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Alkaline Phosphatase Staining
碱性磷酸酶染色   

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Abstract

Two main features characterize pluripotent cells; self-renewal (unlimited cell division) and the ability to give rise to all cells of the adult organism. Given the recent impact of induced pluripotent stem cells (iPSCs) and ongoing use of pluripotent embryonic stem cells ESCs (ESCs) in basic discovery, drug development, and potential use for stem cell therapy and regenerative medicine, methods to definitively distinguish pluripotent cells from their differentiated derivatives are required. This will allow us to better understand the factors that promote their survival, self-renewal, and lineage-specific differentiation.
Undifferentiated embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) may be identified through the use of biomarker and functional assays. Biomarker assays include those for transcript and protein expression of important pluripotency transcription factors (OCT4, SOX2, and NANOG), cell surface markers (SSEA-1, -3, and -4; TRA-1-60, TRA-1-81), and Alkaline Phosphatase (AP) activity (Brambrink et.al., 2008; Ginis et al., 2004). Functional assays include: (1) the ability to generate teratomas consisting of cells from all three germ layers (endoderm, ectoderm, and mesoderm) when transplanted into immunodeficient mice or upon in vitro differentiation; (2) the ability to generate a chimera; and (3) germline transmission (Marti et al., 2013; Buehr et al., 2008). The latter two tests are ethically feasible only for mouse and other non-human pluripotent cells.
In this protocol (Campbell and Rudnicki, 2013) we describe a rapid method to screen for pluripotent cells by AP activity. AP, also known as Basic Phosphatase catalyzes the dephosphorylation of many molecules including nucleotides and proteins. AP activity is high in pluripotent cells but is greatly decreased in more differentiated cell types. The technique described herein may be used to enumerate pluripotent cells during differentiation in the presence or absence of specific genetic manipulations or small chemical modulators. It may also be used to monitor induced pluripotency using defined factors from more differentiated cell types.

Keywords: Pluripotency(多能性), Oct4(4-Oct), Stem cells(干细胞), Alkaline phosphatase(碱性磷酸酶), In vitro assay(体外试验)

Materials and Reagents

  1. Mouse ESCs (mESCs)
  2. 0.1% gelatin (Sigma-Aldrich, catalog number: G1393 )
  3. Murine embryonic fibroblast (MEF) feeder cells
  4. Alkaline Phosphatase Staining Kit (Stemgent, catalog number: 00-0009 )
    1. Fixation Solution
    2. AP Staining Solution A
    3. AP Staining Solution B
  5. 1x PBS (see Recipes)
  6. 1x PBS-T (see Recipes)
  7. 1x PBS-glycerol (see Recipes)

Equipment

  1. 6-well plate or 24-well plate
  2. Aluminum foil
  3. Light microscope with 10x objective

Procedure

  1. Cell Culture
    1. Coat plates with 0.1% gelatin. Warm gelatin to 37 °C. For each well of a 6 well plate, asceptically add 1.5 ml of 0.1% gelatin. Incubate the plates in the tissue culture hood at room temperature for 30 min. Aspirate off the gelatin and allow to dry for 10 min prior to adding media and plating cells.
    2. Seed each well with 4.75 x 105 murine embryonic feeder cells (MEFs) in 2 ml of media. Allow the MEFs to attach overnight prior to plating out the mouse ESCs.
    3. Passage actively dividing cells and culture for 3-5 days to a subconfluent density prior to staining. For mouse ESCs (mESCs) approximately 1 x 105 cells are plated to each well of a 6-well plate (or 1 x 104 cells for 24-well plate) that has been previously coated with 0.1% gelatin and plated with murine embryonic fibroblast (MEF) feeder cells.

  2. Alkaline Phosphatase Staining
    1. Aspirate the culture media from each well and rinse with 1x PBS-T.
    2. Aspirate the rinse solution and fix the cells with Fixation Solution at room temperature for two minutes. Use a sufficient volume to endure complete coverage of the cells. This would be 500 µl for each well of 24-well plate or 2 ml for each well of a 6-well plate. It is important not to exceed the fixation time of two min. Over fixation will result in the loss of AP activity and inaccurate results.
    3. Aspirate the Fixation Solution and rinse with 1x PBS-T. The wells should not be allowed to dry out.
    4. Prepare the Staining Solution by Mixing Solution A and Solution B at a 1:1 ratio. To ensure that there is sufficient volume for staining, prepare (n+1) y volumes of the solution where n is the number of wells, and y is the volume per well. For a 24-well plate y = 500 µl. For a 6-well plate y = 2 ml. The staining solution should be freshly prepared and used within five minutes of preparation.
    5. Aspirate the 1x PBS-T and pipet on the prepared Staining Solution. Cover the plate with aluminum foil in the dark for approximately 15 min. Monitor the color change (pluripotent cells will stain red/purple, MEFs will remain colorless) to avoid non-specific staining.
    6. Remove the staining solution by aspiration and wash the cells two times with 1x PBS. Do not use 1x PBS-T as this may wash out the stain.
    7. Aspirate the 1x PBS. Add 1x PBS-glycerol for long-term storage at 4 °C.

  3. Enumeration of Pluripotent Cells
    1. Count the number of AP stained (red/purple) pluripotent colonies and the differentiated (clear) colonies by eye or using a light microscope with 10x objective. The MEF feeder cells will remain clear.

Recipes

  1. 1x PBS (for 1 L)
    800 ml MilliQ H2O
    8 g NaCl
    0.2 g KCl
    1.44 g Na2PO4
    0.24 g KH2PO4
    Adjust the pH to 7.4 with HCl
    Add MilliQ H2O to a final volume of 1 L
    Filter sterilize and stored at 4 °C until use
  2. 1x PBS-T (1 L)
    800 ml MilliQ H2O
    8 g NaCl
    0.2 g KCl
    1.44 g Na2PO4
    0.24 g KH2PO4
    500 µl Tween-20
    Adjust the pH to 7.4 with HCl
    Add MilliQ H2O to a final volume of 1 L
    Filter sterilize and stored at 4 °C until use
  3. 1x PBS-Glycerol (1 L)
    500 ml MilliQ H2O
    8 g NaCl
    0.2 g KCl
    1.44 g Na2PO4
    0.24 g KH2PO4
    200 ml Glycerol
    Adjust the pH to 7.4 with HCl
    Add MilliQ H2O to a final volume of 1 L
    Filter sterilize and stored at 4 °C until use

Acknowledgments

This work was supported by grants from Genome Canada, the Ontario Genomics Institute, the Stem Cell Network, and the Canadian Institutes of Health Research.

References

  1. Brambrink, T., Foreman, R., Welstead, G. G., Lengner, C. J., Wernig, M., Suh, H. and Jaenisch, R. (2008). Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2(2): 151-159.
  2. Buehr, M., Meek, S., Blair, K., Yang, J., Ure, J., Silva, J., McLay, R., Hall, J., Ying, Q. L. and Smith, A. (2008). Capture of authentic embryonic stem cells from rat blastocysts. Cell 135(7): 1287-1298. 
  3. Campbell, P. A. and Rudnicki, M. A. (2013). Oct4 interaction with Hmgb2 regulates Akt signaling and pluripotency. Stem Cells 31(6): 1107-1120. 
  4. Ginis, I., Luo, Y., Miura, T., Thies, S., Brandenberger, R., Gerecht-Nir, S., Amit, M., Hoke, A., Carpenter, M. K., Itskovitz-Eldor, J. and Rao, M. S. (2004). Differences between human and mouse embryonic stem cells. Dev Biol 269(2): 360-380.
  5. Marti, M., Mulero, L., Pardo, C., Morera, C., Carrio, M., Laricchia-Robbio, L., Esteban, C. R. and Izpisua Belmonte, J. C. (2013). Characterization of pluripotent stem cells. Nat Protoc 8(2): 223-253. 

简介

表征多能细胞的两个主要特征;自我更新(无限细胞分裂)和产生成年生物体的所有细胞的能力。鉴于诱导多能干细胞(iPSC)和多能干胚胎干细胞ESC(ESC)在基础发现,药物开发和干细胞治疗和再生医学的潜在用途中的持续使用的最近的影响,明确区分多能细胞与其需要差异化的衍生物。这将使我们更好地了解促进其生存,自我更新和谱系特异性分化的因素。
未分化的胚胎干细胞(ESC)和诱导多能干细胞(iPSC)可以通过使用生物标志物和功能测定来鉴定。生物标志物测定包括重要多能性转录因子(OCT4,SOX2和NANOG),细胞表面标志物(SSEA-1,-3和-4; TRA-1-60,TRA-1-81)的转录物和蛋白质表达的那些, ,和碱性磷酸酶(AP)活性(Brambrink等人,2008; Ginis等人,2004)。功能测定包括:(1)当移植到免疫缺陷小鼠中或在体外分化时,产生由来自所有三个胚层(内胚层,外胚层和中胚层)的细胞组成的畸胎瘤的能力; (2)产生嵌合体的能力;和(3)种系传递(Marti等人,2013; Buehr等人,2008)。后两个测试仅在小鼠和其他非人多能细胞的伦理上可行。
在本协议中(Campbell和Rudnicki,2013),我们描述了通过AP活性筛选多能细胞的快速方法。 AP,也称为碱性磷酸酶催化包括核苷酸和蛋白质的许多分子的去磷酸化。 AP活性在多能细胞中高,但在更分化的细胞类型中大大降低。本文所述的技术可用于在存在或不存在特异性遗传操作或小化学调节剂的情况下在分化期间计数多能细胞。它还可以用于使用来自更多分化的细胞类型的限定因子来监测诱导多能性

关键字:多能性, 4-Oct, 干细胞, 碱性磷酸酶, 体外试验

材料和试剂

  1. 小鼠ESC(mESC)
  2. 0.1%明胶(Sigma-Aldrich,目录号:G1393)
  3. 小鼠胚胎成纤维细胞(MEF)饲养细胞
  4. 碱性磷酸酶染色试剂盒(Stemgent,目录号:00-0009)
    1. 固定解决方案
    2. AP染色液A
    3. AP染色液B
  5. 1x PBS(请参阅配方)
  6. 1x PBS-T(参见配方)
  7. 1x PBS-甘油(参见配方)

设备

  1. 6孔板或24孔板
  2. 铝箔
  3. 具有10倍物镜的光学显微镜

程序

  1. 细胞培养
    1. 涂有0.1%明胶的板。将明胶温热至37℃。对于6孔板的每个孔,无菌加入1.5ml的0.1%明胶。孵育板组织培养罩在室温下30分钟。吸出明胶,并允许干燥10分钟,然后添加培养基和平板细胞
    2. 在4ml培养基中用4.75×10 5个小鼠胚胎饲养细胞(MEF)接种每个孔。允许MEFs在电镀出小鼠ESC之前连接一夜。
    3. 通道主动分裂细胞和培养3-5天,在染色之前达到亚汇合密度。对于小鼠ESC(mESC),将约1×10 5个细胞接种到6孔板的每个孔(或24孔板的1×10 4个细胞)其先前已涂覆有0.1%明胶并铺有鼠胚胎成纤维细胞(MEF)饲养细胞。

  2. 碱性磷酸酶染色
    1. 从每个孔吸出培养基,用1x PBS-T冲洗。
    2. 吸出   冲洗溶液并在室温下用固定溶液固定细胞 温度2分钟。 使用足够的容量来忍受完成 覆盖的细胞。 这将是24孔的每个孔的500μl 对于6孔板的每个孔为2ml。 重要的不是 超过固定时间两分钟。 过度固定将导致 AP活动丧失和结果不准确。
    3. 吸出固定溶液,并用1x PBS-T冲洗。 井不应该干燥。
    4. 准备   通过以1:1混合溶液A和溶液B的染色溶液 比。 为了确保有足够的染色量,准备 (n + 1)y体积的溶液,其中n是孔的数量,y是 每孔的体积。 对于24孔板y =500μl。 对于6孔板   y = 2ml。 染色溶液应该新鲜制备并使用 在准备的五分钟内。
    5. 吸出1x PBS-T 并在准备的染色溶液上移液。 用盖子盖住 铝箔在黑暗中约15分钟。 监视颜色 改变(多能细胞会染红/紫色,MEFs将保留 无色),以避免非特异性染色
    6. 去除 染色溶液,并用1x洗涤细胞两次 PBS。 不要使用1x PBS-T,因为这可能会洗掉污渍。
    7. 吸出1x PBS。 加入1×PBS-甘油长期储存在4°C。

  3. 多能细胞的计数
    1. 通过目测或使用具有10倍物镜的光学显微镜计数AP染色(红/紫色)多能集落和分化(清除)集落的数目。 MEF饲养细胞将保持透明。

食谱

  1. 1x PBS(1 L)
    800ml MilliQ H 2 O 2/8克NaCl
    0.2克KCl
    1.44g Na 2 PO 4 sub/
    0.24g KH 2 PO 4 sub/
    调节pH至7.4 将MilliQ H <2> O加入到最终体积为1L的
    中 过滤灭菌,并在4°C储存,直到使用
  2. 1x PBS-T(1 L)
    800ml MilliQ H 2 O 2 / 8克NaCl
    0.2克KCl
    1.44g Na 2 PO 4 sub/
    0.24g KH 2 PO 4 sub/
    用HCl
    调节pH至7.4 添加MilliQ H sub 2 O至最终体积为1L
    过滤灭菌,并在4°C储存,直到使用
  3. 1×PBS-甘油(1L)
    500ml MilliQ H 2 O 2 / 8克NaCl
    0.2克KCl
    1.44g Na 2 PO 4 sub/
    0.24g KH 2 PO 4 sub/
    用HCl
    调节pH至7.4 添加MilliQ H sub 2 O至最终体积为1L
    过滤灭菌,并在4°C储存,直到使用

致谢

这项工作得到了来自加拿大基因组,安大略基因组研究所,干细胞网络和加拿大健康研究所的资助。

参考文献

  1. Brambrink,T.,Foreman,R.,Welstead,G.G.,Lengner,C.J.,Wernig,M.,Suh,H.and Jaenisch,R。(2008)。 在直接重编程小鼠体细胞期间多能性标记的连续表达细胞Stem Cell 2(2):151-159。
  2. Buehr,M.,Meek,S.,Blair,K.,Yang,J.,Ure,J.,Silva,J.,McLay,R.,Hall,J.,Ying,QL和Smith, )。 从大鼠胚泡中捕获真正的胚胎干细胞 细胞 135(7):1287-1298。
  3. Campbell,P.A。和Rudnicki,M.A。(2013)。 Oct4与Hmgb2的相互作用调节Akt信号转导和多能性。 干细胞< em> 31(6):1107-1120。 
  4. Ginis,I.,Luo,Y.,Miura,T.,Thies,S.,Brandenberger,R.,Gerecht-Nir,S.,Amit,M.,Hoke,A.,a a target ="_ blank"href ="http://www.ncbi.nlm.nih.gov/pubmed/15110706"> Carpenter,MK,Itskovitz-Eldor,J.和Rao,MS (2004)。 人类和小鼠胚胎干细胞之间的差异。 Dev Biol 269(2):360-380。
  5. Marti,M.,Mulero,L.,Pardo,C.,Morera,C.,Carrio,M.,Laricchia-Robbio,L.,Esteban,C.R。和Izpisua Belmonte, 多能干细胞的表征 Nat Protoc 8( 2):223-253。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Campbell, P. A. (2014). Alkaline Phosphatase Staining . Bio-protocol 4(5): e1060. DOI: 10.21769/BioProtoc.1060.
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