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Synchronize Human Embryonic Stem Cells at Different Cell Cycle Stage
人类胚胎干细胞在细胞分裂周期不同阶段的同步实验   

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Abstract

Pluripotency and the capability for self-renewal are essential characteristics of human embryonic stem cells (hESCs), which hold great potential as a cellular source for tissue replacement. Short cell cycle (15-16 h) compared to somatic cells is another property of hESCs. Efficient synchronization of hESCs at different cell cycle stages is important to elucidate the mechanistic link between cell cycle regulation and cell fate decision. This protocol describes how to establish synchronization of hESCs at different cell cycle stages.

Keywords: Cell cycle(细胞周期), Synchronize(同步), Human embryonic stem cells(人胚胎干细胞)

Materials and Reagents

  1. Human embryonic stem cells (hESCs) Undifferentiated hESCs were cultured on irradiated mouse embryonic fibroblast (MEF) feeders in DMEM/F12 medium supplemented with 20% KnockOut serum replacement, 0.1 mM nonessential amino acids (NEAA), 1 mM GlutaMAXTM-1, 0.1 mM 2-mercaptoethanol (all from Invitrogen) and 8 ng/ml recombinant human FGF2 (Peprotech, catalog number: 100-18 B).
  2. Matrigel (BD Biosciences, catalog number: 354230 )
  3. Nocodazole (Sigma-Aldrich, catalog number: M1404 )
  4. Thymidine (Sigma-Aldrich, catalog number: T1895 )
  5. Aphidicholine (Sigma-Aldrich, catalog number: A0781 )
  6. Phosphate buffered saline (PBS)
  7. Dimethyl sulfoxide (DMSO)
  8. Nocodazole stock solution (see Recipes)
  9. Thymidine stock solution (see Recipes)
  10. Aphidicholine stock solution (see Recipes)

Equipment

  1. Centrifuges (Eppendorf 5415D centrifuge)
  2. Fluorescence activated cell sorter (FACS, FACScan machine, Stanford FACS facility)

Procedure

  1. Synchronize hESCs at G2/M phase
    1. Method I
      1. Passage hESCs to matrigel on the first day. On the second day, change culture medium.
      2. On the third day, add 200 ng/ml nocodazole into fresh medium and incubate for 16 h at 37 °C.
      3. After 16 h, harvest cells, and prepare a small aliquot for cell cycle analysis by FACS.
    2. Method II
      1. Passage hESCs to matrigel on the first day. On the second day, change culture medium.
      2. On the third day, add 2 mM thymidine into fresh medium and incubate for 10 h at 37 °C.
      3. Remove the medium containing thymidine, wash with PBS (store at room temperature) twice to release cells from thymidine arrest.
      4. Add fresh culture medium and incubate for another 10 h at 37 °C.
      5. Add nocodazole to a final concentration at 200 ng/ml. Incubate for 24 h at 37 °C.
      6. Harvest cells, and prepare a small aliquot for cell cycle analysis by FACS.

  2. Synchronize hESCs at G1 phase
    1. Method I
      1. Passage hESCs to matrigel on the first day. On the second day, change culture medium.
      2. On the third day, add 200 ng/ml nocodazole into fresh medium and incubate for 16 h at 37 °C.
      3. Remove the medium containing nocodazole, wash with PBS (store at room temperature) twice.
      4. Add fresh medium supplemented with 10 µg/ml of aphidicholine and incubate for 9-10 h at 37 °C.
      5. Harvest cells, and prepare a small aliquot for cell cycle analysis by FACS.
    2. Method II
      1. Passage hESCs to matrigel on the first day. On the second day, change culture medium.
      2. On the third day, add 10 µg/ml of aphidicholine into fresh medium and incubate for 20 h at 37 °C.
      3. Harvest cells, and prepare a small aliquot sample for cell cycle analysis by FACS.

  3. Synchronize hESCs at S phase
    1. Method I
      1. Synchronize hESCs at G2/M phase as described in A.
      2. Remove the medium containing nocodazole, wash with PBS (store at room temperature) twice.
      3. Add fresh medium to release cells, incubate hESCs at 37 °C. Collect cells every hour, and prepare a small aliquot for cell cycle analysis by FACS. Usually the S phase cell populations appear at 5-6 h after releasing from nocodazole arrest.
    2. Method II
      1. Synchronize hESCs at G1 phase as described in B.
      2. Remove the medium containing aphidicholine, wash with PBS (store at room temperature) twice.
      3. Add fresh medium to release cells, incubate hESCs at 37 °C. Collect cells every hour, and prepare a small aliquot for cell cycle analysis by FACS. Usually the S phase cell populations appear at 2-3 h after releasing from aphidicholine arrest.

Recipes

  1. Nocodazole stock solution
    Dissolve nocodazole in DMSO
    Stock concentration at 5 mg/ml
  2. Thymidine stock solution
    Dissolve thymidine in ddH2O
    Stock concentration at 100 mM
  3. Aphidicholine stock solution
    Dissolve aphidicholine in DMSO
    Stock concentration at 10 mg/ml

Acknowledgments

This work was supported by the California Institute of Regenerative Medicine, Grant RL1-00100.

Reference

  1. Becker, K. A., Ghule, P. N., Therrien, J. A., Lian, J. B., Stein, J. L., van Wijnen, A. J. and Stein, G. S. (2006). Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J Cell Physiol 209(3): 883-893.
  2. Neganova, I., Zhang, X., Atkinson, S. and Lako, M. (2009). Expression and functional analysis of G1 to S regulatory components reveals an important role for CDK2 in cell cycle regulation in human embryonic stem cells. Oncogene 28(1): 20-30.

简介

Pluripotency and the capability for self-renewal are essential characteristics of human embryonic stem cells (hESCs), which hold great potential as a cellular source for tissue replacement. Short cell cycle (15-16 h) compared to somatic cells is another property of hESCs. Efficient synchronization of hESCs at different cell cycle stages is important to elucidate the mechanistic link between cell cycle regulation and cell fate decision. This protocol describes how to establish synchronization of hESCs at different cell cycle stages.

关键字:细胞周期, 同步, 人胚胎干细胞

材料和试剂

  1. 人胚胎干细胞(hESC)将未分化的hESC在补充有20%KnockOut血清替代物,0.1mM非必需氨基酸(NEAA),1mM GlutaMAX TM TM-培养基的DMEM/F12培养基中在经辐射的小鼠胚胎成纤维细胞 0.1mM 2-巯基乙醇(均来自Invitrogen)和8ng/ml重组人FGF2(Peprotech,目录号:100-18B)。
  2. Matrigel(BD Biosciences,目录号:354230)
  3. 诺考达唑(Sigma-Aldrich,目录号:M1404)
  4. 胸苷(Sigma-Aldrich,目录号:T1895)
  5. Aphidicholine(Sigma-Aldrich,目录号:A0781)
  6. 磷酸盐缓冲盐水(PBS)
  7. 二甲基亚砜(DMSO)
  8. 诺考达唑储备溶液(见配方)
  9. 胸苷储备液(见配方)
  10. Aphidicholine储备溶液(见配方)

设备

  1. 离心机(Eppendorf 5415D离心机)
  2. 荧光激活细胞分选仪(FACS,FACScan机,Stanford FACS设备)

程序

  1. 在G2/M阶段同步hESCs
    1. 方法I
      1. 第一天通过hESCs到matrigel。 第二天,更换培养基。
      2. 第三天,将200ng/ml的诺考达唑加入到新鲜培养基中并在37℃下孵育16小时。
      3. 16小时后,收获细胞,并准备小等分用于通过FACS进行细胞周期分析
    2. 方法二
      1. 第一天通过hESCs到matrigel。 第二天,更换培养基。
      2. 第三天,向新鲜培养基中加入2mM胸腺嘧啶并在37℃下孵育10小时
      3. 取出含有胸腺嘧啶核苷的培养基,用PBS洗涤(在室温下储存)两次,以释放胸苷中的细胞。
      4. 加入新鲜培养基,并在37℃下再孵育10小时
      5. 加入nocodazole至终浓度为200ng/ml。 在37℃下孵育24小时
      6. 收获细胞,并准备小等分用于通过FACS的细胞周期分析

  2. 在G1阶段同步hESCs
    1. 方法I
      1. 第一天通过hESCs到matrigel。 第二天,更换培养基。
      2. 第三天,向新鲜培养基中加入200ng/ml的诺考达唑并在37℃下孵育16小时。
      3. 取出含有诺考达唑的培养基,用PBS洗涤(在室温下保存)两次
      4. 加入补充有10μg/ml aphidicholine的新鲜培养基,并在37℃孵育9-10小时。
      5. 收获细胞,并准备小等分用于通过FACS的细胞周期分析
    2. 方法二
      1. 第一天通过hESCs到matrigel。 第二天,更换培养基。
      2. 第三天,向新鲜培养基中加入10μg/ml的aphidicholine,并在37℃下孵育20小时。
      3. 收获细胞,并准备小等分样品用于通过FACS的细胞周期分析

  3. 在S阶段同步hESCs
    1. 方法I
      1. 在G2/M阶段同步hESCs,如A中所述。
      2. 取出含有诺考达唑的培养基,用PBS洗涤(在室温下保存)两次
      3. 添加新鲜培养基释放细胞,孵育hESCs在37℃。 每小时收集细胞,并准备小等分用于通过FACS进行细胞周期分析。 通常S期细胞群体在出现诺卡达唑阻滞后5-6小时出现
    2. 方法二
      1. 按照B中所述同步hESC在G1阶段。
      2. 取出含有aphidicholine的培养基,用PBS洗涤(在室温下保存)两次
      3. 添加新鲜培养基释放细胞,孵育hESCs在37℃。 每小时收集细胞,并准备小等分用于通过FACS进行细胞周期分析。 通常S期细胞群出现在从aphidicholine阻滞释放后2-3小时

食谱

  1. 诺考达唑储备液
    将诺考达唑溶于DMSO中
    5mg/ml的储备浓度
  2. 胸苷储备液
    将胸苷溶解在ddH 2 O中
    库存浓度为100 mM
  3. Aphidicholine储备液
    将aphidicholine溶解在DMSO中
    库存浓度为10mg/ml

致谢

这项工作由加利福尼亚再生医学研究所,批准RL1-00100支持。

参考

  1. Becker,K.A.,Ghule,P.N.,Therrien,J.A.,Lian,J.B.,Stein,J.L.,van Wijnen,A.J.and Stein,G.S。(2006)。 人类胚胎干细胞的自我更新受缩短的G1期细胞周期的支持。 J Cell Physiol 209(3):883-893
  2. Neganova,I.,Zhang,X.,Atkinson,S。和Lako,M。(2009)。 G1至S调节成分的表达和功能分析揭示了CDK2在细胞周期调节中的重要作用 人胚胎干细胞。 癌基因 28(1):20-30
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhu, H. (2012). Synchronize Human Embryonic Stem Cells at Different Cell Cycle Stage. Bio-protocol 2(11): e193. DOI: 10.21769/BioProtoc.193.
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