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Erythroid-Myeloid-Lymphoid cells (EML) are a multipotent haematopoietic cell line of mouse bone marrow origin capable of long-term maintenance in vitro in the presence of SCF (stem cell factor) (Tsai et al., 1994). The self-renewal capacity of the EML cell line is conferred by the presence of a dominant-negative retinoic acid receptor (RAR) originally delivered by retroviral transduction (Tsai et al., 1994), which arrests cells at an early progenitor stage blocked from normal progression into myeloid differentiation. The presence of the RAR trans-gene does not interfere with erythroid differentiation, and it is possible to capture a low percentage of early erythroid, but not myeloid, committed cells in maintenance cultures (Pina et al., 2012; Ye et al., 2005).
Cells can be driven into granulocytic/neutrophil differentiation through the use of high doses of retinoic acid (RA), which overcomes the differentiation block. It should be noted that these pharmacological doses of RA are not compatible with erythroid differentiation, and it is hence not viable to obtain robust erythroid and myeloid differentiation in the same assay. Indeed, colonies scored as mixed-lineage in CFC assays are a mixture of undifferentiated and erythroid cells (Tsai et al., 1994). Nevertheless, robust single-lineage erythroid and neutrophil differentiation can be obtained in liquid culture under defined cytokine conditions, as specified below.

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EML Erythroid and Neutrophil Differentiation Protocols
骨髓淋巴样红细胞(EML)中的红血球和噬中性细胞分化实验流程

细胞生物学 > 细胞分离和培养 > 细胞分化
作者: Cristina Pina
Cristina PinaAffiliation: Department of Haematology, University of Cambridge, Cambridge, UK
For correspondence: cp533@medschl.cam.ac.uk
Bio-protocol author page: a1418
Cristina Fugazza
Cristina FugazzaAffiliation: Dipartimento de Biotecnologie e Bioscienze, Universita degli Studi de Milano-Bicocca, Milan, Italy
Bio-protocol author page: a1419
 and Tariq Enver
Tariq EnverAffiliation: UCL Cancer Institute, University College, London, UK
Bio-protocol author page: a1420
Vol 4, Iss 12, 6/20/2014, 3703 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1151

[Abstract] Erythroid-Myeloid-Lymphoid cells (EML) are a multipotent haematopoietic cell line of mouse bone marrow origin capable of long-term maintenance in vitro in the presence of SCF (stem cell factor) (Tsai et al., 1994). The self-renewal capacity of the EML cell line is conferred by the presence of a dominant-negative retinoic acid receptor (RAR) originally delivered by retroviral transduction (Tsai et al., 1994), which arrests cells at an early progenitor stage blocked from normal progression into myeloid differentiation. The presence of the RAR trans-gene does not interfere with erythroid differentiation, and it is possible to capture a low percentage of early erythroid, but not myeloid, committed cells in maintenance cultures (Pina et al., 2012; Ye et al., 2005).
Cells can be driven into granulocytic/neutrophil differentiation through the use of high doses of retinoic acid (RA), which overcomes the differentiation block. It should be noted that these pharmacological doses of RA are not compatible with erythroid differentiation, and it is hence not viable to obtain robust erythroid and myeloid differentiation in the same assay. Indeed, colonies scored as mixed-lineage in CFC assays are a mixture of undifferentiated and erythroid cells (Tsai et al., 1994). Nevertheless, robust single-lineage erythroid and neutrophil differentiation can be obtained in liquid culture under defined cytokine conditions, as specified below.

[Abstract]

Materials and Reagents

  1. IMDM (powder or liquid)
  2. Horse serum (HS) (may require batch testing if low cell viability)
  3. Foetal bovine serum (FBS)
  4. L-Glutamine
  5. Penicillin/Streptomycin (P/S)
  6. SCF-conditioned medium (SCF-CM) (see Notes)
  7. Recombinant human erythropoietin (e.g. epoietin alpha, Amgen) (obtained through hospital pharmacy under appropriate local guidelines)
  8. Recombinant mouse interleukin-3 (IL-3) (e.g. Pepro Tech, catalog number: 213-13 )
  9. Recombinant mouse granulocyte-monocyte colony-stimulating factor (GM-CSF) (e.g. Pepro Tech, catalog number: 315-03 )
  10. All-Trans Retinoic Acid (ATRA) (e.g. Sigma-Aldrich, catalog number: 302-79-4 , reconstitute in ethanol)
  11. Culture supplements and antibiotics (L-Glutamine and Penicillin/Streptomycin)
  12. Monoclonal anti-mouse antibodies for flow cytometry
    1. C-kit/CD117 (clone 2B8) (e.g. PE-Cy7, eBioscience, catalog number: 25-1171 ) (suggested use at 1:100 dilution)
    2. CD34 (clone RAM34) (e.g. Alexa-Fluor 647, eBioscience, catalog number:  51-0341 ) (suggested use at 1:100 dilution)
    3. Mac-1/CD11b (clone M1/70) (e.g. PE, eBioscience, catalog number: 12-0112 ) (suggested use at 1:100 dilution)
    4. Gr1/Ly6C (clone RB6-8C5) (e.g. FITC, eBioscience, catalog number: 11-5931 ) (suggested use at 1:100 dilution)
    5. Sca-1/Ly6A/E (clone D7) (e.g. Pacific blue, BioLegend, catalog number: 122520 ) (suggested use at 1:50 dilution)
      Note: Stain on ice, in culture medium, for 20 min; wash with 10-20x volume of medium; pellet cells at 400 x g for 5 min. Re-suspend in 300-500 μl of medium for FACS analysis.
  13. Trypan blue

Equipment

  1. T175 tissue culture flasks with filtered cap (for production of conditioned medium)
  2. T75 or T25 tissue culture flasks with filtered cap, or 6-well plates
    Note: For EML cultures, the size of the tissue culture vial is determined by the cell number seeded at the cell densities indicated in the protocol.
  3. Cell culture incubator

Procedure

  1. Maintenance culture conditions
    1. EML cells maintenance culture conditions are IMDM with 5% HS, 2 mM L-glutamine and 1x penicillin/streptomycin (P/S) and 10-15% of SCF-conditioned medium (SCF-CM) obtained from BHK-MKL cells.
      Note: The original cultivars of EML cells were grown in medium supplemented with rat SCF at a final concentration of 200 ng/ml (Tsai et al., 1994).
    2. Cells should be seeded at 2 x 104 cells/ml and split to the initial dilution when a cell density of 105-2 x 105/ml (absolute maximum) is reached. This typically means splitting the cultures every 2 or sometimes 3 days. Cultures die rapidly at higher cell densities.
      Note: The dead cell fraction in maintenance cultures, as judged by Trypan blue exclusion, should not exceed 5-8% (10% is acceptable if not indicative of a culture exhaustion trend.).
    3. Cultures can be maintained for at least 20 passages post-thaw without any significant changes in biology.

  2. Erythroid differentiation conditions
    1. Use EML cells in logarithmic growth phase from a maintenance culture, and plate them at a seeding concentration of 2 x 104 cells/ml in fresh maintenance culture medium + recombinant human erythropoietin (EPO) at a final concentration of 10 U/ml.
    2. EML erythroid differentiation cultures are very sensitive to cell crowding, so opt for increasing culture volumes rather than initial cell densities if high numbers of cells are required.
    3. Culture for 2 days. After this initial period, wash cells 2-3x in IMDM with 5% HS + 2 mM L-glutamine and 1x P/S, and re-seed at 2 x 104 cells/ml in IMDM, 5% HS, 2 mM L-glutamine and 1x P/S, with 2.5% SCF-CM and 10 U/ml EPO.
    4. Differentiate cells for a total of 7 days (so, 5 additional days), with fresh EPO (10 U/ml) added on day 5 to the existing culture.
    5. It is expected to observe an average 20-fold expansion in cell numbers during the culture period, with cell viability decreasing gradually as cells differentiate, to a minimum of 40-50% at day 7.
    6. The extent of cell differentiation can be determined by flow cytometry staining with C-Kit, Sca1 and CD34, with accumulation of differentiated Sca1loCD34-C-kitlo/- cells (referred to as Kit-, see Figure 1 for representative plot) (Pina et al., 2012). It is expected that 20-30% of the culture become Kit- by day 6 of differentiation.


      Figure 1. Erythroid differentiation of EML cells. A representative plot of flow cytometry analysis at day 4 is shown; c-kit-CD34- cells are Sca1lo/-. In this instance, cultures were seeded at day 0 with a population of cells with culture-reconstituting (or self-renewal) potential (Pina et al., 2012).

    7. When erythroid differentiation cultures are seeded with committed erythroid cells (e.g. sorted Sca1loCD34- cells cultured as per B1 above) (Pina et al., 2012), 60-80% of the culture are Kit- by day 4 of differentiation, reflecting faster differentiation kinetics from committed cells.
    8. Not unexpectedly, cultures seeded with erythroid-committed cells are less proliferative, with a 10-fold expansion in cell numbers expected by day 4, after which the cultures plateau and become less viable.

  3. Neutrophil differentiation conditions
    1. Use EML cells in logarithmic growth phase from a maintenance culture, and plate them at a seeding concentration of 2 x 104 cells/ml in fresh maintenance culture medium + 10 ng/ml of recombinant mouse IL-3 and 10 μM ATRA.
    2. Viability is less affected by cell crowding than in erythroid differentiation, and it is thus possible to seed cultures at higher densities (0.5-1 x 105) if high cell numbers are required.
    3. After 2 days, wash cells 2-3x in IMDM with 5% HS + 2 mM L-glutamine and 1x P/S, and re-seed at 2 x 104 cells/ml in IMDM, 5% HS, 2 mM L-glutamine and 1x P/S, with 2.5% SCF-CM + 10 ng/ml of recombinant mouse GM-CSF and 10 μM ATRA.
    4. Keep the culture growing for a total of 7 days (so, 5 additional days), with fresh GM-CSF (10 ng/ml) added on day 5 to the existing culture.
    5. It is expected to observe an average 10-fold expansion in cell numbers during the culture period, with viabilities close to 80% or higher throughout the duration of the culture.
    6. The extent of cell differentiation can be determined by flow cytometry after staining with Gr-1 and Mac-1 antibodies (Pina et al., 2012), with accumulation of granulocytic Gr1+Mac-1+ cells to an expected proportion of 40-50% by days 4-5 of culture (see Figure 2 for representative plot). Proportions can drop thereafter, although absolute numbers of differentiated cells continue to increase.


      Figure 2. Neutrophil differentiation of EML cells. A representative plot of flow cytometry analysis at day 6 is shown.

Notes

  1. SCF-conditioned medium (SCF-CM)
    1. One possible source are BHK cells expressing the murine kit ligand protein (MKL, or SCF).
    2. Conditioned medium can be produced in T175 flasks with a total production volume of 60 ml/flask.
    3. Culture the cells in IMDM with 10% FBS, 2 mM L-Glutamine and 1x Penicillin/Streptomycin until they reach 80% confluence.
    4. At this stage replace with fresh medium and culture for an additional 48 h, at which point the supernatant is collected, floating cells removed by pelletting at 400 x g, 5 min, and the medium filtered through a 0.2 μm mesh.
    5. Conditioned medium can be aliquoted and stored for several months at -20 °C without loss of activity.

Acknowledgments

The protocol herein described was originally reported in Pina et al. (2012). Its development and implementation was financially supported by the Medical Research Council of the United Kingdom, Leukaemia and Lymphoma Research, EuroSyStem and STEMEXPAND.

References

  1. Orford, K., Kharchenko, P., Lai, W., Dao, M. C., Worhunsky, D. J., Ferro, A., Janzen, V., Park, P. J. and Scadden, D. T. (2008). Differential H3K4 methylation identifies developmentally poised hematopoietic genes. Dev Cell 14(5): 798-809.
  2. Pina, C., Fugazza, C., Tipping, A. J., Brown, J., Soneji, S., Teles, J., Peterson, C. and Enver, T. (2012). Inferring rules of lineage commitment in haematopoiesis. Nat Cell Biol 14(3): 287-294.
  3. Tsai, S., Bartelmez, S., Sitnicka, E. and Collins, S. (1994). Lymphohematopoietic progenitors immortalized by a retroviral vector harboring a dominant-negative retinoic acid receptor can recapitulate lymphoid, myeloid, and erythroid development. Genes Dev 8(23): 2831-2841.
  4. Ye, Z. J., Kluger, Y., Lian, Z. and Weissman, S. M. (2005). Two types of precursor cells in a multipotential hematopoietic cell line. Proc Natl Acad Sci U S A 102(51): 18461-18466.

材料和试剂

  1. IMDM(粉末或液体)
  2. 马血清(HS)(如果细胞存活率低,可能需要批次测试)
  3. 胎牛血清(FBS)
  4. L-谷氨酰胺
  5. 青霉素/链霉素(P/S)
  6. SCF条件培养基(SCF-CM)(见注释)
  7. 重组人促红细胞生成素(例如,epoietin alpha,Amgen)(通过医院药房在适当的本地指导下获得)
  8. 重组小鼠白细胞介素-3(IL-3)(例如Pepro Tech,目录号:213-13)
  9. 重组小鼠粒细胞 - 单核细胞集落刺激因子(GM-CSF)(例如Pepro Tech,目录号:315-03)
  10. 全反式视黄酸(ATRA)(例如Sigma-Aldrich,目录号:302-79-4,在乙醇中重建)
  11. 文化补充剂和抗生素(L-谷氨酰胺和青霉素/链霉素)
  12. 用于流式细胞术的单克隆抗小鼠抗体
    1. C-kit/CD117(克隆2B8)(例如PE-Cy7,eBioscience,目录号:25-1171)(建议以1:100稀释使用)
    2. CD34(克隆RAM34)(例如Alexa-Fluor 647,eBioscience,目录号:51-0341)(建议以1:100稀释使用)
    3. Mac-1/CD11b(克隆M1/70)(例如PE,eBioscience,目录号:12-0112)(建议以1:100稀释使用)
    4. Gr1/Ly6C(克隆RB6-8C5)(例如FITC,eBioscience,目录号:11-5931)(建议以1:100稀释使用)
    5. Sca-1/Ly6A/E(克隆D7)(例如Pacific blue,BioLegend,目录号:122520)(建议以1:50稀释使用)
      注意:   在冰上,在培养基中,染色20分钟; 用10-20倍体积的洗涤   中; 沉淀细胞在400×g离心5分钟。 重悬在300-500μl的 介质用于FACS分析
  13. 台盼蓝

设备

  1. 具有过滤盖(用于生产条件培养基)的T175组织培养瓶
  2. T75或T25组织培养瓶中,过滤的盖或6孔板 注意:对于EML培养物,组织培养瓶的尺寸由在方案中指示的细胞密度下接种的细胞数确定。
  3. 细胞培养孵化器

程序

  1. 维持培养条件
    1. EML细胞维持培养条件是具有5%HS,2mM的IMDM L-谷氨酰胺和1×青霉素/链霉素(P/S)和10-15% SCF条件培养基(SCF-CM)。
      注意: 将EML细胞的原始品种在补充的培养基中生长 与大鼠SCF的最终浓度为200ng/ml(Tsai等,1994)。
    2. 细胞应以2×10 4个细胞/ml接种,并分开至初始 当细胞密度为10 5 s -1 x 10 -5 s/ml(绝对最大值)时的稀释度为 到达。 这通常意味着每2或分裂文化 有时3天。 培养物在更高的细胞密度下快速死亡。
      注意:   维持培养中的死细胞分数,由台盼判断 蓝色排除,不应超过5-8%(如果不是10%是可以接受的 表示培养物耗尽趋势。
    3. 培养物可以在解冻后保持至少20代,而生物学没有任何显着变化。

  2. 红细胞分化条件
    1. 使用来自维持培养的对数生长期的EML细胞, 并以2×10 4个细胞/ml的接种浓度接种它们 维持培养基+重组人促红细胞生成素(EPO)   最终浓度为10U/ml
    2. EML红系分化 文化对细胞拥挤非常敏感,所以选择增加 培养体积而不是初始细胞密度,如果大量 需要单元格。
    3. 文化2天。 之后这个初始 期间,在含有5%HS + 2mM L-谷氨酰胺和1x的IMDM中洗涤细胞2-3x P/S,并以2×10 4个细胞/ml在IMDM,5%HS,2mM L-谷氨酰胺 和1x P/S,具有2.5%SCF-CM和10U/ml EPO。
    4. 区分 细胞共7天(因此,另外5天),用新鲜的EPO(10 U/ml)在第5天加入到现有培养物中
    5. 预期   观察培养期间细胞数量的平均20倍扩增   期间,细胞活力随细胞逐渐减少 分化,在第7天至少为40-50%。
    6. 程度 细胞分化可以通过流式细胞术染色来确定 C-Kit,Sca1和CD34,具有分化的积累 Sca1 -/- CD34-C-kit -/- 细胞(称为Kit - ,参见图1的 代表性图)(Pina等人,2012)。 预计20-30%的 在分化的第6天,培养物变成Kit -

      图1。 EML细胞的红细胞分化。 流动的代表图 显示了第4天的细胞计数分析; c-kit CD34 - 细胞是Sca1 -/- 。 在   在这种情况下,在第0天用一群细胞接种培养物   具有培养 - 重建(或自我更新)潜力(Pina等人, 2012)。

    7. 当接种红细胞分化培养物时 与定向的红细胞(例如分选的Sca1 lo CD34 - 细胞培养 根据上述B1)(Pina等人,2012),60-80%的培养物是Kit - 第4天的分化,反映更快的分化动力学 从定型细胞。
    8. 不出意料,种子的文化 红细胞定型的细胞较少增殖,具有10倍 第4天预期的细胞数量的扩增,之后培养物 高原并且变得不太可行。

  3. 嗜中性粒细胞分化条件
    1. 使用来自维持培养的对数生长期的EML细胞, 并以2×10 4个细胞/ml的接种浓度接种它们 维持培养基+ 10ng/ml重组小鼠IL-3和10 μMATRA。
    2. 生存力受细胞拥挤的影响小于 红细胞分化,因此有可能种子培养物 如果需要高细胞数,则密度更高(0.5-1×10 5
    3. 2天后,用含5%HS + 2mM L-谷氨酰胺的IMDM洗涤细胞2-3次 和1×P/S,并以2×10 4个细胞/ml重新接种在IMDM,5%HS,2mM L-谷氨酰胺和1×P/S,具有2.5%SCF-CM + 10ng/ml重组小鼠   GM-CSF和10μMATRA。
    4. 保持文化增长总 的7天(因此,另外5天),加入新鲜的GM-CSF(10ng/ml)   第5天对现有文化
    5. 它预计将观察到 在培养期期间细胞数量的平均10倍扩增, 在整个持续时间内具有接近80%或更高的存活率 文化。
    6. 可以确定细胞分化的程度 通过用Gr-1和Mac-1抗体染色后的流式细胞术(Pina等   2012),其中粒细胞Gr1 + sup/+ Mac-1 + sup细胞的积累至 预期比例为40-50%,培养4-5天(见图2 代表性图)。 比例可以后来,虽然绝对   分化细胞数持续增加。


      图2.EML细胞的嗜中性粒细胞分化。显示在第6天的流式细胞术分析的代表性图。

笔记

  1. SCF条件培养基(SCF-CM)
    1. 一种可能的来源是表达鼠试剂盒配体蛋白(MKL或SCF)的BHK细胞
    2. 条件培养基可以在总产量为60ml /瓶的T175烧瓶中生产
    3. 在含有10%FBS,2mM L-谷氨酰胺和1×青霉素/链霉素的IMDM中培养细胞,直到它们达到80%汇合。
    4. 在这一阶段,更换新鲜培养基和文化另外 48小时,此时收集上清液,漂浮细胞 通过在400×g下沉淀5分钟除去,并将培养基过滤   0.2μm网。
    5. 条件培养基可以等分并在-20℃下储存几个月而不丧失活性。

致谢

本文所述的方案最初在Pina等人(2012)中报道。 其发展和实施得到英国医学研究委员会,白血病和淋巴瘤研究,EuroSyStem和STEMEXPAND的财政支持。

参考文献

  1. Orford,K.,Kharchenko,P.,Lai,W.,Dao,M.C.,Worhunsky,D.J.,Ferro,A.,Janzen,V.,Park,P.J.and Scadden,D.T。(2008)。 差异性H3K4甲基化识别发育上有意义的造血基因。 Dev Cell 14(5):798-809。
  2. Pina,C.,Fugazza,C.,Tipping,A.J.,Brown,J.,Soneji,S.,Teles,J.,Peterson,C.and Enver,T。(2012)。 推荐谱系承诺规则 haematopoiesis。 Nat Cell Biol 14(3):287-294。
  3. Tsai,S.,Bartelmez,S.,Sitnicka,E。和Collins,S。(1994)。 通过含有显性负性视黄酸受体的逆转录病毒载体永生化的淋巴造血祖细胞可以重现淋巴样,骨髓样, 和红细胞发育。 Dev 8(23):2831-2841。
  4. Ye,Z.J.,Kluger,Y.,Lian,Z.and Weissman,S.M。(2005)。 多潜能造血细胞系中的两种类型的前体细胞 Proc Natl Acad Sci USA 102(51):18461-18466。
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How to cite this protocol: Pina, C., Fugazza, C. and Enver, T. (2014). EML Erythroid and Neutrophil Differentiation Protocols. Bio-protocol 4(12): e1151. DOI: 10.21769/BioProtoc.1151; Full Text



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