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Reprogram Murine Epiblast Stem Cells by Epigenetic Inhibitors
通过表观遗传抑制剂重新编程鼠外胚层干细胞

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Abstract

Pluripotent stem cells in the naïve state are highly useful in regenerative medicine and tissue engineering. A robust reprogramming of the primed murine Epiblast Stem Cells (EpiSCs) to naïve pluripotency is feasible via chemical-only approach. This protocol described a method to reprogram murine EpiSCs by MM-401 treatment, which blocks histone H3K4 methylation by MLL1/KMT2A.

Keywords: Naïve pluripotentcy(原始多能), EpiSCs(EpiSCs), MLL1(MLL1), H3K4 methylation(H3K4甲基化), Chemical inhibitor(化学抑制剂)

Background

Previous protocols on EpiSC reprogramming depended on the genetic manipulations of transcriptional factors or chemical inhibition of the signaling pathways, albeit with varying efficiency and duration. Based on a recent mechanistic study that links the MLL1 complex to the naïve state, this protocol provides a straightforward and robust method to restore naïve pluripotency from EpiSCs via targeting MLL1 mediated H3K4 methylation and subsequent transcription regulation. The reprogramming efficiency is significantly higher than previous published method, achieving 50% conversion rate in two weeks.

Materials and Reagents

  1. Tissue culture plate, 24 well (Coring, Falcon®, catalog number: 353047 )
  2. 15 ml conical tube (Coring, Falcon®, catalog number: 352196 )
  3. MEF feeder cells (University of Michigan, Transgenic Animal Model Core, https://www.med.umich.edu/tamc/list.html#reagents)
  4. 0.2% gelatin solution (Sigma-Aldrich, catalog number: G1890 )
  5. PBS
  6. Collagenase type IV (Thermo Fisher Scientific, GibcoTM, catalog number: 17104-019 )
  7. 0.05% trypsin (Thermo Fisher Scientific, GibcoTM, catalog number: 25300-054 )
  8. The VectorTM alkaline phosphatase (AP) staining kit (Vector Laboratories, catalog number: SK-5100 )
  9. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11995-065 )
  10. Fetal bovine serum (FBS) (Atlas Biologicals, catalog number: F-0500-D )
  11. L-glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-081 )
  12. 100x non-essential amino acids (Thermo Fisher Scientific, GibcoTM, catalog number: 11140-050 )
  13. 100x sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360-070 )
  14. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
    Note: This product has been discontinued.
  15. KnockOutTM D-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 10829018 )
  16. KnockOutTM serum replacement (KSR) (Thermo Fisher Scientific, GibcoTM, catalog number: 10828028 )
  17. Glutamax (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
  18. Fibroblast growth factor basic (FGF2), human recombinant (R&D Systems, catalog number: 233-FB )
  19. ESGRO® leukemia inhibitory factor (LIF) (EMD Millipore, catalog number: ESG1107 )
  20. MM-401
  21. DMSO
  22. Mouse embryonic fibroblasts (MEFs) culture medium (see Recipes)
  23. EpiSCs culture medium (see Recipes)
  24. Embryonic stem cells (ESCs) culture medium (see Recipes)
  25. 100 mM MM-401 (see Recipes)
  26. MM-401 reversion medium (see Recipes)
  27. MM-401 maintenance medium (see Recipes)

Equipment

  1. 1 ml pipette
  2. Tissue culture incubator (IsotempTM Microbiogical Incubator) (Fisher Scientific, model: Fisher ScientificTM IsotempcatalogTM Microbiogical Incubator, catalog number: S28670 ), humidified, 37 °C and 5% CO2 in air
  3. Stereomicroscope (Olympus, model: DP73 )
  4. Centrifuge (Eppendorf, model: 5810 R ; Swing-bucket rotor: Eppendorf, model: A-4-81 )

Procedure

A schematic summary of the reprogramming procedure described in this protocol can be found in Figure 1.


Figure 1. The schematic view of general procedures for the reversion of EpiSCs by MM-401 treatment

  1. Preparation of MEF feeder cells: Irradiated MEF feeder cells are seeded at 1 x 105 cell/well in MEF medium in a 24-well plate (pre-coated with 0.2% gelatin solution).
  2. EpiSC culture: EpiSCs are routinely cultured following previously described protocol (Chenoweth and Tesar, 2010). For chemical treatment experiments, EpiSCs are cultured in the 24-well plate and enzymatically passaged every third day (see Figure 2).


    Figure 2. Representative phase image of EpiSCs at the third day of culture before splitting. Scale bar = 50 μm.

    To passage,
    1. Remove EpiSC culture medium from each well and wash briefly with PBS. Discard PBS and add 150 μl of collagenase type IV or 0.05% trypsin to each well, and incubate at room temperature for up to 4 min.
    2. Add 1 ml of EpiSC culture medium (without FGF2) to each well and gently pipet with a 1-ml pipette. Combine suspensions from 1 to 2 wells into a 15-ml conical tube. Add EpiSC culture medium (without FGF2) to a final volume of 5 ml to each tube for centrifugation.
    3. Separate EpiSCs from MEFs (Optional):
      1. For EpiSCs passaging in small cluster, separate clusters by centrifugation at 8 x g for 15 sec (25 °C). The EpiSC clusters will loosely pellet while the individual MEFs remain in the supernatant.
      2. For EpiSCs passaging in single cell, separate EpiSCs by centrifugation at 8 x g for 3 min (25 °C). A mixture of the MEFs and EpiSCs will loosely pallet while the individual single EpiSC will remain in the supernatant.
      3. Examine the separated EpiSCs after centrifugation under the microscope. Single EpiSCs and remaining MEF feeder cells can be distinguished by size (see Figure 3). 


        Figure 3. Representative image shows the EpiSCs and MEF feeders (collected from 2 well of 24-well plate) before and after centrifugation under the microscope. A and A’. EpiSCs and MEF feeder before separation; B and B’. MEF feeder cells. Black arrow, MEF feeder; Open arrow, EpiSC cells; Open circle, MEF feeder cells, which are still 2-3-fold larger than single EpiSC cell. Scale bar = 100 μm. 
         
  3. Plating of EpiSCs for reversion: EpiSCs were passaged in single cell or small clumps on MEF feeder cells (as described in step 1) in MM-401 reversion medium.
    1. Centrifuge the EpiSCs (from step 2b or 2c), aspirate and discard the supernatant. Resuspend EpiSC pellet in MM-401 reversion medium. For the 24-well plate, add 400 μl MM-401 reversion medium to each well.
    2. For EpiSCs passaging in small cluster, replate cells to a new well with 1:4-1:5 dilutions. For EpiSCs passaging in single cells, plate the cells at 20,000/cm2 (approximately 4 x 104 cell/well for the 24-well plate, see Figure 4).


      Figure 4. Representative image of single EpiSCs plated at the density of 20,000 cell/cm2. Scale bar = 200 μm.

  4. Establish MM-401-reverted ESC line
    1. Morphologically distinct reverted naïve colonies become evident over the next 3-6 days. AKP is used to stain the colonies to evaluate reversion efficiency. Strong AKP staining distinguishes the reverted naïve colonies from EpiSCs that have weak AKP staining (see Figure 5). AKP activity is detected using VectorTM alkaline phosphatase (AP) staining kit according to the manufacturer’s instructions.


      Figure 5. AKP staining of EpiSCs and reverted naïve colonies after MM-401 treatment as indicated on top. Left: Flat EpiSC clones with weak AKP staining before MM401 induction; Right: Two representative images of EpiSCs taken at 72 h of 100 μM MM-401 treatment. The reverted cells (indicated by arrows) are morphologically distinct from EpiSCs. The colonies are dome-shaped with intense AKP staining. Scale bar = 100 μm

    2. The reversion culture is passaged by a brief exposure (4-5 min) to 0.05% trypsin/EDTA with gentle pipetting into single cells. Cells can be re-seeded at 20,000-50,000/cm2. MM-401 is replenished to reach 50-100 μM final concentrations in each passage.
    3. Reverted ESC (rESC) lines can be established by clone picking or en masse passaging. The established rESC line can be maintained, passaged, cryopreserved and thawed by the standard protocol for conventional mESCs.
    4. rESC line can be expanded in MM-401 maintenance medium or ESC culture medium without MM-401 after passage 6.

Notes

  1. For reversion, MM-401 (50-100 μM final concentration) can be added to culture medium immediately or 2-3 days after EpiSCs forming clones.
  2. Mediums containing MM-401 are recommended to be pre-warmed at room temperature (RT) for 15 min.
  3. For MM-401 request: please contact yalid@med.umich.edu.

Recipes

  1. Mouse embryonic fibroblasts (MEFs) medium
    DMEM supplemented with:
    10% FBS
    2 mM 1x L-glutamine
    1x nonessential amino acids
    1x sodium pyruvate
    0.1 mM 2-mercaptoethanol
  2. EpiSC culture medium
    KnockOutTM D-MEM supplemented with:
    20% KSR
    2 mM Glutamax
    1x nonessential amino acids
    1x sodium pyruvate
    0.1 mM 2-mercaptoethanol
    10 ng/ml FGF2
    Note: The medium can be stored at 4 °C for up to 2 weeks.
  3. Embryonic stem cells (ESCs) culture medium
    KnockOutTM D-MEM supplemented with:
    20% FBS
    2 mM Glutamax
    1x nonessential amino acids
    1x sodium pyruvate
    0.1 mM 2-mercaptoethanol
    103 U/ml LIF
    Note: The medium can be stored at 4 °C for up to 2 weeks.
  4. 100 mM MM-401
    MM-401 (Molecular Mass: 700.75) is dissolved in DMSO at the final concentration of 100 mM
    Note: It is stored at -20 °C or -80 °C.
  5. MM-401 reversion medium
    ESC culture medium supplemented with 100 μM MM-401
    Note: The medium can be stored at 4 °C for up to 2 weeks.
  6. MM-401 maintenance medium
    ESC culture medium supplemented with 20 μM MM-401
    Note: The medium can be stored at 4 °C for up to 2 weeks.

Acknowledgments

This protocol was originally published as part of Zhang et al. (2016). The authors wish to thank all present and past members developing/functional testing MM-401 in the Dr. Yali Dou, Dr. Sundeep Kalantry and Dr. Shaomeng Wang laboratories. Special thanks are obligated to Dr. Saunders and Ms. Hughes at the University of Michigan for technical support, and Dr. Brady and Dr. Tesar for sharing materials.
This work is supported by National Institute of General Medical Sciences (NIGMS) (GM082856), Leukemia and Lymphoma Society and the National Cancer Institute (CA117307-04) to Dr. Yali Dou.

References

  1. Chenoweth, J. G. and Tesar, P. J. (2010). Isolation and maintenance of mouse epiblast stem cells. Methods Mol Biol 636: 25-44.
  2. Zhang, H., Gayen, S., Xiong, J., Zhou, B., Shanmugam, A. K., Sun, Y., Karatas, H., Liu, L., Rao, R. C., Wang, S., Nesvizhskii, A. I., Kalantry, S. and Dou, Y. (2016). MLL1 inhibition reprograms epiblast stem cells to naive pluripotency. Cell Stem Cell 18(4): 481-494.

简介

初生状态下的多能干细胞在再生医学和组织工程中非常有用。 通过化学方法,将引发的鼠上皮细胞干细胞(EpiSCs)的鲁棒重新编程成为初始多能性是可行的。 该方案描述了通过MM-401处理重编程小鼠EpiSCs的方法,其通过MLL1 / KMT2A阻断组蛋白H3K4甲基化。

背景 以前关于EpiSC重编程的方案取决于转录因子的遗传操作或信号通路的化学抑制,尽管效率和持续时间不同。 基于最近将MLL1复合物与初始状态联系起来的机制研究,该方案提供了一种直观而有力的方法,通过靶向MLL1介导的H3K4甲基化和随后的转录调节来恢复EpiSC的初始多能性。 重编程效率明显高于以前公布的方法,在两周内达到50%的转化率。

关键字:原始多能, EpiSCs, MLL1, H3K4甲基化, 化学抑制剂

材料和试剂

  1. 组织培养板24孔(Coring,Falcon ®,目录号:353047)
  2. 15 ml锥形管(Coring,Falcon ®,目录号:352196)
  3. MEF饲养细胞(密歇根大学,转基因动物模型核心, https://www.med.umich.edu/tamc/list.html#reagents
  4. 0.2%明胶溶液(Sigma-Aldrich,目录号:G1890)
  5. PBS
  6. 胶原酶IV型(Thermo Fisher Scientific,Gibco TM,目录号:17104-019)
  7. 0.05%胰蛋白酶(Thermo Fisher Scientific,Gibco TM,目录号:25300-054)
  8. Vector TM碱性磷酸酶(AP)染色试剂盒(Vector Laboratories,目录号:SK-5100)
  9. Dulbecco改性Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:11995-065)
  10. 胎牛血清(FBS)(Atlas Biologicals,目录号:F-0500-D)
  11. L-谷氨酰胺(200mM)(Thermo Fisher Scientific,Gibco TM,目录号:25030-081)
  12. 100倍非必需氨基酸(Thermo Fisher Scientific,Gibco TM,目录号:11140-050)
  13. 100x丙酮酸钠(Thermo Fisher Scientific,Gibco TM,目录号:11360-070)
  14. 2-巯基乙醇(Sigma-Aldrich,目录号:M7522)
    注意:本产品已停产。
  15. KnockOut TM D-MEM(Thermo Fisher Scientific,Gibco TM,目录号:10829018)
  16. KnockOut TM 血清置换(KSR)(Thermo Fisher Scientific,Gibco TM,目录号:10828028)
  17. Glutamax(Thermo Fisher Scientific,Gibco TM ,目录号:35050061)
  18. 成纤维细胞生长因子基因(FGF2),人重组(R& D Systems,目录号:233-FB)
  19. ESGRO ®白血病抑制因子(LIF)(EMD Millipore,目录号:ESG1107)
  20. MM-401
  21. DMSO
  22. 小鼠胚胎成纤维细胞(MEFs)培养基(参见食谱)
  23. EpiSCs培养基(见食谱)
  24. 胚胎干细胞(ESCs)培养基(参见食谱)
  25. 100 mM MM-401(见食谱)
  26. MM-401反转介质(见配方)
  27. MM-401维护介质(参见配方)

设备

  1. 1 ml移液器
  2. 组织培养培养箱(Isotemp TM 微生物孵育器)(Fisher Scientific,型号:Fisher Scientific TM Isotempcatalog TM微生物培养箱,目录号:S28670),在空气中加湿37℃和5%CO 2
  3. 立体显微镜(Olympus,型号:DP73)
  4. 离心机(Eppendorf,型号:5810 R;摆动转子:Eppendorf,型号:A-4-81)

程序

本协议中描述的重新编程程序的原理图可以在图1中找到。


图1.通过MM-401治疗恢复EpiSCs的一般程序的示意图

  1. MEF饲养细胞的制备:将照射的MEF饲养细胞以1×10 5个细胞/孔接种在24孔板(预先用0.2%明胶溶液包被)的MEF培养基中。
  2. EpiSC文化:EpiSCs按照以前描述的方案进行常规培养(Chenoweth和Tesar,2010)。对于化学处理实验,将EpiSCs在24孔板中培养并每三天进行酶促传代(参见图2)。


    图2.分裂前培养第三天的EpiSCs的代表性相图。比例尺=50μm。

    为了通过,
    1. 从每个孔中取出EpiSC培养基,并用PBS短暂洗涤。弃去PBS,每孔加入150μlIV型胶原酶或0.05%胰蛋白酶,室温孵育4 min。
    2. 向每个孔中加入1ml EpiSC培养基(不含FGF2),并用1 ml移液管轻轻移液。将1至2个孔的悬浮液合并成15ml锥形管。将EpiSC培养基(不含FGF2)添加至每个管的终体积为5ml,用于离心。
    3. 与MEF分开的EpiSC(可选):
      1. 对于在小簇中传代的EpiSC,通过在8×g下离心15秒(25℃)来分离簇。 EpiSC簇将松散沉淀,而单个MEF保留在上清液中
      2. 对于在单细胞中传代的EpiSC,通过以8×g离心3分钟(25℃)分离EpiSC。 MEF和EpiSC的混合物将松散地托盘,而单个EpiSC将保留在上清液中。
      3. 在显微镜下离心后检查分离的EpiSC。单个EpiSC和剩余的MEF饲养细胞可以通过大小来区分(参见图3)。 


        图3.代表性的图像显示了在显微镜下离心之前和之后的EpiSCs和MEF进料器(从24孔板的2孔收集)。 A和A'。 EpiSCs和MEF进料器分离前; B和B'。 MEF饲养细胞。黑色箭头,MEF进料器;打开箭头,EpiSC细胞;开环,MEF饲养细胞,仍比单个EpiSC细胞大2-3倍。比例尺=100μm。 
         
  3. 用于逆转的EpiSC的电镀:将EpiSC在MM-401逆转培养基中在MEF饲养细胞(如步骤1所述)中的单细胞或小团块中传代。
    1. 离心EpiSC(从步骤2b或2c),吸出并丢弃上清液。将EpiSC沉淀重悬于MM-401反转培养基中。对于24孔板,向每个孔中加入400μlMM-401逆转培养基。
    2. 对于在小簇中传播的EpiSC,将细胞用1:4-1:5稀释液再次移植到新的孔中。对于在单细胞中传代的EpiSC,对于24孔板,以20,000/cm 2(约4×10 4个/孔)细胞平板化细胞,参见图4)。


      图4.以20,000个细胞/cm 2的密度电镀的单个EpiSC的代表性图像比例尺=200μm。

  4. 建立MM-401恢复的ESC行
    1. 在接下来3-6天内,形态上不同的恢复天真的殖民地变得明显。 AKP用于染色菌落以评估回复效率。强AKP染色将恢复的初始殖民地与具有弱AKP染色的EpiSCs区分开(参见图5)。根据制造商的说明书,使用Vector TM 碱性磷酸酶(AP)染色试剂盒检测AKP活性。


      图5.如上所述,MM-401处理后EpiSCs和回归初始菌落的AKP染色。左侧:在MM401诱导之前具有弱AKP染色的平面EpiSC克隆;右图:在200μMMM-401处理72小时时拍摄的EpiSC的两个代表性图像。回归细胞(由箭头指示)在形态上与EpiSCs不同。殖民地是具有强烈AKP染色的圆顶形。比例尺= 100μm

    2. 通过短暂曝光(4-5分钟)将0.05%胰蛋白酶/EDTA通过轻微移液到单个细胞中传代逆转培养物。细胞可以以20,000-50,000/cm 2重新接种。补充MM-401在每个通道中达到50-100μM终浓度。
    3. 恢复ESC(rESC)线可以通过克隆采集或一次通过建立。已建立的rESC系统可以通过常规mESCs的标准方案进行维护,传代,冷冻保存和解冻。
    4. 通过6号后,可以在不含MM-401的MM-401维护培养基或ESC培养基中扩增rESC系。

笔记

  1. 为了逆转,可以立即或在EpiSC形成克隆后2-3天将MM-401(50-100μM终浓度)加入到培养基中。
  2. 含有MM-401的介质建议在室温(RT)下预热15分钟。
  3. 对于MM-401请求:请联系 yalid@med.umich.edu

食谱

  1. 小鼠胚胎成纤维细胞(MEFs)培养基
    DMEM补充:
    10%FBS
    2 mM 1x L-谷氨酰胺
    1x非必需氨基酸
    1x丙酮酸钠
    0.1mM 2-巯基乙醇
  2. EpiSC培养基
    KnockOut TM D-MEM补充:
    20%KSR
    2 mM Glutamax
    1x非必需氨基酸
    1x丙酮酸钠
    0.1mM 2-巯基乙醇
    10ng/ml FGF2
    注意:介质可以在4°C下储存长达2周。
  3. 胚胎干细胞(ESCs)培养基
    KnockOut TM D-MEM补充:
    20%FBS
    2 mM Glutamax
    1x非必需氨基酸
    1x丙酮酸钠
    0.1mM 2-巯基乙醇
    10 3 U/ml LIF
    注意:介质可以在4°C下储存长达2周。
  4. 100 mM MM-401
    将MM-401(分子量:700.75)溶解在DMSO中,终浓度为100mM 注意:保存在-20°C或-80°C。
  5. MM-401逆转媒体
    补充有100μMMM-401的ESC培养基
    注意:介质可以在4°C下储存长达2周。
  6. MM-401维护媒体
    补充有20μMMM-401的ESC培养基
    注意:介质可以在4°C下储存长达2周。

致谢

该协议最初作为Zhang等人的一部分发布。 (2016)。作者希望感谢所有现任和过往成员在Yali Dou博士,Sundeep Kalantry博士和Shaomeng Wang博士实验室开发/功能测试MM-401。特别感谢Saunders博士和休斯大学的密歇根大学的技术支持,Brady博士和Tesar博士分享材料。
这项工作得到了国家综合医学研究所(NIGMS)(GM082856),白血病和淋巴瘤协会和国家癌症研究所(CA117307-04)对Yali Dou博士的支持。

参考文献

  1. Chenoweth,JG和Tesar,PJ(2010)。  隔离和维持小鼠上皮干细胞。 Methods Mol Biol 636:25-44。
  2. Zhang,H.,Gayen,S.,Xiong,J.,Zhou,B.,Shanmugam,AK,Sun,Y.,Karatas,H.,Liu,L.,Rao,RC,Wang,S.,Nesvizhskii, AI,Kalantry,S.and Dou,Y.(2016)。 
  • English
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引用:Zhang, H. and Dou, Y. (2017). Reprogram Murine Epiblast Stem Cells by Epigenetic Inhibitors. Bio-protocol 7(5): e2168. DOI: 10.21769/BioProtoc.2168.
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