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ES cells (ESCs) are pluripotent and offer a good tool to study early embryonic development. Intestinal cells are derived from the definitive endoderm. In contrast to adult tissue stem cells, embryonic development and differentiation from ES cells have not been as well investigated in the intestine. There are four differentiated cell types of non-proliferative epithelial cells: enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Intestinal stem cells (ISCs) and progenitor cells reside in crypts, proliferate vigorously, and function as the source of differentiated epithelial cells. Here, we describe a protocol, in which differentiated cell types of the intestine are derived from mouse ESCs. In this protocol, we describe a protocol to differentiate mouse ES cells into Cdx2-expressing intestinal endoderm efficiently by co-culturing with M15, a mouse mesonephric cell line, and treatment with two chemical compounds. The chemical compounds used are BIO and DAPT. BIO is a Gsk3 inhibitor, that activate Wnt signaling pathway, and DAPT is a-secretase inhibitor that inhibit Notch signaling pathway. BIO and DAPT treatment yielded all representative cell lineages, enterocytes, goblet cells, enteroendocrine cells and paneth cells, to be derived from mouse ESCs.

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Intestinal Differentiation of Mouse ESCs
小鼠胚胎干细胞在肠道中的分化

干细胞 > 胚胎干细胞 > 维持和分化
作者: Soichiro Ogaki
Soichiro OgakiAffiliation: Department of Stem Cell Biology, Kumamoto University, Kumamoto, Japan
Bio-protocol author page: a1078
 and Shoen Kume
Shoen KumeAffiliation: Department of Stem Cell Biology, Kumamoto University, Kumamoto, Japan
For correspondence: skume@kumamoto-u.ac.jp
Bio-protocol author page: a1079
Vol 3, Iss 24, 12/20/2013, 2340 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1011

[Abstract] ES cells (ESCs) are pluripotent and offer a good tool to study early embryonic development. Intestinal cells are derived from the definitive endoderm. In contrast to adult tissue stem cells, embryonic development and differentiation from ES cells have not been as well investigated in the intestine. There are four differentiated cell types of non-proliferative epithelial cells: enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Intestinal stem cells (ISCs) and progenitor cells reside in crypts, proliferate vigorously, and function as the source of differentiated epithelial cells. Here, we describe a protocol, in which differentiated cell types of the intestine are derived from mouse ESCs. In this protocol, we describe a protocol to differentiate mouse ES cells into Cdx2-expressing intestinal endoderm efficiently by co-culturing with M15, a mouse mesonephric cell line, and treatment with two chemical compounds. The chemical compounds used are BIO and DAPT. BIO is a Gsk3 inhibitor, that activate Wnt signaling pathway, and DAPT is a-secretase inhibitor that inhibit Notch signaling pathway. BIO and DAPT treatment yielded all representative cell lineages, enterocytes, goblet cells, enteroendocrine cells and paneth cells, to be derived from mouse ESCs.

[Abstract]

Materials and Reagents

  1. Mouse embryonic stem cells (ESCs)
  2. M15 cells (ECACC: catalog number: 95102517 )
  3. Mouse embryonic fibroblast (MEF)
  4. 0.1% gelatin
  5. PBS
  6. Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco®, catalog number: 11995-075)
  7. DMEM low glucose (Gibco®, catalog number: 11885-084 )
  8. Fetal Bovine Serum (FBS) (Hyclone, catalog number: SH30310.03 )
  9. 200 mM L-glutamine (L-Gln) (Nacalai Tesque, catalog number: 16948-04 )
  10. 5,000 units/ml mixture of penicillin and streptomycin (PS) (Nacalai Tesque, catalog number: 26252-94 )
  11. 10 mM MEM Non-Essential Amino Acids Solution (NEAA) (Gibco®, catalog number: 11140-050 )
  12. 2-mercaptoethanol (2-ME) (Sigma-Aldrich, catalog number: M7522 )
  13. D-(+)-Glucose (Sigma-Aldrich, catalog number: G5146 )
  14. KnockOut Serum Replacement (KSR) (Gibco®, catalog number: 10828 )
  15. Recombinant human LIF (Wako Pure Chemical Industries, catalog number: 129-05601 )
  16. Recombinant human Activin A (R&D Systems, catalog number: 338-AC )
  17. Recombinant human bFGF (Pepro Tech, catalog number: 100-18B )
  18. BIO (Calbiochem®, catalog number: 361550 )
  19. DAPT (Wako Pure Chemical Industries, catalog number: 041-30983 )
  20. Mitomycin C (MMC) (Sigma-Aldrich, catalog number: M4287 )
  21. 0.05% Trypsin-EDTA (Life Technologies, catalog number: 2014-11)
  22. 0.25% Trypsin-EDTA (Life Technologies, catalog number: 25200-072 )
  23. EF medium (see Recipes)
  24. Maintenance Medium (see Recipes)
  25. EB medium (see Recipes)
  26. Endoderm Medium (see Recipes)
  27. Intestinal Medium (see Recipes)

Equipment

  1. 6 well plate (Corning, catalog number: 3516)
  2. 100 mm dish (Corning, catalog number: 430167 )
  3. 60 mm dish (BD Biosciences, Falcon®, catalog number: 353004 )
  4. Centrifuge
  5. 37 °C 5% CO2 Cell culture incubator

Procedure

  1. Preparation of M15 cells
    1. M15 cells (5x106) are plated to a 100 mm dish with 10 ml EF medium.
    2. M15 cells are incubated in the 5% CO2 incubator at 37 °C.
    3. When M15 cells are confluent (for 2-3 days), passage from a 100 mm dish to five 100 mm dishes.
      1. M15 cells are washed by PBS.
      2. M15 cells are trypsinized with 1 ml 0.05% Trypsin-EDTA at 37 °C for 5 min.
      3. M15 cells are pelleted at 190 x g for 5 min.
      4. M15 cells are plated to five 100 mm dishes.
    4. When M15 cells are confluent, M15 cells are incubated with 10 μg/ml MMC containing EF medium in the 5% CO2 incubator at 37 °C for 2 h. M15 cells are treated with MMC to stop M15 cell from proliferation.
    5. MMC-treated M15 cells are washed by PBS.
    6. MMC-treated M15 cells are trypsinized with 1 ml 0.05% Trypsin-EDTA at 37 °C for 5 min.
    7. M15 cells are pelleted at 190 x g for 5 min.
    8. Resuspend and freeze MMC-treated M15 cells into cryovials in 22% FBS and 20% DMSO containing DMEM at 1 x 107 cells per tube at -150 °C.

  2. Preparation of MEF
    1. MEF (5x106) are plated to a 100 mm dish with 10 ml EF medium.
    2. MEF are incubated in the 5% CO2 incubator at 37 °C.
    3. When MEF are confluent (for 2-3 days), passage from a 100 mm dish to five 100 mm dishes.
      1. MEF are washed by PBS.
      2. MEF are trypsinized with 1 ml 0.05% Trypsin-EDTA at 37 °C for 5 min.
      3. MEF are pelleted at 190 x g for 5 min.
      4. MEF cells are plated to five 100 mm dishes.
    4. When MEF cells are confluent, MEF are incubated with 10 μg/ml MMC containing EF medium in the 5% CO2 incubator at 37 °C for 2 h. MEF are treated with MMC to stop M15 cell from proliferation.
    5. MMC-treated MEF are washed by PBS.
    6. MMC-treated MEF are trypsinized with 1 ml 0.05% Trypsin-EDTA at 37 °C for 5 min.
    7. M15 cells are pelleted at 190 x g for 5 min.
    8. Resuspend and freeze MMC-treated M15 cells into cryovials in 22% FBS and 20% DMSO containing DMEM at 2 x 106 cells per tube at -150 °C.

  3. Maintenance of mouse ESCs
    1. 2 x 106 MMC-treated MEF cells are plated to a gelatin-precoated 60 mm dish with EF medium.
    2. MMC-treated MEF cells are incubated in the 5% CO2 incubator at 37 °C overnight.
    3. Mouse ESCs (5x105) are plated on MEF-coated dish with Maintenance Medium, and are incubated in the 5% CO2 incubator at 37 °C (day 0) until passage (3 days).
    4. Medium is changed every day.

  4.  Intestinal differentiation of mouse ESCs
    1. 1 x 107 MMC-treated M15 cells are plated to two gelatin-coated 6 well plates and are incubated in the 5% CO2 incubator at 37 °C overnight.
    2. ESCs plated on MEF-coated dishes (step B3) are washed by PBS.
    3. ESCs are trypsinized with 1 ml 0.25% Trypsin-EDTA at 37 °C for 5 min.
    4. ESC cells are pelleted at 190 x g for 5 min.
    5. The supernatant is removed and 1 x 106 cells/ml resuspended in the EB Medium.
    6. 2 x 104 ESCs in 2 ml EB medium are plated to a M15-pre-coated well (day 0).
    7. Medium is changed to Endoderm Medium at day 1 and 3.
    8. Medium is changed to the Intestinal Medium at day 5.
    9. Medium is changed with 2 ml Intestinal Medium at day 7, 9, 11, 13, 15, 17 and 19 (Figure 1).


      Figure 1. Mouse ESC-derived Cdx2 (red)-expressing intestinal endoderm at day12 (left) and PAS-positive mucus secreting functional goblet cell at day 20 (Right). Scale bar indicates 100 μm.

Recipes

  1. EF medium
    DMEM high glucose containing 10% FBS
    2 mM L-Gln
    50 units/ml PS
  2. Maintenance Medium
    DMEM high glucose containing 10% FBS
    2 mM L-Gln
    50 units/ml PS
    100 μM NEAA
    100 μM 2-ME
    LIF
  3. EB Medium
    DMEM high glucose containing 10% FBS
    2 mM L-Gln
    50 units/ml PS
    100 μM NEAA
    100 μM 2-ME
  4. Endoderm Medium
    EB medium with 10 ng/ml Activin A
    5 ng/ml bFGF
  5. Intestinal Medium
    2 ml DMEM low glucose with 1 mg/ml D-(+)-Glucose
    10% KSR
    2 mM L-Gln
    50 units/ml PS
    100 μM NEAA
    100 μM 2-ME
    5 μM BIO
    10 μM DAPT

Acknowledgments

This protocol is adapted from Ogaki et al. (2013). This work was supported by a Grant-in-Aid (#21390280 to S.K. and #21790671 to N.S.), and in part by a Global COE grant (Cell Fate Regulation Research and Education Unit, to S.K.), and a grant from the Project for Realization of Regenerative Medicine from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan.

References

  1. Ogaki, S., Shiraki, N., Kume, K. and Kume, S. (2013). Wnt and Notch signals guide embryonic stem cell differentiation into the intestinal lineages. Stem Cells 31(6): 1086-1096.

材料和试剂

  1. 小鼠胚胎干细胞(ESC)
  2. M15细胞(ECACC:目录号:95102517)
  3. 小鼠胚胎成纤维细胞(MEF)
  4. 0.1%明胶
  5. PBS
  6. Dulbecco改良的Eagle培养基(DMEM)高葡萄糖(Gibco ,目录号:11995-075)
  7. DMEM低葡萄糖(Gibco ,目录号:11885-084)
  8. 胎牛血清(FBS)(Hyclone,目录号:SH30310.03)
  9. 200mM L-谷氨酰胺(L-Gln)(Nacalai Tesque,目录号:16948-04)
  10. 5,000单位/ml青霉素和链霉素(PS)的混合物(Nacalai Tesque,目录号:26252-94)
  11. 10mM MEM非必需氨基酸溶液(NEAA)(Gibco ,目录号:11140-050)
  12. 2-巯基乙醇(2-ME)(Sigma-Aldrich,目录号:M7522)
  13. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G5146)
  14. KnockOut血清替代品(KSR)(Gibco ®,目录号:10828)
  15. 重组人LIF(Wako Pure Chemical Industries,目录号:129-05601)
  16. 重组人激活素A(R& D Systems,目录号:338-AC)
  17. 重组人bFGF(Pepro Tech,目录号:100-18B)
  18. BIO(Calbiochem ®,目录号:361550)
  19. DAPT(Wako Pure Chemical Industries,目录号:041-30983)
  20. 丝裂霉素C(MMC)(Sigma-Aldrich,目录号:M4287)
  21. 0.05%胰蛋白酶-EDTA(Life Technologies,目录号:2014-11)
  22. 0.25%胰蛋白酶-EDTA(Life Technologies,目录号:25200-072)
  23. EF介质(见配方)
  24. 维护介质(参见配方)
  25. EB介质(参见配方)
  26. 内胚层培养基(见配方)
  27. 肠介质(见配方)

设备

  1. 6孔板(Corning,目录号:3516)
  2. 100mm皿(Corning,目录号:430167)
  3. 60mm培养皿(BD Biosciences,Falcon ,目录号:353004)
  4. 离心机
  5. 37℃5%CO 2细胞培养箱

程序

  1. M15细胞的制备
    1. 将M15细胞(5×10 6个)接种到具有10ml EF培养基的100mm皿中。
    2. 将M15细胞在37℃下在5%CO 2培养箱中温育
    3. 当M15细胞汇合(2-3天)时,从100mm培养皿传代至5个100mm培养皿
      1. M15细胞用PBS洗涤
      2. M15细胞用1ml 0.05%胰蛋白酶-EDTA在37℃下胰蛋白酶消化5分钟
      3. M15细胞在190×g下沉淀5分钟。
      4. 将M15细胞接种到5个100mm培养皿中。
    4. 当M15细胞汇合时,将M15细胞与含有EF培养基的10μg/ml MMC在5%CO 2培养箱中在37℃下温育2小时。 M15细胞用MMC处理以阻止M15细胞增殖
    5. MMC处理的M15细胞用PBS洗涤
    6. MMC处理的M15细胞用1ml 0.05%胰蛋白酶-EDTA在37℃下胰蛋白酶消化5分钟。
    7. M15细胞在190×g下沉淀5分钟。
    8. 在-150℃下将MMC处理的M15细胞重悬浮并冷冻在含有每管1×10 7个细胞的含有DMEM的22%FBS和20%DMSO的冷冻管中。
  2. MEF的制备
    1. 将MEF(5×10 6个)接种到具有10ml EF培养基的100mm培养皿中。
    2. MEF在37℃下在5%CO 2培养箱中温育
    3. 当MEF汇合(2-3天)时,从100mm培养皿通过到5个100mm培养皿
      1. MEF用PBS洗涤
      2. MEF用1ml 0.05%胰蛋白酶-EDTA在37℃下胰蛋白酶消化5分钟
      3. MEF在190×g下沉淀5分钟。
      4. 将MEF细胞接种到5个100mm培养皿中。
    4. 当MEF细胞汇合时,将MEF与含有EF培养基的10μg/ml MMC在5%CO 2培养箱中在37℃下温育2小时。 MEF用MMC处理以阻止M15细胞增殖
    5. MMC处理的MEF用PBS洗涤
    6. MMC处理的MEF用1ml 0.05%胰蛋白酶-EDTA在37℃下胰蛋白酶消化5分钟。
    7. M15细胞在190×g下沉淀5分钟。
    8. 在-150℃下将MMC处理的M15细胞重悬浮并冷冻在含有每管2×10 6个细胞的含有DMEM的22%FBS和20%DMSO的冷冻管中。
  3. 维持小鼠ESC
    1. 将2×10 6个MMC处理的MEF细胞接种到具有EF培养基的明胶预包被的60mm培养皿中。
    2. 将MMC处理的MEF细胞在5%CO 2培养箱中在37℃温育过夜。
    3. 将小鼠ESC(5×10 5)接种在具有维持培养基的MEF包被的皿上,并在37℃在5%CO 2培养箱中孵育(第0天)直到 (3天)。
    4. 每天更换培养基。

  4.  小鼠胚胎干细胞的肠分化
    1. 将1×10 7个MMC处理的M15细胞接种到两个明胶包被的6孔板中,并在5%CO 2培养箱中在37℃温育过夜。 br />
    2. 接种在MEF包被的培养皿上的ESC(步骤B3)用PBS洗涤
    3. 将ESC用1ml 0.25%胰蛋白酶-EDTA在37℃下胰蛋白酶消化5分钟
    4. ESC细胞在190×g下沉淀5分钟。
    5. 除去上清液,并将1×10 6个细胞/ml重悬于EB培养基中。
    6. 将2ml EB培养基中的2×10 4个ESC接种到M15预包被的孔(第0天)。
    7. 在第1天和第3天将培养基更换为内胚层培养基。
    8. 培养基在第5天更换为肠介质。
    9. 在第7,9,11,13,15,17和19天用2ml肠介质改变培养基(图1)。


      图1.在第12天(左)和在第20天(右)的PAS阳性粘液分泌功能杯状细胞的小鼠ESC-衍生的Cdx2(红色)表达肠内胚层。 比例尺表示100μm。

食谱

  1. EF介质
    含有10%FBS的DMEM高葡萄糖 2 mM L-Gln
    50单位/ml PS
  2. 维护介质
    含有10%FBS的DMEM高葡萄糖 2 mM L-Gln
    50单位/ml PS
    100μMNEAA
    100μM2-ME
    LIF
  3. EB中
    含有10%FBS的DMEM高葡萄糖 2 mM L-Gln
    50单位/ml PS
    100μMNEAA
    100μM2-ME
  4. 内胚层中层
    EB培养基与10ng/ml激活素A
    5 ng/ml bFGF
  5. 肠介质
    2ml含有1mg/ml D - (+) - 葡萄糖的DMEM低葡萄糖 10%KSR
    2 mM L-Gln
    50单位/ml PS
    100μMNEAA
    100μM2-ME
    5μMBIO
    10μMDAPT

致谢

该协议改编自Ogaki等人(2013)。 这项工作得到了Grant-in-Aid(#21390280至S.K.和#21790671至N.S.)的支持,部分由全球COE资助(Cell Fate Regulation Research and Education Unit, 5 ng/ml bFGF

  • 肠介质
    2ml含有1mg/ml D - (+) - 葡萄糖的DMEM低葡萄糖 10%KSR
    2 mM L-Gln
    50单位/ml PS
    100μMNEAA
    100μM2-ME
    5μMBIO
    10μMDAPT
  • 致谢

    该协议改编自Ogaki等人(2013)。 这项工作得到了Grant-in-Aid(#21390280至S.K.和#21790671至N.S.)的支持,部分由全球COE资助(Cell Fate Regulation Research and Education Unit,...

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    How to cite this protocol: Ogaki, S. and Kume, S. (2013). Intestinal Differentiation of Mouse ESCs. Bio-protocol 3(24): e1011. DOI: 10.21769/BioProtoc.1011; Full Text



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