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Mouse Embryonic Stem Cell Differentiation to Hematopoietic Precursors
小鼠胚胎干细胞(ES)分化造血前体   

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参见作者原研究论文

本实验方案简略版
The Journal of Biological Chemistry
May 2003

Abstract

Embryonic stem cells are derived from inner cell mass of an embryo that can differentiate into every cell type in the body. Clinically, cultured red blood cell supply is of great interest. However, some of the hurdles need to be overcome. This protocol describes a two step protocol to form embryoid body and differentiate them in to hematopoietic lineage cells, especially erythroid cells.

Materials and Reagents

  1. Mouse embryonic stem cell (ES cells)
  2. 200 mM L-Glutamine (100 ml) (Life Technologies, Gibco®, catalog number: 320-5030AG )
  3. Methylcellulose (STEMCELL Technologies, catalog number: 03134 )
  4. Gelatin (STEMCELL Technologies, catalog number: 07903 )
  5. Transferrin (Human) (Boehringer-Mannheim) (Roche Diagnostics, catalog number: 652202 )
  6. MTG (Sigma-Aldrich, catalog number: M6145 )
  7. Plasma derived serum (PDS) (Animal technologies) special order from company. Typically serum contains some PDGF, which inhibit differentiation (Must call company, animal technologies, Inc)
  8. Epo (Amgen) (Thermo Fisher Scientific, catalog number: 50948385 )
  9. Protein Free Hybridoma Media II (PFHM-II) (Life Technologies, Gibco®, catalog number: 12040-077 )
  10. Ascorbic acid
  11. Penicillin/Streptomycin (Pen/Strep) (Thermo Fisher Scientific, catalog number: SV30010 )
  12. Fetal bovine serum (FBS)
  13. Fetal calf serum (FCS)
  14. Phosphate buffered saline (PBS)
  15. Trypsin
  16. EDTA
  17. NaHCO3
  18. Dulbecco's modified eagle medium (DMEM)
  19. ES-IMDM (see Recipes)
  20. Differentiation medium (see Recipes)
  21. Secondary differentiation media (see Recipes)
  22. Iscove’s modified dulbecco medium (IMDM) (Life Technologies, Gibco®, catalog number: 12200-036 , 1 L/pack) (see Recipes)
  23. Cellulase (Sigma-Aldrich, catalog number: C-1794 ) (see Recipes)

Equipment

  1. Standard table-top centrifuges
  2. T25 flask
  3. Snap cap tube
  4. Incubator
  5. Water bath
  6. Gauge needle
  7. 50 ml Falcon tube
  8. 0.22 µm filter

Procedure

  1. Primary differentiation of ES cells (making Embryoid Bodies)
    1. Two days prior to setting up differentiation, split cells into ES-IMDM medium without feeder cells in gelatinized T25 flask (this dilutes out most of the STO cells). Add 4 x 105 ES cells per T25 flask.
    2. Change medium the next day (ES cells grow faster therefore use up nutrients faster; doubles every 8 h).
    3. Set up differentiation as follows.
      1. Aspirate medium from the flask.
      2. Add 1 ml of trypsin/EDTA, swirl around, and quickly remove.
      3. Add 1 ml of trypsin/EDTA and wait until cells start to detach. It usually takes about 1-2 min. Do not over-trypsinize cells.
      4. Deactivate the trypsin by adding 1 ml FCS (differentiation serum) and 4 ml IMDM and pipette up and down to make single cell suspensions. It is important not to have cell clumps. Transfer to 15 ml snap cap tube.
      5. Centrifuge for 5-10 min at 1,000 rpm.
      6. Resuspend the pellet in 10 ml IMDM (w/o FCS; this removes LIF and any other factors that might interfere with differentiation). Spin down cells for 5-10 min at 1,000 rpm.
      7. Resuspend the pellet in 5 ml IMDM (w/ differentiation serum) and count the VIABLE ES cell number using eosin (0.2% eosin in 1× PBS). Make sure to count ES cells only.
      8. Add 5,000-6,000 ES cells/ml of differentiation media for day 2.75-3 EBs. For ChIP purpose, I prepared 8-10 dishes and prepared enough media for one extra dish. Add 4,000-5,000 cells/ml for day 4-5 EBs. Add 500-2,000 cells/ml for day 6-10 EBs. Add more cells (7-8,000 cells/ml for day 2.75-3 EBs) if they differentiate poorly. Too many cells could result in poor differentiation of EBs. You don’t want the cells to use up the nutrients before they reach the certain days.
      9. Primary differentiation to derive EBs is typically done in 10 ml/10 cm dish. Use bacterial petri dish (Valmark (good), Fisher (poor) VWR (not recommended)). Do not use tissue culture dishes because all the cells will adhere and will not form EBs. If you have a way to constantly shake your dish in the incubator this improves efficiency, otherwise shake your dish 2-3 times a day until the EBs are ready.

  2. Hematopoietic precursor analysis (secondary differentiation)
    1. Embryoid body (EB) harvest.
      1. EBs in liquid: Transfer media containing EBs into 50 ml tubes. Wash the dish with IMDM and pool. Let it sit at room temperature (RT) for about 10-20 min. EBs will settle down to the bottom of the tube.
      2. EBs in methylcellulose: Add equal volume of cellulase (2 units/ml, final 1 unit/ml) and incubate 20 min at 37 °C. Collect EBs in 50 ml tubes. Wash the dish with IMDM. Let it sit at RT for about 5-10 min. EBs will settle down to the bottom of the tube.
    2. While the EBs are settling down prepare replating media enough for one extra plate (if you need 10 plates prepare media for 11 plates) look at the recipe for details.
    3. Aspirate off as much media as possible minimizing the EB loss, add 3 ml trypsin and incubate for 3 min at 37 °C (use water bath). Very important to keep the time. It is directly related with survival and differentiation potential of ES cells. If you are harvesting for FACS analysis, 7.5 mM EDTA should be used to dissociate the EBs. This is more harsh treatment, however, receptors are kept intact.
    4. Quickly vortex briefly and add 1 ml FCS (differentiation serum).
    5. Dissociate by passing 8-10 times through a 20 gauge needle. Transfer to 15 ml tube and spin for 5-10 min at 1,000 rpm. Use collagenase for older EBs (i.e. >day 9). When collagenase is used, incubate cells for 1 h at 37 °C.
    6. Resuspend the pellet in 0.3-1 ml of IMDM (w/ 10% FCS). Smaller the volume the better. Count the viable cells. At this point, there should be no cell clumps.
    7. Add EBs to methylcellulose replating media. Shake well and let it sit for few minutes for bubbles to rise up. Using the 16 gauge blunt-end needle dispence the media+EBs on to appropriate dishes (you can use any bacterial dish for this). Always prepare bit more than what you need. Methylcellulose is sticky.
      1. Small scale differentiation: Add 3-6 x 104 EBs ml-1 of methylcellulose replating media. Add 1.25 ml methylcellulose mix to each 35 mm bacterial dishes for blast colony assay. Otherwise, add 1 ml each. Prepare three replica dishes for each sample, i.e. make 4.5 ml methylcellulose replating media for blast colony assay and make 4 ml methylcellulose replating media for erythroid and other myeloid colony assay.
      2. For ChIP: Add 1-1.5 x 105 EBs/ml of methylcellulose replating media. Add 10 ml methylcellulose mix to each 10 cm dishes. 10 x 10 cm plates will yield enough cells for 4 IPs (8-10 x 107).
    8. Place the plates in 245 mm square dish and a small 35 mm dish with water to maintain the humidity. This prevents the gel from drying up and cracking.

  3. Harvest
    1. Prepare celluase + PBS solution (2 μg/ml)
    2. Add same volume of cellulase solution to each plate so the final concentration of cellalase is 1 μg/ml.
    3. Incubate at 37 °C for 30-40 min until methocell is watery.
    4. Scrape plates and collect cells in 50 ml Falcon tube. Vortex and spin at 1,000 rpm, 5 min.
    5. Wash with fresh media or PBS and spin at 1,000 rpm, 5 min.

Recipes

  1. IMDM
    For 1 L
    1 pack IMDM powder
    10 ml/liter Pen/Strep
    3.024 g/L NaHCO3
    Filter thru 0.22 µm filter
    If using premade liquid media -> add 5.05 ml of P/S
  2. ES-IMDM
    This media is essentially the same as ES-DMEM but use IMDM (richer media) instead of DMEM. Used for gelatin+ES cell culture. This is used for gelatin+ES cells and for primary differentiation to generate EBs.
  3. Differentiation medium
    15% pre-selected FCS in IMDM with the followings
    1% ascorbic acid (5 mg/ml): Make ascorbic acid fresh each time you set up a differentiation. Dissolve ascorbic acid (5 mg/ml) in H2O and filter sterilize (0.22 µm).
    1% L-glutamine: Once thawed use it for 1 wk.
    3 µl/ml MTG (MTG is diluted by adding 26 µl of MTG to 2 ml IMDM).
  4. Cellulase
    2 U/ml in 1× PBS filter sterilize with 0.45 µm filter.
  5. Secondary differentiation media (methylcellulose mix)a (all % is v/v)

    Erythroid
    115 ml for 10 plates
    Methylcelluloseb
    44%
    46 ml
    PDS
    10%
    11.5 ml
    Ascorbic acid (5 mg/ml)
    0.25%
    287 µl
    L-glutamine
    1%
    1.15 ml
    Transferrin
    1%
    1.15 ml
    MTG (26 µl per 2 ml)c
    3 µl/ml
    345 µl
    Epo
    2 unit/ml
    5.75 µl
    PFHM-IId
    5%
    5.75 ml
    IMDM
    To 100%
    48.8 ml

    1. If efficiency of EB plating decreases, replace one or more of the following with fresh aliquots or lots: PFHM II, IMDM, MTG, FBS.
    2. Prewarm methylcellulose to 37 °C to decrease viscosity. Discard after it has been thawed and stored at 4 °C for >1 month. If desired, methylcellulose can be spun down at 6,000 rpm for 10 min to remove debris (transfer to new conical when spin is complete). Methocell you get from stem cell tech. is 2.5%. Calculations are made so that final methocell is 1.1%.
    3. Close MTG and PFHM II caps immediately after use to prevent oxidation. When opening a new 50 ml aliquots of PFHM II, sub-aliquot into five 10 ml aliquots to prevent oxidation (cover conical tubes with foil to protect from light).
    4. Various Factors added include bFGF (10 ng/ml), activin A (2-10 ng/ml), BMP-2 (2-10 ng/ml), and BMP-4 (2-10 ng/ml).  Add KL (final 1%) and IL-3 (final 1%) for >day 6 EBs. Feed for > day 9 EBs on day 6 (4-5 ml per 10 cm petridish). Make the same methyl cellulose cocktail with 20% methylcellulose instead of 44%.

Acknowledgments

This protocol was adapted from Im et al., (2003). Funding for this work was obtained from the NIH.

References

  1. Zhang, W. J., Chung, Y. S., Eades, B. and Choi, K. (2003). Gene targeting strategies for the isolation of hematopoietic and endothelial precursors from differentiated ES cells. Methods Enzymol 365: 186-202.
  2. Im, H., Park, C., Feng, Q., Johnson, K. D., Kiekhaefer, C. M., Choi, K., Zhang, Y. and Bresnick, E. H. (2003). Dynamic regulation of histone H3 methylated at lysine 79 within a tissue-specific chromatin domain. J Biol Chem 278(20): 18346-18352.

简介

胚胎干细胞是从小鼠囊胚期内细胞团分离得到。这些内细胞团可以分化成身体内每一种细胞类型。红细胞培养在临床上引起了极大兴趣。然而,有一些障碍需要克服。这份指南介绍了两部分内容,胚体形成和将其分化到造血系细胞,特别是红系细胞。

材料和试剂

  1. 小鼠胚胎干细胞(ES细胞)
  2. 200mM L-谷氨酰胺(100ml)(Life Technologies,Gibco ,目录号:320-5030AG)
  3. 甲基纤维素(STEMCELL Technologies,目录号:03134)
  4. 明胶(STEMCELL Technologies,目录号:07903)
  5. 转铁蛋白(人)(Boehringer-Mannheim)(Roche Diagnostics,目录号:652202)
  6. MTG(Sigma-Aldrich,目录号:M6145)
  7. 血浆来源血清(PDS)(动物技术)特殊订单从公司。 通常血清含有一些抑制分化的PDGF(必须叫公司,动物技术公司)
  8. Epo(Amgen)(Thermo Fisher Scientific,目录号:50948385)
  9. 无蛋白的杂交瘤培养基II(PFHM-II)(Life Technologies,Gibco ,目录号:12040-077)
  10. 抗坏血酸
  11. 青霉素/链霉素(Pen/Strep)(Thermo Fisher Scientific,目录号:SV30010)
  12. 胎牛血清(FBS)
  13. 胎牛血清(FCS)
  14. 磷酸盐缓冲盐水(PBS)
  15. 胰蛋白酶
  16. EDTA
  17. NaHCO 3
  18. Dulbecco改良的Eagle培养基(DMEM)
  19. ES-IMDM(参见配方)
  20. 分化介质(参见配方)
  21. 次级分化培养基(参见配方)
  22. Iscove改良的dulbecco培养基(IMDM)(Life Technologies,Gibco ,目录号:12200-036,1L/pack)(参见Recipes)
  23. 纤维素酶(Sigma-Aldrich,目录号:C-1794)(参见Recipes)

设备

  1. 标准台式离心机
  2. T25烧瓶
  3. 卡口管
  4. 孵化器
  5. 水浴
  6. 计量针
  7. 50ml Falcon管
  8. 0.22μm过滤器

程序

  1. ES细胞的初级分化(制备类胚体)
    1. 在建立分化之前两天,将细胞分裂成在糊化T25烧瓶中没有饲养细胞的ES-IMDM培养基(这稀释了大多数STO细胞)。 每个T25烧瓶中加入4×10 5个ES细胞
    2. 第二天更换培养基(ES细胞生长更快,因此使用更快的营养成分;每8小时增加一倍)
    3. 设置差异如下。
      1. 从烧瓶中吸出培养基。
      2. 加入1ml胰蛋白酶/EDTA,涡旋,迅速除去
      3. 加入1毫升胰蛋白酶/EDTA,等待细胞开始分离。 它通常需要约1-2分钟。 不要过度胰蛋白酶消化细胞。
      4. 通过加入1毫升FCS(分化血清)和4毫升IMDM停止胰蛋白酶,吸移管向上和向下,使单细胞悬浮液。 重要的是不要有细胞团。 转移到15毫升卡通管。
      5. 以1,000 rpm离心5-10分钟。
      6. 将沉淀重悬在10ml IMDM(w/o FCS;这可以去除LIF和任何其他可能干扰分化的因素)。以1,000 rpm速度旋转细胞5-10分钟。
      7. 将沉淀重悬在5ml IMDM(w /分化血清)中,并使用伊红(0.2%曙红在1×PBS中)计数VIABLE ES细胞数。确保只计数ES细胞。
      8. 加入5,000-6,000个ES细胞/ml分化培养基,用于2.75-3EB。对于ChIP目的,我准备8-10盘和准备足够的媒体一个额外的菜。添加4,000-5,000个细胞/ml的第4-5天EB。加入500-2,000个细胞/ml,用于6-10天的EB。添加更多的细胞(7-8,000个细胞/ml,2.75-3 EB),如果他们分化差。太多的细胞可能导致EB的差的分化。你不想让细胞在达到某些日子之前用完营养。
      9. 通常在10ml/10cm培养皿中进行初级分化以得到EB。使用细菌培养皿(Valmark(好),Fisher(差)VWR(不推荐))。不要使用组织培养皿,因为所有的细胞将粘附,不会形成EB。如果你有办法不断地摇动你的菜在孵化器,这会提高效率,否则摇动你的菜,每天2-3次,直到EB准备好。

  2. 造血前体分析(二次分化)
    1. 胚状体(EB)收获。
      1. 液体中的EB:将含有EB的介质转移到50ml管中。 用IMDM和游泳池洗菜。 使其在室温(RT)下静置约10-20分钟。 EB将沉降到管的底部。
      2. EB在甲基纤维素中:加入等体积的纤维素酶(2单位/ml,最终1单位/ml)并在37℃下孵育20分钟。 收集EB在50ml管中。 用IMDM洗菜。 让它在室温下放置约5-10分钟。 EB将沉降到管的底部。
    2. 当EB放置准备复制介质足够一个额外的板(如果你需要10板准备介质为11板)看细节的食谱。
    3. 吸出尽可能多的媒体尽可能最小化EB损失,添加3毫升胰蛋白酶,并在37℃孵育3分钟(使用水浴)。非常重要的是保持时间。它与ES细胞的存活和分化潜能直接相关。如果你收获的FACS分析,7.5 mM EDTA应该用于解离的EB。这是更苛刻的治疗,但是,受体保持完好
    4. 快速涡旋,加入1ml FCS(分化血清)
    5. 通过用20号针头通过8-10次来解离。转移到15毫升管和旋转5-10分钟,在1000转。对较老的EB使用胶原酶( >第9天)。当使用胶原酶时,在37℃孵育细胞1小时
    6. 将沉淀重悬在0.3-1ml IMDM(w/10%FCS)中。体积越小越好。计数活细胞。在这一点上,应该没有细胞团块。
    7. 将EB加入甲基纤维素复制培养基中。摇匀,让它坐几分钟,气泡起来。使用16号钝端针将培养基+ EB放在适当的培养皿上(可以使用任何细菌培养皿)。总是准备比你需要的更多。甲基纤维素是粘性的。
      1. 小规模分化:添加甲基纤维素复制培养基的3-6×10 4次EBs ml -1 -1次。加入1.25毫升甲基纤维素混合物到每个35毫米细菌盘用于胚芽集落测定。否则,每个添加1ml。为每个样品制备三个复制皿,即,制备4.5ml甲基纤维素复制培养基用于胚细胞集落测定,并制备4ml用于红细胞和其它骨髓集落测定的甲基纤维素复制培养基。
      2. 对于ChIP:加入1-1.5×10 5 EBs/ml的甲基纤维素复制培养基。向每个10cm培养皿中加入10ml甲基纤维素混合物。 10×10cm板将产生足够的用于4个IP(8-10×10 7个)的细胞。
    8. 将板放置在245mm方形盘和小的35mm盘中用水保持湿度。这防止凝胶干燥和开裂。

  3. 收成
    1. 制备纤维素酶+ PBS溶液(2μg/ml)
    2. 向每个板中加入相同体积的纤维素酶溶液,使得细胞酶的最终浓度为1μg/ml
    3. 在37℃下孵育30-40分钟,直到细胞水化
    4. 刮板和收集细胞在50毫升Falcon管。 涡旋并以1,000rpm旋转5分钟
    5. 用新鲜培养基或PBS洗涤,并以1,000rpm,5分钟旋转

食谱

  1. IMDM
    对于1 L
    1包IMDM粉
    10ml /升Pen/Strep
    3.024g/L NaHCO 3 3/
    过滤通过0.22μm过滤器
    如果使用预制液体介质 - 加入5.05ml P/S
  2. ES-IMDM
    这种媒体基本上与ES-DMEM相同,但使用IMDM(更丰富的媒体)而不是DMEM。 用于明胶+ ES细胞培养。 这用于明胶+ ES细胞和原代分化以产生EB
  3. 分化媒介
    在IMDM中15%预选的FCS,具有以下
    1%抗坏血酸(5 mg/ml):每次设置分化时,使抗坏血酸新鲜。 将抗坏血酸(5mg/ml)溶解在H 2 O中并过滤除菌(0.22μm)。
    1%L-谷氨酰胺:一旦解冻,使用1周。
    3μl/ml MTG(MTG通过向2ml IMDM中加入26μlMTG而稀释)。
  4. 纤维素酶
    2U/ml在1×PBS过滤器中用0.45μm过滤器灭菌。
  5. 二级分化培养基(甲基纤维素混合物)a(所有%为v/v)

    红细胞
    10个平板上有115毫升
    甲基纤维素 b
    44%
    46 ml
    PDS
    10%
    11.5 ml
    抗坏血酸(5mg/ml)
    0.25%
    287微升
    L-谷氨酰胺 1%
    1.15 ml
    转移酶
    1%
    1.15 ml
    MTG(26μl每2ml)
    3μl/ml
    345μl
    Epo
    2单位/ml
    5.75微升
    PFHM-II d
    5%
    5.75 ml
    IMDM
    到100%
    48.8 ml

    1. 如果EB电镀效率降低,请用新鲜的等分试样或批次替换以下一种或多种:PFHM II,IMDM,MTG,FBS。
    2. 预热甲基纤维素至37℃以降低粘度。在其已经解冻并在4℃储存> 1个月后丢弃。如果需要,甲基纤维素可以以6,000rpm离心10分钟以除去碎片(当旋转完成时转移到新的锥形)。 Methocell你从干细胞技术。是2.5%。计算结果为最终测定值为1.1%
    3. 在使用后立即关闭MTG和PFHM II盖以防止氧化。当打开一个新的50毫升的PFHM II等分试样,亚等分成五个10毫升等分试样,以防止氧化(覆盖锥形管用箔保护光)。
    4. 添加的各种因子包括bFGF(10ng/ml),激活素A(2-10ng/ml),BMP-2(2-10ng/ml)和BMP-4(2-10ng/ml)。对于>第6天EB加入KL(最终1%)和IL-3(最终1%)。 Feed的>在第6天(4-5ml/10cm培养皿)的第9天EB。用20%甲基纤维素代替44%制备相同的甲基纤维素鸡尾酒

致谢

该协议改编自Imem et al。,(2003)。 这项工作的资金来自NIH。

参考文献

  1. Zhang,W.J.,Chung,Y.S.,Eades,B.and Choi,K。(2003)。 基因靶向策略,用于从分化的ES细胞分离造血和内皮前体。 em> Methods Enzymol 365:186-202
  2. Im,H.,Park,C.,Feng,Q.,Johnson,K.D.,Kiekhaefer,C.M.,Choi,K.,Zhang,Y.and Bresnick,E.H。(2003)。 在组织特异性染色质结构域内赖氨酸79甲基化的组蛋白H3的动态调节。 J Biol Chem 278(20):18346-18352
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Im, H. (2012). Mouse Embryonic Stem Cell Differentiation to Hematopoietic Precursors. Bio-protocol 2(7): e144. DOI: 10.21769/BioProtoc.144.
  2. Im, H., Park, C., Feng, Q., Johnson, K. D., Kiekhaefer, C. M., Choi, K., Zhang, Y. and Bresnick, E. H. (2003). Dynamic regulation of histone H3 methylated at lysine 79 within a tissue-specific chromatin domain. J Biol Chem 278(20): 18346-18352.
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