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Intestinal Differentiation of Human ESCs
人胚胎干细胞在肠道中的分化   

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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 human 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 human ESCs. The protocol for human ESCs is principally very similar with that for the mouse ESCs, with some modifications.

Keywords: Intestine(肠), Embryonic stem cells(胚胎干细胞), differentiation(分化), endoderm(内胚层), human(人类)

Materials and Reagents

  1. Human 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. KnockOut DMEM/F12 (Gibco®, catalog number: 12660-012 )
  9. RPMI1640 (Gibco®, catalog number: 11875-093 )
  10. Fetal Bovine Serum (FBS) (Hyclone, catalog number: SH30310.03 )
  11. 200 mM L-glutamine (L-Gln) (Nacalai Tesque, catalog number: 16948-04 )
  12. 5,000 units/ml mixture of penicillin and streptomycin (PS) (Nacalai Tesque, catalog number: 26252-94 )
  13. 10 mM MEM Non-Essential Amino Acids Solution (NEAA) (Gibco®, catalog number: 11140-050 )
  14. 2-mercaptoethanol (2-ME) (Sigma-Aldrich, catalog number: M7522 )
  15. D-(+)-Glucose (Sigma-Aldrich, catalog number: G5146 )
  16. KnockOut Serum Replacement (KSR) (Gibco®, catalog number: 10828 )
  17. B27 supplements (Gibco®, catalog number: 17504044 )
  18. Recombinant human Activin A (R&D Systems, catalog number: 338-AC )
  19. Recombinant human bFGF (Pepro Tech, catalog number: 100-18B )
  20. BIO (Calbiochem®, catalog number: 361550 )
  21. DAPT (Wako Pure Chemical Industries, catalog number: 041-30983 )
  22. Y-27432 (Wako Pure Chemical Industries, catalog number: 688000 )
  23. Mitomycin C (MMC) (Sigma-Aldrich, catalog number: M4287 )
  24. 0.05% Trypsin-EDTA (Life Technologies, catalog number: 2014-11 )
  25. 0.25% Trypsin-EDTA (Life Technologies, catalog number: 25200-072 )
  26. EF medium (see Recipes)
  27. hESC Medium (see Recipes)
  28. EB medium (see Recipes)
  29. hESCs Endoderm Medium (see Recipes)
  30. Intestinal Medium (see Recipes)

Equipment

  1. 24 well plate (Corning, catalog number: 3526 )
  2. 10 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. 5x106 M15 cells 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 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 with 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 human ESCs
    1. 1 x 105 MMC-treated MEF cells are plated to a gelatin-coated 60 mm dish with EF medium.
      Note: Lower number of MEF cells is used compared to that of the mouse ESCs.
    2. MMC-treated MEF cells are incubated in the 5% CO2 incubator at 37 °C overnight.
    3. 5x106 Human ESCs are plated on MEF-coated dish with hESC Medium in the 5% CO2 incubator at 37 °C (day 0), until passage (4-7 days).
    4. Medium is changed daily.

  4. Intestinal differentiation of human ESCs
    1. 1 x 107 MMC-treated M15 cells are plated to two gelatin-coated 24 well plates in the 5% CO2 incubator at 37 °C overnight.
    2. ESCs plated on MEF-coated dishes (step C3) are treated hESC Medium with 10 μM Y-27632, which is a ROCK inhibitor and is added to inhibit apoptosis, in the 5% CO2 incubator at 37 °C overnight (day 1).
    3. ESCs are washed by PBS.
    4. ESCs are trypsinized with 1 ml 0.25% Trypsin-EDTA at 37 °C for 5 min.
    5. ESC cells are pelleted at 190 x g for 5 min.
    6. The supernatant is removed and 1 x 106 cells/ml resuspended in EB medium.
    7. 5 x 104 ESCs in 500 μl EB medium are plated to a M15-pre-coated well (day 0).
    8. Medium is changed to hESCs Endoderm Medium at day 1, 3, 5, 7 and 9.
      Note: Human ES cells-derived definitive endodermal induction is different with that of the mouse ESCs.
    9. Medium is changed to 2 ml Intestinal Medium at day 10.
    10. Medium is changed with 2 ml Intestinal Medium at every two days (Figure 1).


      Figure 1. Human ESC-derived enterocyte at day 30. Cells expressing the intestinal transcription factor CDX2 are stained positive for alkaline phosphatase (ALP), a marker for the absorptive enterocytes. Red: CDX2, Black: ALP. Scale bar indicates 100 μm.

Recipes

  1. EF medium
    DMEM high glucose containing 10% FBS
    2 mM L-Gln
    50 units/ml PS
  2. hESC Medium
    DMEM/F12 containing 20% FBS
    2 mM L-Gln
    50 units/ml PS
    100 μM NEAA
    100 μM 2-ME
    5 ng/ml bFGF
  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. hESCs Endoderm Medium
    RPMI1260 with 100 ng/ml Activin A
    2% B27 supplements
    2 mM L-Gln
    50 units/ml PS
    100 μM NEAA
    100 μM 2-ME
  5. Intestinal Medium
    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.

简介

ES细胞(ESC)是多能性的,并且提供了研究早期胚胎发育的良好工具。肠细胞来源于定形内胚层。与成体组织干细胞相反,胚胎发育和胚胎干细胞的分化在肠道中没有得到很好的研究。存在四种分化的细胞类型的非增殖性上皮细胞:肠细胞,杯状细胞,肠内分泌细胞和Paneth细胞。肠干细胞(ISCs)和祖细胞驻留在隐窝中,有力地增殖,并且作为分化的上皮细胞的来源。在这里,我们描述了一种协议,其中分化的细胞类型的肠来自人类胚胎干细胞。在这个协议中,我们描述一个协议,通过与M15,小鼠中肾细胞系共同培养和用两种化合物治疗有效地将小鼠ES细胞分化为表达Cdx2的肠内胚层。使用的化学化合物是BIO和DAPT。 BIO是Gsk3抑制剂,激活Wnt信号通路,DAPT是抑制Notch信号通路的α-分泌酶抑制剂。 BIO和DAPT处理产生来自人ESC的所有代表性细胞谱系,肠细胞,杯状细胞,肠内分泌细胞和paneth细胞。人ESC的方案主要与用于小鼠ESC的方案非常相似,具有一些修改。

关键字:肠, 胚胎干细胞, 分化, 内胚层, 人类

材料和试剂

  1. 人胚胎干细胞(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. KnockOut DMEM/F12(Gibco ,目录号:12660-012)
  9. RPMI1640(Gibco ,目录号:11875-093)
  10. 胎牛血清(FBS)(Hyclone,目录号:SH30310.03)
  11. 200mM L-谷氨酰胺(L-Gln)(Nacalai Tesque,目录号:16948-04)
  12. 5,000单位/ml青霉素和链霉素(PS)的混合物(Nacalai Tesque,目录号:26252-94)
  13. 10mM MEM非必需氨基酸溶液(NEAA)(Gibco ,目录号:11140-050)
  14. 2-巯基乙醇(2-ME)(Sigma-Aldrich,目录号:M7522)
  15. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G5146)
  16. KnockOut血清替代品(KSR)(Gibco ®,目录号:10828)
  17. B27补充剂(Gibco ®,目录号:17504044)
  18. 重组人激活素A(R& D Systems,目录号:338-AC)
  19. 重组人bFGF(Pepro Tech,目录号:100-18B)
  20. BIO(Calbiochem ®,目录号:361550)
  21. DAPT(Wako Pure Chemical Industries,目录号:041-30983)
  22. Y-27432(和光纯药工业,目录号:688000)
  23. 丝裂霉素C(MMC)(Sigma-Aldrich,目录号:M4287)
  24. 0.05%胰蛋白酶-EDTA(Life Technologies,目录号:2014-11)
  25. 0.25%胰蛋白酶-EDTA(Life Technologies,目录号:25200-072)
  26. EF介质(见配方)
  27. hESC Medium(请参阅配方)
  28. EB介质(参见配方)
  29. hESCs内胚层培养基(见配方)
  30. 肠介质(见配方)

设备

  1. 24孔板(Corning,目录号:3526)
  2. 10mm圆盘(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5×10 6个M15细胞接种到具有10ml EF培养基的100mm培养皿中
    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细胞重悬浮并冷冻于含有22%FBS和含有DMEM的20%DMSO的冷冻管中,每管1×10 7个细胞。
  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. 维持人类ESC
    1. 将1×10 5个MMC处理的MEF细胞接种到具有EF培养基的明胶包被的60mm皿中。
      注意:与小鼠ESC相比,使用较低数量的MEF细胞。
    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5×10 6个人ESC在37℃下在5%CO 2培养箱中的hESC培养基(第0天)上铺板在MEF包被的皿上,直到传代(4-7 天)。
    4. 每天更换培养基。

  4. 人类ESC的肠分化
    1. 将1×10 7个MMC处理的M15细胞在37℃下在5%CO 2培养箱中接种到两个明胶包被的24孔板上过夜。
    2. 在5%CO 2培养箱中在37℃下,用10μMY-27632(其为ROCK抑制剂并加入以抑制凋亡)处理铺在MEF包被的培养皿上的ESC(步骤C3) ℃过夜(第1天)。
    3. 用PBS洗涤ESC
    4. 将ESC用1ml 0.25%胰蛋白酶-EDTA在37℃下胰蛋白酶消化5分钟
    5. ESC细胞在190×g下沉淀5分钟。
    6. 除去上清液,并将1×10 6个细胞/ml重悬浮于EB培养基中。
    7. 将5×10 4个在500μlEB培养基中的ESC接种到M15预包被的孔(第0天)。
    8. 在第1,3,5,7和9天将培养基更换为hESCs内胚层培养基。
      注意:人ES细胞衍生的定形内胚层诱导与小鼠ESC的不同。
    9. 在第10天将培养基更换为2ml肠道培养基。
    10. 每两天用2ml肠内培养基更换培养基(图1)。


      图1.第30天时人ESC诱导的肠细胞 。表达肠转录因子CDX2的细胞对于碱性磷酸酶(ALP)染色为阳性,碱性磷酸酶(ALP)是吸收性肠细胞的标志物。红色:CDX2,黑色:ALP。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. hESC中等
    含有20%FBS的DMEM/F12 2 mM L-Gln
    50单位/ml PS
    100μMNEAA
    100μM2-ME
    5 ng/ml bFGF
  3. EB介质
    含有10%FBS的DMEM高葡萄糖 2 mM L-Gln
    50单位/ml PS
    100μMNEAA
    100μM2-ME
  4. hESCs内胚层培养基
    RPMI1260与100ng/ml激活素A
    2%B27补充剂
    2 mM L-Gln
    50单位/ml PS
    100μMNEAA
    100μM2-ME
  5. 肠介质
    DMEM低葡萄糖与1mg/ml D - (+) - 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)。 这项工作得到了一个Grant-in-Aid(#21390280到SK和#21790671到NS)和部分由全球COE赠款(Cell Fate监管研究和教育单位,SK)的支持,并从项目 从教育,文化,体育,科学技术部(MEXT)日本实现再生医学。

参考文献

  1. Ogaki,S.,Shiraki,N.,Kume,K。和Kume,S。(2013)。 Wnt和Notch信号指导胚胎干细胞分化为肠系谱。 Stem Cells 31(6):1086-1096。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ogaki, S. and Kume, S. (2013). Intestinal Differentiation of Human ESCs. Bio-protocol 3(24): e1010. DOI: 10.21769/BioProtoc.1010.
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