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iPS Cell Induction from Human Non-T, B cells from Peripheral Blood
用人外周血液中的非T淋巴细胞、B细胞诱导生成全能干细胞   

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Abstract

The generation of iPS cells gives an opportunity to use patient-specific somatic cells which are a valuable source for disease modeling and drug discovery. To promote these studies, it is important to make iPS cells from easily accessible and less invasive tissues like blood. Here, we describe the basic method to generate human iPS cells from adult peripheral blood. After the isolation of mononuclear cells, a combination of cytokines stimulates the expansion of hematopoietic stem/progenitor population, which is the main target of this protocol. The cells are transduced with plasmid mixture encoding reprogramming factors. In most cases, the plasmids are lost during the establishment of iPS clones.

Materials and Reagents

  1. Fresh anti-coagulated blood (~10 ml)
  2. PBS without Ca2+ and Mg2+ (Nacalai tesque, catalog number: 14249-95 )
  3. Ficoll-paque Plus (GE Healthcare, catalog number: 17-1440-02 )
  4. StemSpan H3000 (STEMCELL Technologies, catalog number: 0 9800 )
  5. Recombinant human Interleukin (IL)-6 (100 μg/ml) (PeproTech, catalog number: AF-200-06 )
  6. Recombinant human Stem Cell Factor (SCF) (300 μg/ml) (PeproTech, catalog number: AF-300-07 )
  7. Recombinant human Thrombopoietin (TPO) (300 μg/ml) (PeproTech, catalog number: AF-300-18 )
  8. Recombinant human Flt3 ligand (300 μg/ml) (PeproTech, catalog number: AF-300-19 )
  9. Recombinant human Interleukin (IL)-3 (10 μg/ml) (PeproTech, catalog number: AF-200-03 )
  10. Recombinant human basic Fibroblast Growth Factor (bFGF) (10 μg/ml) (Wako, catalog number: 064-04541 )
  11. Amaxa Human CD34+ cell Nucleofector Kit (Lonza, catalog number: VPA-1003 )
  12. MEF feeder (Repro Cell, catalog number: RCHEFC003 )
  13. Matrigel growth factor reduced (BD Biosciences, catalog number: 356231 )
  14. Primate ES Cell Medium (Repro Cell, catalog number: RCHEMD001 )
  15. Essential 6 (Life Technologies, catalog number: A1516401 )
  16. Gelatin (Sigma-Aldrich, catalog number: G1890 )
  17. Dulbecco’s Modified Eagle’s Medium (DMEM) High glucose with stable L-glutamine (Nacalai tesque, catalog number: 08459-35 )
  18. Fetal Bovine Serum (FBS) (Life Technologies, catalog number: 10437-028 )
  19. Plasmid set 2 (Life Technologies, catalog number: A15960 )
  20. Blood medium (see Recipes)
  21. Plasmid mixture (see Recipes)
  22. Transfection mixture (see Recipes)
  23. iPS medium 1 (see Recipes)
  24. iPS medium 2 (see Recipes)
  25. 0.1% Gelatin solution (see Recipes)
  26. MEF medium (see Recipes)

Equipment

  1. Nucleofector 2b (Lonza, catalog number: AAB-1001 )
  2. 6-well tissue culture plate (BD Biosciences, Falcon®, catalog number: 353046 )
  3. 15 ml conical tube (BD Biosciences, Falcon®, catalog number: 352196 )
  4. 50 ml conical tube (BD Biosciences, Falcon®, catalog number: 352070 )
  5. 37 °C 5% CO2 Cell culture incubator
  6. Microscope
  7. Centrifuge

Procedure

Day 0

  1. Culture medium preparation:
    1. Add 2 ml of blood medium containing cytokines to a well of 6-well plate.
    2. Add 2 ml of PBS in the remaining wells of the plate to prevent excess evaporation of the medium and store the plate at 37 °C, 5% CO2.
  2. Purification of mononuclear cells:
    1. To 10 ml of anti-coagulated blood (EDTA), add 10 ml of PBS.
    2. In two 15 ml tubes, add 5 ml Ficoll-Paque and gently add 10 ml of blood + PBS.
    3. Spin tubes at 400 x g for 30 min at 18 °C. Use slow acceleration and slow brake.
    4. Remove the plasma (around 2 ml) for the top fraction without disrupting the mononuclear cells at the interface.
    5. Transfer the cells at interface (1-2 ml) to a 15 ml tube and mix well with 12 ml of PBS.
    6. Spin at 200 x g for 10 min at 18 °C (slow brake).
    7. Resuspend cells in 3 ml of H3000 medium and count cells.
    8. Prepare aliquots of 3 x 106 cells in 1.5 ml tube.
    9. Spin at 200 x g for 10 min at 18 °C (slow brake).
    10. Aspirate supernatant.
  3. Plating:
    1. Resuspend the cells in the medium prepared in step 1.
    2. Store the plate at 37 °C, 5% CO2 for around 6 days. Medium change is not needed during this culture period.


Day 5

  1. Gelatin coat:
    1. Add 1 ml/well of 0.1% gelatin solution to a 6-well plate.
    2. Incubate the plate for at least half an hour at 37 °C.
  2. Preparation of MEF feeder cells:
    1. Thaw MEF feeder cells at 37 °C and count the cell number.
    2. Aspirate excess gelatin solution prepared above.
    3. Seed the MEF feeder cells at 3 x 105 cell/well in MEF medium.


Day 6

  1. Culture medium preparation:
    1. Aspirate the medium from MEF feeder cells.
    2. Add 2 ml/well of blood medium containing cytokines to the plate.
  2. Harvest the cultured cells:
    1. Resuspend the cells in the medium and count cells. Number of live cells is usually around 1 x 106.
    2. Harvest the floating cells into 15 ml tube.
    3. Spin at 200 x g for 10 min at 18 °C (slow brake). During spin, prepare transfection mixture.
  3. Nucleofection:
    1. Aspirate the supernatant of the cells completely by hand using a pipette.
    2. Add transfection mixture and suspend cells, be careful not to create any bubbles.
    3. Perform nucleofection using program U-008.
  4. Plating, 105 to 104 cells per well:
    1. Immediately following nucleofection, add 800 μl of H3000 to the electroporation cuvette, and harvest the cells. Metal ions in the nucleofection solution are toxic to cells!
    2. Plate the cells to MEF feeder plate ranging from 5 x 105 cells to 5 x 104 cells per well.


Day 8, 10, and 12

  1. Add additional 1.5 ml of iPS medium 1 per well. Do not aspirate the existing medium.


Day 14-

  1. Replace medium with 1.5 ml of iPS medium 1 per well.
    * Medium replacement is performed every 2 days.


Day 25 to 35

  1. Pick colonies of about 2 mm diameter.

Notes

  1. Frozen peripheral blood mononuclear cells (PBMC) can be used with this protocol. Culture the thawed PBMC directly in blood medium on day 0.
  2. iPSCs can be established in non-feeder condition, but the efficiency is low. For non-feeder condition, use Matrigel coated plate and iPS medium 2 instead of MEF feeder and iPS medium 1. Followings are the procedure to make Matrigel coated plate.
    1. Thaw Matrigel at 4 °C and dilute it equivalent to 2 mg of protein in 6 ml of cooled DMEM.
    2. Add 1 ml/well of the solution to a 6-well plate.
    3. Store at RT for 1 h.
    4. Wash the plate once with PBS before cell seeding.

Recipes

  1. Blood medium
    StemSpan H3000, containing following cytokines:
    10 ng/ml IL-3
    100 ng/ml IL-6
    300 ng/ml SCF
    300 ng/ml TPO
    300 ng/ml Flt3 ligand
    Use immediately after preparation
  2. Plasmid mixture (Addgene, http://www.addgene.org/Shinya_Yamanaka)
    Use following plasmid mixtures. Set 1 shows higher efficiency. In set 2, we omitted WPRE sequence and replaced shRNA against p53 with dominant negative form of mouse p53, which exist in set 1. Store at -20 °C. Set 2 can be purchased from Life Technologies.
    Plasmid set 1
    pCXLE-hOCT3/4-shp53-F
    0.83 g
    pCXLE-hSK
    0.83 g
    pCXLE-hUL
    0.83 g
    pCXWB-EBNA1
    0.5 g
    Plasmid set 2
    pCE-hOCT3/4
    0.63 g
    pCE-hSK
    0.63 g
    pCE-hUL
    0.63 g
    pCE-mp53DD
    0.63 g
    pCXB-EBNA1
    0.5 g
  3. Transfection mixture
    Amaxa CD34 Solution
    81.8 μl
    Supplement
    18.2 μl
    Plasmid mixture (set 1 or 2)
    3 μl (3 μg)
    Use immediately after preparation
  4. iPS medium 1
    Primate ES Cell Medium containing 4 ng/ml bFGF
    Store at 4 °C for 1 week in dark
  5. iPS medium 2
    Essential 6 containing 100 ng/ml bFGF
    Store at 4 °C for 1 week in dark
  6. 0.1% (w/v) Gelatin solution
    1. Dissolve 1 g gelatin powder in 100 ml dH2O (1% w/v, 10x concentration), autoclave and store at 4 °C.
    2. To prepare 0.1% (1x) gelatin solution, warm the 10x gelatin stock at 37 °C, add 50 ml of this to 450 ml dH2O. Filter the solution with a bottle-top filter (0.22 μm) and store at 4 °C.
  7. MEF medium
    DMEM containing 10% FBS

Acknowledgments

We thank T. Aoi, K. Takahashi, M. Nakagawa and Y. Yoshida for scientific discussion; M. Narita, T. Ichisaka and M. Ohuchi for technical assistance; R. Kato, E. Nishikawa, S. Takeshima, and Y. Ohtsu for administrative assistance; and Drs. H. Niwa (RIKEN) and J. Miyazaki (Osaka University) for the CAG promoter. This study was supported in part by a grant from the Program for Promotion of Fundamental Studies in Health Sciences of National Institute of Biomedical Innovation, a grant from the Leading Project of Ministry of Education, Culture, Sports, Science and Technology (MEXT), a grant from Funding Program for World-Leading Innovative Research and Development on Science and Technology (FIRST Program) of Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research of Japan Society for the Promotion of Science and MEXT (to S.Y.), and Grants-in-Aid for Scientific Research for Young Scientists B (to K.O.).

References

  1. Mack, A. A., Kroboth, S., Rajesh, D. and Wang, W. B. (2011). Generation of induced pluripotent stem cells from CD34+ cells across blood drawn from multiple donors with non-integrating episomal vectors. PLoS One 6(11): e27956. 
  2. Okita, K., Matsumura, Y., Sato, Y., Okada, A., Morizane, A., Okamoto, S., Hong, H., Nakagawa, M., Tanabe, K., Tezuka, K., Shibata, T., Kunisada, T., Takahashi, M., Takahashi, J., Saji, H. and Yamanaka, S. (2011). A more efficient method to generate integration-free human iPS cells. Nat Methods 8(5): 409-412.
  3. Okita, K., Yamakawa, T., Matsumura, Y., Sato, Y., Amano, N., Watanabe, A., Goshima, N. and Yamanaka, S. (2013). An efficient nonviral method to generate integration‐free human‐induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells 31(3): 458-466. 

简介

iPS细胞的产生提供了使用患者特异性体细胞的机会,其是用于疾病建模和药物发现的有价值的来源。 为了促进这些研究,重要的是使iPS细胞从容易获得和侵入性较小的组织如血液。 在这里,我们描述从成人外周血生成人类iPS细胞的基本方法。 在分离单核细胞后,细胞因子的组合刺激造血干/祖细胞群的扩增,这是该方案的主要目标。 用编码重编程因子的质粒混合物转导细胞。 在大多数情况下,质粒在iPS克隆的建立期间丢失。

材料和试剂

  1. 新鲜抗凝血液(约10ml)
  2. 没有Ca 2+和Mg 2+的PBS(Nacalai tesque,目录号:14249-95)的
  3. Ficoll-paque Plus(GE Healthcare,目录号:17-1440-02)
  4. StemSpan H3000(STEMCELL Technologies,目录号:09800)
  5. 重组人白细胞介素(IL)-6(100μg/ml)(PeproTech,目录号:AF-200-06)
  6. 重组人干细胞因子(SCF)(300μg/ml)(PeproTech,目录号:AF-300-07)
  7. 重组人血小板生成素(TPO)(300μg/ml)(PeproTech,目录号:AF-300-18)
  8. 重组人Flt3配体(300μg/ml)(PeproTech,目录号:AF-300-19)
  9. 重组人白细胞介素(IL)-3(10μg/ml)(PeproTech,目录号:AF-200-03)
  10. 重组人碱性成纤维细胞生长因子(bFGF)(10μg/ml)(Wako,目录号:064-04541)
  11. Amaxa人CD34 +细胞Nucleofector Kit(Lonza,目录号:VPA-1003)
  12. MEF进料器(Repro Cell,目录号:RCHEFC003)
  13. 减少Matrigel生长因子(BD Biosciences,目录号:356231)
  14. Primate ES细胞培养基(Repro Cell,目录号:RCHEMD001)
  15. Essential 6(Life Technologies,目录号:A1516401)
  16. 明胶(Sigma-Aldrich,目录号:G1890)
  17. Dulbecco改良的Eagle培养基(DMEM)具有稳定的L-谷氨酰胺的高葡萄糖(Nacalai tesque,目录号:08459-35)
  18. 胎牛血清(FBS)(Life Technologies,目录号:10437-028)
  19. 质粒组2(Life Technologies,目录号:A15960)
  20. 血液介质(见配方)
  21. 质粒混合物(参见配方)
  22. 转染混合物(参见配方)
  23. iPS介质1(参见配方)
  24. iPS介质2(参见配方)
  25. 0.1%明胶溶液(见配方)
  26. MEF介质(参见配方)

设备

  1. 核转染体2b(Lonza,目录号:AAB-1001)
  2. 6孔组织培养板( BD Biosciences,Falcon , 程序

    第0天

    1. 培养基制备:
      1. 向6孔板的孔中加入2ml含有细胞因子的血液培养基。
      2. 在板的剩余孔中加入2ml PBS以防止培养基的过度蒸发,并将板在37℃,5%CO 2下保存。
    2. 单核细胞的纯化:
      1. 向10ml抗凝血(EDTA)中加入10ml PBS
      2. 在两个15毫升管中,加入5毫升Ficoll-Paque,轻轻加入10毫升血液+ PBS
      3. 在40℃下旋转管在18℃下30分钟。 使用慢加速和慢刹车。
      4. 去除血浆(约2毫升)的顶部部分,而不破坏界面上的单核细胞
      5. 将细胞在界面(1-2毫升)转移到15毫升管,与12毫升PBS充分混合
      6. 在18℃下以200转/分钟的速度旋转10分钟(慢速制动)
      7. 重悬细胞在3毫升H3000培养基和计数细胞
      8. 在1.5ml管中制备3×10 6个细胞的等分试样
      9. 在18℃下以200×g离心10分钟(慢速制动)。
      10. 吸出上清液。
    3. 电镀:
      1. 将细胞重悬在步骤1中制备的培养基中。
      2. 将板在37℃,5%CO 2储存约6天。 在此培养期间不需要中等变化。


    第5天

    1. 明胶包衣:
      1. 将1ml /孔的0.1%明胶溶液加入6孔板中
      2. 在37℃下孵育平板至少半小时。
    2. MEF饲养细胞的制备:
      1. 解冻MEF饲养细胞在37°C,并计数细胞数量
      2. 吸出上面制备的过量明胶溶液
      3. 将MEF饲养细胞以3×10 5个细胞/孔接种在MEF培养基中


    第6天

    1. 培养基制备:
      1. 从MEF饲养细胞吸出培养基。
      2. 加入2毫升/孔含有细胞因子的血液培养基
    2. 收获培养的细胞:
      1. 重悬细胞在培养基中并计数细胞。 活细胞数通常约为1×10 6
      2. 收获浮动细胞到15毫升管
      3. 在18℃下以200×g离心10分钟(慢速制动)。 在旋转期间,准备转染混合物
    3. 核转染:
      1. 用移液管用手完全吸出细胞的上清液。
      2. 加入转染混合物并悬浮细胞,小心不要产生任何气泡。
      3. 使用程序U-008进行核转染
    4. 电镀,每孔10 10至10 10个细胞:
      1. 在核转染后立即,向电穿孔比色杯中加入800μlH3000,并收获细胞。 核转染溶液中的金属离子对细胞有毒!
      2. 将细胞以每孔5×10 5个细胞至5×10 4个细胞平板接种到MEF饲养板上。


    第8,10和12天

    1. 每孔加入另外的1.5ml iPS培养基1。 不要吸入现有介质。


    第14天 -

    1. 用每孔1.5ml iPS培养基1更换培养基。
      *每2天进行一次中等更换。


    第25至35天

    1. 挑选约2mm直径的菌落。

    笔记

    1. 冷冻的外周血单核细胞(PBMC)可以与该方案一起使用。 在第0天将解冻的PBMC直接培养在血液培养基中。
    2. iPSCs可以在非饲养条件下建立,但效率低。 对于非进料条件,使用Matrigel包被板和iPS培养基2代替MEF进料器和iPS培养基1.以下是制备Matrigel包被板的程序。
      1. 解冻Matrigel在4°C,稀释它相当于2毫克的蛋白质在6毫升冷却的DMEM。
      2. 将1ml /孔的溶液加入6孔板中
      3. 在室温储存1小时。
      4. 在细胞接种前用PBS洗涤板一次。

    食谱

    1. 血液介质
      StemSpan H3000,含有以下细胞因子:
      10 ng/ml IL-3
      100ng/ml IL-6
      300 ng/ml SCF
      300 ng/ml TPO
      300ng/ml Flt3配体 准备后立即使用
    2. 质粒混合物(Addgene, http://www.addgene.org/Shinya_Yamanaka
      使用以下质粒混合物。 组1显示更高的效率。 在集合2中,我们省略了WPRE序列并且取代针对p53的shRNA与显示阴性形式的小鼠p53,其存在于组1中。存储在-20℃。 组2可以购买 来自Life Technologies。
      质粒组1
      pCXLE-hOCT3/4-shp53-F
      0.83克
      pCXLE-hSK
      0.83克
      pCXLE-hUL
      0.83克
      pCXWB-EBNA1
      0.5克
      质粒组2
      pCE-hOCT3/4
      0.63克
      pCE-hSK
      0.63克
      pCE-hUL
      0.63克
      pCE-mp53DD
      0.63克
      pCXB-EBNA1
      0.5克
    3. 转染混合物
      Amaxa CD34解决方案
      81.8微升
      补充
      18.2μl
      质粒混合物(组1或2)
      3μl(3μg)
      准备后立即使用
    4. iPS介质1
      含有4ng/ml bFGF的灵长类ES细胞培养基 在4℃下储存1周,在暗处
    5. iPS中等2
      含有100ng/ml bFGF的Essential 6 在4℃下储存1周,在暗处
    6. 0.1%(w/v)明胶溶液
      1. 将1g明胶粉末溶解在100ml dH 2 O(1%w/v,10x浓度)中,高压灭菌并在4℃下储存。
      2. 为了制备0.1%(1x)明胶溶液,在37℃下加热10x明胶储备液,将其加入到450ml dH 2 O中。用瓶顶过滤器(0.22μm)过滤溶液,并在4℃下保存
    7. MEF培养基
      含有10%FBS的DMEM

    致谢

    我们感谢T. Aoi,K. Takahashi,M. Nakagawa和Y. Yoshida的科学讨论; M. Narita,T. Ichisaka和M.Ohuchi的技术援助; R. Kato,E. Nishikawa,S.Takeshima,and Y.Ohtsu for administrative assistance;和Drs。 H.Niwa(RIKEN)和J. Miyazaki(大阪大学)的CAG启动子。这项研究得到了国家生物医学创新研究所健康科学基础研究计划资助,教育,文化,体育,科学和技术部领导项目(MEXT)的资助,日本社会科学促进科学技术世界领先创新研究与发展(FIRST计划)资助计划,日本科学促进科学研究计划资助计划),以及青年科学家B(KO)科学研究助学金。

    参考文献

    1. Mack,A.A.,Kroboth,S.,Rajesh,D。和Wang,W.B。(2011)。 从具有非整合游离载体的多个供体获得的血液中从CD34 +细胞产生诱导多能干细胞。 PLoS One 6(11):e27956。 
    2. Okita,A.,Morizane,A.,Okamoto,S.,Hong,H.,Nakagawa,M.,Tanabe,K.,Tezuka,K。, Shibata,T.,Kunisada,T.,Takahashi,M.,Takahashi,J.,Saji,H.and Yamanaka,S。(2011)。 一种更有效的方法来生成无集成的人类iPS细胞。 Nat Methods 8(5):409-412。
    3. Okita,K.,Yamakawa,T.,Matsumura,Y.,Sato,Y.,Amano,N.,Watanabe,A.,Goshima,N.and Yamanaka, 一种有效的非病毒方法,用于从脐带血和外周血中产生无整合的人诱导多能干细胞细胞。 干细胞 31(3):458-466。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Okita, K. (2013). iPS Cell Induction from Human Non-T, B cells from Peripheral Blood. Bio-protocol 3(18): e907. DOI: 10.21769/BioProtoc.907.
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