock solution
    Dilute the stock with water (Final concentration is 10 ug/ml) before use
  5. Glass wash solution
    Reagents
    Volume
    Final conc.
    EtOH (99.5%)
    24 ml
    74.6%
    HCl (12.1 mol/l)
    8 ml
    3.0 mol/l
  6. Differentiation-inducing medium
    Reagents
    Volume
    Final conc.
    DMEM/F12 Ham
    487.83 ml

    EGF
    500 μl
    50 ng/ml
    BSA
    1.67 ml
    0.1%
    N2 supplement
    5 ml
    1%
    Penicillin-streptomycin
    5 ml
    100 IU/ml and 100 μg/ml, respectively

Acknowledgments

This protocol was adapted from our previously published paper: Iwamaru et al. (2013). This work was supported by a Grant-in-Aid from the BSE and Other Prion Disease Control Project of the Ministry of Agriculture, Forestry, and Fisheries of Japan and by a Grant-in-Aid for Young Scientists (category B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References

  1. Iwamaru, Y., Takenouchi, T., Imamura, M., Shimizu, Y., Miyazawa, K., Mohri, S., Yokoyama, T. and Kitani, H. (2013). Prion replication elicits cytopathic changes in differentiated neurosphere cultures. J Virol 87(15): 8745-8755.
  2. Iwamaru, Y., Takenouchi, and Kitani, H. (2014). Isolation and culture of neurospheres for the study of pathogenesis of prion disease. Bio-Protocol 3(6): e1081.

简介

朊病毒疾病是人类和动物中可传播的,致命的,神经变性疾病。 朊病毒疾病中神经变性的分子基础很大程度上不清楚。 开发能够监测朊病毒诱导的细胞毒性的细胞模型将是更好地理解朊病毒发病机制的有前途的方法。 一种候选细胞测定是基于神经球的模型,其含有神经干细胞(NSC)。 未分化和分化的NSCs都被证明是容许朊病毒感染,并且报道了分化的神经球培养物中的朊病毒诱导的细胞病变变化(Iwamaru等人,2013)。 该协议描述了诱导NSCs从过度表达朊病毒蛋白(tga20小鼠)的转基因小鼠分化成易感染朊病毒感染的培养物的过程。

关键字:神经干细胞, 分化培养, 朊病毒感染, 细胞病变作用

材料和试剂

  1. 小鼠适应的朊病毒感染的小鼠脑(在约6个月龄时脑内接种小鼠适应的瘙痒病的终末期CD-1雌性小鼠)
  2. 乙醇(99.5%EtOH)(Nacalai Tesque,目录号:14713-95)
  3. 不含Ca和Mg的Dulbecco磷酸盐缓冲盐水(D-PBS)(Nacalai Tesque,目录号:14249-95)
  4. Hank's平衡盐溶液(HBSS)(Sigma-Aldrich,目录号:H8264)
  5. Dulbecco改良Eagle培养基/营养F-12 Ham(DMEM/F12 Ham)(Sigma-Aldrich,目录号:D8437)
  6. N-2补充剂(Life Technologies,目录号:17502-048)
  7. 牛血清白蛋白溶液(30%BSA的D-PBS溶液)(Sigma-Aldrich,目录号:A9576)
  8. Accutase(Innovative Cell Technologies,目录号:AT-104)
  9. 99%丙酮(Nacalai Tesque,目录号:00310-95)
  10. 胚胎移植用水(无菌过滤)(Sigma-Aldrich,目录号:W1503)
  11. 35%盐酸(HCl)(Nacalai Tesque,目录号:18321-05)
  12. 青霉素 - 链霉素(Sigma-Aldrich,目录号:P0781)
  13. 表皮生长因子人(EGF)(Sigma-Aldrich,目录号:E9644)(参见Recipes)
  14. 成纤维细胞生长因子 - 碱性人(bFGF)(Sigma-Aldrich,目录号:F0291)(参见配方)
  15. 神经球基础培养基(见配方)
  16. 纤连蛋白(Sigma-Aldrich,目录号:F4759)(参见Recipes)
  17. 玻璃清洗溶液(见配方)
  18. 分化诱导介质(参见配方)

设备

  1. Falcon 15ml锥形离心管(Corning,目录号:352096)
  2. Falcon 50ml圆锥形离心管(Corning,目录号:352070)
  3. Corning primaria 6孔透明多孔板(Corning,目录号:353846)
  4. 具有HydroCell表面的Nunc 60mm皿(Thermo Fisher Scientific,目录号:174912)
  5. 玻璃盖玻片(22×22mm)(Asahi Techno Glass Corporation,目录号:2918)
  6. ART 1000 REACH提示(Thermo Fisher Scientific,目录号:2079)
  7. 多珠冲击器细胞破碎机(Yasui Kikai Corporation)
  8. 2ml螺旋盖微量离心管 - 独立式(Sarstedt K.K.,目录号:72.664.002)
  9. 氧化锆球(直径1 mm)(As One YTZ1)
  10. 塑料方形盘(Eiken Chemical,型号:AW2000)
  11. 1.5ml螺旋盖微量离心管(Sarstedt K.K.,目录号:72.687.028J)
  12. 密封超声波仪(Biorupter ,型号:UCD250)
  13. 离心机(Kubota Corporation,型号:5220)
  14. CO 2培养箱(设定在37℃和5%CO 2 -95%空气)下培育。
  15. 交互振荡器(Taitec,型号:NR-1)
  16. 生物安全柜(ORIENTAL GIKEN,型号:LAD-1300XB3)
  17. 深冷冻(设定在-80℃)
  18. 细钳(NAPOX A-5,110mm; NAPOX MA-45,110mm)
  19. C-Chip一次性血细胞计数器(iN CYTO,DHC-N01 Neubauer Improved)
  20. 电平衡(Mettler Toledo,型号:AB104-S)

程序

  1. 玻璃盖玻片的制备
    1. 浸入24玻璃盖玻片在含有玻璃洗涤溶液(25-30毫升)的塑料方盘,在25°C下12-16小时,使用往复振荡器以50 rpm的温和摇动(图1)。


      图1.在往复振动器上的塑料方形盘中的玻璃盖玻片的清洗过程

    2. 将盖玻片放置在50ml锥形管中,用35ml丙酮洗涤盖玻片1分钟,每次3分钟,倒置管。
    3. 在生物安全柜中,在空气干燥30秒后,将单个盖玻片短暂地通过火焰,并在6孔板的每个孔的底部放置一个盖玻片。
    4. 在生物安全柜中,用1.5ml纤连蛋白溶液包被盖玻片,并在37℃孵育12-16小时。吸入纤维连接蛋白溶液后,将空气干燥的盖玻片放置在孔底部30分钟(图2)。


      图2.在生物安全柜中放置在6孔板中的纤维连接蛋白包被的玻璃盖玻片的空气干燥

  2. 制备朊病毒感染的脑匀浆
    已知朊病毒,人和动物中的致命神经疾病的感染剂,对于灭活或消除微生物的常规物理和化学程序是高度耐受的。为了最小化生物危险风险,在处理朊病毒感染的组织之前,您必须与您部门的生物安全官员联系,并遵守生物安全法规。所需的个人防护设备,净化方法,废物管理和程序应参考每个机构的生物安全法规。例如,请参见其他地方发表的朊病毒实验指南(Biosafety in Microbiological and Biomedical Laboratories第5版: http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf )。
    1. 在生物安全柜中,用电平衡称重大脑,沿着中线将其切断,并将含有脑干和小脑的每个半球置于含有氧化锆球(约800mg)的2ml螺旋盖微型离心管中,如图所示在图3中

      图3.朊病毒感染的脑组织的解剖和加载到含有氧化锆球的螺旋微型离心管中

    2. 在4℃下加入1ml无菌D-PBS,并通过多珠震动器在3000rpm下在25℃下匀化1分钟。再次重复此均质化过程。
    3. 将脑匀浆放入15毫升锥形管,并加入适当体积的无菌D-PBS在4℃下调整为10%重量/体积。
    4. 等分(250微升)在1.5毫升螺旋盖微量离心管,并储存在-80℃下使用。在25°C解冻后,使用密封的超声波仪在内容物超声处理30秒,然后使用
  3. NSC培养物的制备
    1. 在室温下以200xg离心5分钟从转基因小鼠中过表达鼠朊病毒蛋白(tga20小鼠)的神经球悬浮液(约100-150μm直径)离心5分钟(Iwamaru等, em>,2013; Iwamaru ,,2014)。弃去上清液,重悬于2ml Accutase中,并在37℃下孵育10分钟。
    2. 轻轻地吸取上下使用ART 1000到达提示将神经球分离成单个细胞。加入12毫升HBSS,并通过使用C-Chip一次性计数细胞悬液(10微升)的细胞密度 血细胞计数器。在25℃下以200×g离心5分钟,弃去上清液。在神经球基础培养基中以100,000个细胞/ml的浓度重悬沉淀。将2ml细胞悬浮液分配到含有在步骤A4中制备的玻璃盖玻片的6孔​​板的孔中。
    3. 在37℃在CO 2培养箱中孵育两天后,向培养物中加入1ml Neurosphere基础培养基。
    4. 孵育另外一至两天,直到培养物达到90%的汇合

      图4.未分化神经干细胞培养物在90%汇合时的差异干涉对比显微照片(比例尺=100μm)

    5. 在25℃下用2ml HBSS洗涤培养物,用精细镊子轻轻握住玻璃盖玻片的边缘,并将其转移到新的6孔板中,该孔含有2ml每孔不含bFGF(分化诱导培养基)的Neurosphere基础培养基。
    6. 每2天更换2ml分化诱导培养基。
    7. 分化6天后,将该培养基替换为在2ml的分化诱导培养基中以1:1,000稀释的朊病毒感染的脑匀浆。
    8. 感染两天后,用相同稀释度的新鲜制备的朊病毒感染的脑匀浆替换2ml培养基,继续孵育另外两天。
    9. 每2天更换分化诱导培养基,直到进行朊病毒的生化和感染性测定。

食谱

  1. 表皮生长因子人(EGF)
    以50μg/ml溶于含有1%BSA的无菌DMEM/F12 Ham中溶解 分装(100μl)并贮存在-20℃下 在6个月内使用
  2. 成纤维细胞生长因子 - 碱性人(bFGF)
    以50μg/ml溶于含有1%BSA的无菌DMEM/F12 Ham中溶解 分装(50μl)并储存在-20℃下
    在6个月内使用
  3. 神经球基础培养基
    试剂

    最终浓度。
    DMEM/F12 Ham
    487.33 ml

    EGF
    500微升
    50 ng/ml
    bFGF
    500微升
    50 ng/ml
    BSA
    1.67 ml
    0.1%
    N2补助
    5 ml
    1%
    青霉素 - 链霉素 5 ml
    100 IU/ml和100μg/ml,分别为
  4. 纤连蛋白
    将1mg纤连蛋白溶解于1ml无菌水中作为储备溶液
    在使用前用水稀释原液(最终浓度为10ug/ml)
  5. 玻璃清洗溶液
    试剂

    最终浓度。
    EtOH(99.5%)
    24 ml
    74.6%
    HCl(12.1mol/l)
    8 ml
    3.0 mol/l
  6. 分化诱导培养基
    试剂

    最终浓度。
    DMEM/F12 Ham
    487.83 ml

    EGF
    500微升
    50 ng/ml
    BSA
    1.67 ml
    0.1%
    N2补助
    5 ml
    1%
    青霉素 - 链霉素 5 ml
    100 IU/ml和100μg/ml,分别为

致谢

这个协议改编自我们以前发表的文章: Iwamaru (2013)。 这项工作得到了日本农业,林业和渔业部BSE和其他朊病毒病防治项目的资助,以及来自该部的青年科学家资助(B类) 日本教育,文化,体育,科学和技术。

参考文献

  1. Iwamaru,Y.,Takenouchi,T.,Imamura,M.,Shimizu,Y.,Miyazawa,K.,Mohri,S.,Yokoyama,T.and Kitani,H。 Prion replication elicits 分化的神经球培养物中的细胞病变改变。病毒学87(15):8745-8755。
  2. Iwamaru,Y.,Takenouchi和Kitani,H。(2014)。 神经球的分离和培养用于研究朊病毒病的发病机理 /em> 3(6):e1081。
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引用:Iwamaru, Y., Takenouchi, T. and Kitani, H. (2014). Neural Stem Cell Differentiation and Prion Infection. Bio-protocol 4(6): e1082. DOI: 10.21769/BioProtoc.1082.
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