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Mouse ESC Differentiation to Nkx2.1+ Lung and Thyroid Progenitors
小鼠ESC分化成Nkx2.1+肺和甲状腺祖细胞   

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Abstract

The de novo derivation of lung progenitors from pluripotent stem cells provides the opportunity to model early lung development in vitro and allows easy access to cells for tissue engineering or basic cell biology studies. This detailed protocol allows the generation of lung and thyroid progenitors from mouse embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC) lines. When used together with a published Nkx2.1-GFP knock-in ESC line, the protocol allows tracking and purification of lung and thyroid progenitors by sorting on the GFP reporter based on the induction of the earliest known marker of lung and thyroid cell fate, Nkx2.1. After sorting, a pure population of Nkx2.1+ cells can then be replated for further expansion, differentiation, and maturation in culture in serum-free conditions.

Keywords: Embryonic Stem Cells(胚胎干细胞), Directed Differentiation(定向分化), Lung Progenitors(肺细胞), Thyroid Progenitors(甲状腺细胞), Nkx2-1(NKX2-1)

Materials and Reagents

  1. Mouse ESCs or iPSCs carrying a GFP reporter knocked in to the Nkx2.1 locus (Nkx2.1-GFP ESCs) (Longmire et al., 2012)
  2. 1x 0.05% Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 25300-054 )
  3. Defined Fetal Bovine Serum (Hyclone, catalog number: SH30070.03 )
  4. IMDM powder (Life Technologies, Invitrogen™, catalog number: 12200-036 )
  5. NaHCO3 (Sigma-Aldrich, catalog number: S-5761 )
  6. Pen/Strep (Life Technologies, Invitrogen™, catalog number: 15140-148 ) (10,000 U Penicillin and 10 mg Streptomycin per ml)
  7. Cellgro water (VWR, catalog number: 45000-672 )
  8. Ham’s F-12 (Cellgro, catalog number: 10-080-CV )
  9. B-27 supplement with RA (Life Technologies, Invitrogen™, catalog number: 17504-044 )
  10. N-2 supplement (Life Technologies, Invitrogen™, catalog number: 17502-048 )
  11. BSA Fraction V 7.5% in PBS (Life Technologies, Invitrogen™, catalog number: 15260-037 )
  12. 1-thioglycerol (MTG) (Sigma, M6145-25ml )
  13. 200 mM L-Glutamine (Life Technologies, Invitrogen™, catalog number: 25030-081 )
  14. Ascorbic Acid (Sigma-Aldrich, catalog number: A4544-25G )
  15. 1M HEPES (Gibco, 15630-080 )
  16. CaCl2 (Sigma-Aldrich, catalog number: C4901 )
  17. BSA (Sigma-Aldrich, catalog number: A9418-10G )
  18. 100x ITS supplement (BD Biosciences, catalog number: 354352 )
  19. mNoggin (R&D Systems, catalog number: 1967-NG-025 )
  20. SB431542 (Sigma-Aldrich, catalog number: S4317 )
  21. mWnt3a (R&D Systems, catalog number: 1324-WN-010 )
  22. hBMP4 (R&D Systems, catalog number: 314-BP-050 )
  23. hEGF (R&D Systems, catalog number: 236-EG-01M )
  24. mFGF2 (R&D Systems, catalog number: catalog number: 3139-FB-025 )
  25. mFGF7 (R&D Systems, catalog number: 5028-KG-025 )
  26. hFGF10 (R&D Systems, catalog number: 345-FG-025 )
  27. Heparin sodium salt (Sigma-Aldrich, catalog number: H4784-250mg )
  28. Dexamethasone (Sigma-Aldrich, catalog number: D4902 )
  29. 8‐Br‐cAMP (Sigma-Aldrich, catalog number: B7880 )
  30. IBMX (Sigma-Aldrich, catalog number: I5879 )
  31. DMSO, Hybri-Max (Sigma-Aldrich, catalog number: D2650 )
  32. Ethanol (Sigma-Aldrich, catalog number: E7023 )
  33. Activin A (R&D Systems, catalog number: 338-AC )
  34. PBS (Life Technologies, Gibco®, catalog number: 14190-250 )
  35. 0.1% Gelatin in ultrapure water (EMD Millipore, catalog number: ES-006-B )
  36. Cxcr4 Antibody: APC Rat anti-mouse CD184 (Cxcr4) (BD-Pharmigen, catalog number: 558644 )
  37. cKit Antibody: PE Rat anti-mouse CD117 (cKit) (BD-Pharmigen, catalog number: 553355 )
  38. APC Isotype: APC Rat IgG2b, κ (BD-Pharmigen, catalog number: 553991 )
  39. PE Isotype: PE Rat IgG2b, κ (BD-Pharmigen, catalog number: 553989 )
  40. IMDM (see Recipes)
  41. Serum free differentiation medium (SFD) (see Recipes)
  42. Complete serum free differentiation medium (cSFDM) (see Recipes)
  43. BASE medium for DCI+K (see Recipes)
  44. Anteriorization medium (see Recipes)
  45. Ventralization medium (see Recipes)
  46. DCI+K medium (see Recipes)
  47. Preparation of 10x cAMP+IBMX stock (see Recipes)

Equipment

  1. P100 Petri dish (100 mm x 15 mm Bacteriological Petri Dish, nontreated polystyrene, BD Falcon, catalog number: 351029 )
  2. P150 Petri dish (150 mm x 15 mm Bacteriological Petri Dish, nontreated polystyrene, BD Falcon, catalog number: 351058 )
  3. 12 x 75 mm, 5 ml polystyrene round bottom test tube with a cell strainer cap (BD, 352235 )
  4. 1.5-ml Eppendorf Snap-Cap microcentrifuge tubes (Thermo Fisher Scientific, catalog number: 05-402-25 )
  5. Centrifuges
  6. LSRII flow cytometer
  7. 0.22μm filter

Procedure

  1. Timeline
    1. Timepoint: 0 h
      1. Nkx2-1-GFP mouse ESCs or iPSCs are cultured in 2i_LIF (serum-free, feeder-free) conditions. For each experiment, a new vial of passage 23 is thawed and cells are used after two passages.
      2. Seed 500,000 Nkx2-1-GFP mouse ESCs or iPSCs (in suspension) per P100 Petri dish with 12.5 ml/dish cSFDM.
      3. During this period of time ESCs or iPSCs will form embryoid bodies (EBs) between 100-300 μm that will remain in suspension.
    2. Timepoint: 60 h
      1. Collect EBs from one dish in a 50-ml conical, let EBs settle for 1-2 min and carefully remove most of the supernatant containing dead cells and debris.
      2. Spin 5 min, 300 x g, 4 °C.
      3. Aspirate off supernatant.
      4. Add 1 ml of 0.05% trypsin/EDTA per tube, incubate at 37 °C water bath for 1 minute while swirling the tube.
      5. Disaggregate EBs to form single cell suspension by gentle trituration.
      6. Add 1 ml serum to block the trypsin.
      7. Add 4 ml IMDM.
      8. Spin 5 min, 300 x g, 4°C.
      9. Resuspend the cells in 5 ml cSFDM.
      10. Count cells.
      11. Plate 0.5-1 x 106 cells (in suspension) per P100 or P150 Petri dish in 25 ml cSFDM supplemented with 50 ng/ml Activin A for definitive endoderm induction and EB formation.
    3. Timepoint: 120 h (Day 5)
      1. Perform staining for surrogate definitive endoderm markers (Cxcr4/cKit, see FACS protocol) to confirm efficient definitive endoderm induction (more than 40-50% of cells should be Cxcr4+/cKit+).
      2. Collect EBs and wash in IMDM.
      3. Spin 5 min, 300 x g, 4 °C.
      4. Resuspend in 10-15 ml of Anteriorization media and plate in suspension in new P100 Petri dish.
    4. Timepoint: 144 h (Day 6)
      1. Collect EBs.
      2. Wash in IMDM.
      3. Spin 5 min, 300 x g, 4 °C.
      4. Get cell count by trypsinizing a 1 ml aliquot of EBs to get single cells and calculating total cell number equivalent.
      5. Resuspend in ventralization media.
      6. Plate the EB equivalent of 50,000 cells/cm2 on gelatin--coated plates or dishes (e.g. 100,000/well of a 24--well plate). For coating procedure see step 2 below.
      7. Change media every other day.
      8. GFP+ will start emerging by Days 8-9.
    5. Timepoint: Day 15
      1. Remove ventralization media and add appropriate volume of trypsin (e.g. 1 ml per well of a 6-well plate or 3-4 ml per P100 dish).
      2. Transfer cells from several wells in a 50-ml conical, create a single cell suspension by gentle trituration and inactivate with an equal volume of FBS.
      3. Resuspend in PBS+ (PBS+2% FBS).
      4. Sort GFP+ cells.
      5. Spin 7 min, 500 x g, 4 °C (note the change in centrifuge settings).
      6. Replate 25,000 cells/cm2 (e.g. 50,000 cells/well of a 24-well plate) in cSFDM supplemented with FGF2 (250 ng/ml), FGF10 (100 ng/ml) and heparin salt (100 ng/ml).
      7. Change media every other day for 7 days.
    6. Timepoint: Day 22
      1. Remove media and rinse with PBS.
      2. Switch to DCI+K media.
      3. Harvest cells on Day 25 (procedure same as on Day 15).

  2. Gelatin coating
    Apply 0.1% gelatin (dissolved in ultrapure water) for 25-30 min at room temperature (e.g. 1 ml in a well of a 6-well plate). Aspirate gelatin, rinse with PBS and aspirate again. The plate is now ready to use.

  3. FACS for Cxcr4/cKit
    1. Cell count
      1. Remove 1 ml of EBs, spin down and trypsinize (0.5 ml trypsin), count cells.
      2. Based on previous cell count, remove a culture volume that corresponds to 2‐2.5 x 106 cells.
      3. Repeat procedure for undifferentiated ES cells.
    2. Preparation of cells for staining (all steps on ice)
      1. Spin down EBs, resuspend in 1 ml trypsin (60 sec at 37 °C), monodisperse with a P1000 pipette.
      2. Inactivate with 1 ml serum, spin down (5 min, 300 x g, 4 °C) and wash once with 5 ml IMDM.
      3. Resuspend in 500 μl PBS+ (PBS+2% FBS), cell concentration should be 0.4 - 0.5 x 106 cells/100 μl.
      4. Prepare 2 x 5 Eppendorf tubes (unstained, isotypes, Cxcr4, cKit, Cxcr4/cKit (double)), mark each tube series (D5 endoderm or undifferentiated ES cells) and transfer 100 μl of each culture to each tube.
    3. Cell staining
      1. Add the appropriate antibodies per tube (e.g. no antibodies in the “unstained” tube, both antibodies in the “double” tube).
        Antibodies used for this protocol as of 08/15/12:
        Isotype: APC Rat IgG2b, κ
        Isotype: PE Rat IgC2b, κ
        Cxcr4: APC Rat α-mouse CD184
        cKit: PE Rat α-mouse CD117
      2. Vortex briefly and transfer on ice for 30 min (cover with aluminum foil, vortex once again at 15 min).
      3. Add 1 ml PBS+ per tube, spin at 300 x g for 5 min in a tabletop centrifuge, carefully aspirate supernatant.
      4. Resuspend pellet in 500 μl PBS+.
      5. Transfer to FACS polystyrene tubes with the cell strainer cap.
      6. Take cells to LSRII for analysis.

Recipes

  1. IMDM
    1 packet IMDM powder
    3.02 g NaHCO3
    10 ml Pen/Strep
    1 L Cellgro water
    Check pH (acceptable range 6.9≤ pH ≤7.3)
  2. Serum free differentiation medium (SFD)
    IMDM-375 ml
    Ham’s F-1-125 ml
    B-27 supplement with RA-5 ml
    N-2 supplement-2.5 ml
    BSA 7.5% in PBS-3.3 ml
  3. Complete serum free differentiation medium (cSFDM)
    SFD -100 ml
    MTG 300 μl of stock (Stock: 26 μl MTG to 2 ml IMDM)
    200 mM L-Glut -1 ml
    Ascorbic Acid -1 ml of stock (Stock: 5 mg/ml distilled water, prepare fresh!)
  4. BASE medium for DCI+K
    Ham’s F-12-243.7 ml
    1.0 M HEPES (pH 7.4)-3.75 ml
    1.0 M CaCl2-200 μl
    BSA-0.625 g
    100x ITS supplement-2.5 ml
  5. Anteriorization medium
    cSFDM-10 ml
    mNoggin -100 ng ml (Stock: 10 μg/ml)
    SB431542 -10 μM (Stock: 10 mM in DMSO)
  6. Ventralization medium
    cSFDM – 10 ml
    mWnt3a - 100 ng/ml (Stock: 100 μg/ml)
    hBMP4 - 10 ng/ml (Stock: 10 μg/ml)
    hEGF - 20 ng/ml (Stock: 20 μg/ml)
    mFGF2 - 250 ng/ml (Stock: 100 μg/ml)
    mFGF7 – 10 ng/ml (Stock: 10 μg/ml)
    hFGF10 - 10 ng/ml (Stock: 10 μg/ml)
    Heparin sodium salt - 100 ng/ml (Stock: 1 mg/ml)
  7. DCI+K medium
    BASE media - 25 ml
    Dexamethasone - 50 nM (Stock: 250 μM in ethanol)
    KGF (FGF7) - 10 ng/ml (Stock: 10 μg/ml)
    cAMP+IBMX - 0.1 mM (Stock: 1 mM cAMP+1 mM IBMX)
  8. Preparation of 10x cAMP+IBMX stock
    Dissolve 22.22 mg IBMX in 1 ml of DMSO (0.1 M IBMX stock, store at -20 °C)
    To prepare the 1 mM cAMP+1mM IBMX (10x) stock, dissolve 21.5 mg 8BrcAMP in 49.5 ml of BASE media and add 0.5 ml of IBMX stock
    0.22 μm filter
    Store at 4 °C for up to 4 weeks

Acknowledgments

This protocol was originally published as part of: Longmire et al. (2012). The authors wish to thank all members of the Kotton laboratory for helpful discussions and editing. DNK is supported by NIH PO1 HL047049-16A1, 1RC2HL101535-01, 1R01 HL095993-01, 1R01 HL108678, a USAMRRA Award, an Alpha-1 Foundation Award, and an ARC award from the Evans Center for Interdisciplinary Research at Boston University. TAL is supported by NIH training grant T32 HL007035. LI is supported by R01 HL111574 and an ATS/ChILD Foundation Award.

References

  1. Gonzales, L. W., Guttentag, S. H., Wade, K. C., Postle, A. D. and Ballard, P. L. (2002). Differentiation of human pulmonary type II cells in vitro by glucocorticoid plus cAMP. Am J Physiol Lung Cell Mol Physiol 283(5): L940 - 951.
  2. Longmire, T. A., Ikonomou, L., Hawkins, F., Christodoulou, C., Cao, Y., Jean, J. C., Kwok, L. W., Mou, H., Rajagopal, J., Shen, S. S., Dowton, A. A., Serra, M., Weiss, D. J., Green, M. D., Snoeck, H. W., Ramirez, M. I. and Kotton, D. N. (2012). Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell 10(4): 398-411.
  3. Wray, J., Kalkan, T., and Smith, A.G. (2010). Revolutionizing Drug Discovery with Stem Cell Technology. Biochem Soc Trans 38, 1027-1032.

简介

来自多能干细胞的肺祖细胞的新发现提供了在体外模拟早期肺发育的机会,并且容易获得用于组织工程或基础细胞生物学研究的细胞。 该详细方案允许从小鼠胚胎干细胞(ESC)或诱导多能干细胞(iPSC)系生成肺和甲状腺祖细胞。 当与公布的Nkx2.1-GFP敲入ESC系一起使用时,该方案允许通过基于对最早已知的肺和甲状腺细胞命运的标记物的诱导在GFP报告基因上进行分选来跟踪和纯化肺和甲状腺祖细胞, Nkx2.1。 在分选后,然后可以在无血清条件下培养纯化的Nkx2.1 +细胞群,以进一步扩增,分化和成熟。

关键字:胚胎干细胞, 定向分化, 肺细胞, 甲状腺细胞, NKX2-1

材料和试剂

  1. 携带敲入Nkx2.1基因座(Nkx2.1-GFP ESC)的GFP报告基因的小鼠ESC或iPSC(Longmire等人,2012)
  2. 1x 0.05%胰蛋白酶-EDTA(Life Technologies,Gibco ,目录号:25300-054)
  3. 定义的胎牛血清(Hyclone,目录号:SH30070.03)
  4. IMDM粉末(Life Technologies,Invitrogen TM,目录号:12200-036)
  5. NaHCO 3(Sigma-Aldrich,目录号:S-5761)
  6. Pen/Strep(Life Technologies,Invitrogen TM,目录号:15140-148)(10,000U青霉素和10mg链霉素/ml)
  7. Cellgro水(VWR,目录号:45000-672)
  8. Ham's F-12(Cellgro,目录号:10-080-CV)
  9. B-27补充RA(Life Technologies,Invitrogen TM,目录号:17504-044)
  10. N-2补充剂(Life Technologies,Invitrogen TM,目录号:17502-048)
  11. BSA中的7.5%BSA(Life Technologies,Invitrogen TM,目录号:15260-037)
  12. 1-硫代甘油(MTG)(Sigma,M6145-25ml)
  13. 200mM L-谷氨酰胺(Life Technologies,Invitrogen TM,目录号:25030-081)
  14. 抗坏血酸(Sigma-Aldrich,目录号:A4544-25G)
  15. 1M HEPES(Gibco,15630-080)
  16. CaCl 2(Sigma-Aldrich,目录号:C4901)
  17. BSA(Sigma-Aldrich,目录号:A9418-10G)
  18. 100x ITS补充剂(BD Biosciences,目录号:354352)
  19. mNoggin(R& D Systems,目录号:1967-NG-025)
  20. SB431542(Sigma-Aldrich,目录号:S4317)
  21. mRnt3a(R& D Systems,目录号:1324-WN-010)
  22. hBMP4(R& D Systems,目录号:314-BP-050)
  23. hEGF(R& D Systems,目录号:236-EG-01M)
  24. mFGF2(R& D Systems,目录号:目录号:3139-FB-025)
  25. mFGF7(R& D Systems,目录号:5028-KG-025)
  26. hFGF10(R& D Systems,目录号:345-FG-025)
  27. 肝素钠盐(Sigma-Aldrich,目录号:H4784-250mg)
  28. 地塞米松(Sigma-Aldrich,目录号:D4902)
  29. 8-Br-cAMP(Sigma-Aldrich,目录号:B7880)
  30. IBMX(Sigma-Aldrich,目录号:I5879)
  31. DMSO,Hybri-Max(Sigma-Aldrich,目录号:D2650)
  32. 乙醇(Sigma-Aldrich,目录号:E7023)
  33. 激活素A(R& D Systems,目录号:338-AC)
  34. PBS(Life Technologies,Gibco ,目录号:14190-250)
  35. 0.1%明胶在超纯水(EMD Millipore,目录号:ES-006-B)中
  36. Cxcr4抗体:APC大鼠抗小鼠CD184(Cxcr4)(BD-Pharmigen,目录号:558644)
  37. cKit抗体:PE大鼠抗小鼠CD117(cKit)(BD-Pharmigen,目录号:553355)
  38. APC同种型:APC大鼠IgG 2b,κ(BD-Pharmigen,目录号:553991)
  39. PE同种型:PE大鼠IgG 2b,κ(BD-Pharmigen,目录号:553989)
  40. IMDM(请参阅配方)
  41. 无血清分化培养基(SFD)(参见配方)
  42. 完全无血清分化培养基(cSFDM)(参见配方)
  43. DCI + K的BASE介质(参见配方)
  44. 前置介质(参见配方)
  45. 中和介质(见配方)
  46. DCI + K介质(见配方)
  47. 制备10x cAMP + IBMX储液(见配方)

设备

  1. P100培养皿(100mm×15mm细菌培养皿,未处理的聚苯乙烯,BD Falcon ,目录号:351029)
  2. P150培养皿(150mm×15mm细菌培养皿,未处理的聚苯乙烯,BD Falcon ,目录号:351058)
  3. 12×75mm,具有细胞过滤帽(BD,352235)的5ml聚苯乙烯圆底试管
  4. 1.5-ml Eppendorf Snap-Cap微量离心管(Thermo Fisher Scientific,目录号:05-402-25)
  5. 离心机
  6. LSRII流式细胞仪
  7. 0.22μm过滤器

程序

  1. 时间线
    1. 时间点:0小时
      1. Nkx2-1-GFP小鼠ESC或iPSC在2i_LIF(无血清,无饲养细胞)条件下培养。 对于每个实验,将第23个通道的新小瓶解冻,并在两次传代后使用细胞
      2. 每个具有12.5ml /皿cSFDM的P100培养皿种子500,000Nkx2-1-GFP小鼠ESC或iPSC(悬浮液)。
      3. 在此期间,ESC或iPSC将形成100-300μm之间的将保持悬浮的胚状体(EB)。
    2. 时间点:60小时
      1. 从一个盘中收集EB在一个50毫升锥形,让EB定居1-2分钟,小心删除大部分含有死细胞和碎片的上清液。
      2. 旋转5分钟,300×g <4℃,4℃
      3. 吸出上清液。
      4. 每管加入1ml 0.05%胰蛋白酶/EDTA,在37℃水浴中孵育1分钟,同时涡旋管。
      5. 通过温和研磨分解EB以形成单细胞悬浮液
      6. 加入1ml血清封闭胰蛋白酶
      7. 加入4ml IMDM。
      8. 旋转5分钟,300×g <4℃,4℃
      9. 将细胞重悬于5ml cSFDM中
      10. 计数单元格。
      11. 在每个P100或P150培养皿中,在补充有50ng/ml激活素A的25ml cSFDM中平板0.5-1×10 6个细胞(悬浮液),用于定形内胚层诱导和EB形成。
    3. 时间点:120小时(第5天)
      1. 对替代定形内胚层标记物(Cxcr4/cKit,参见FACS方案)进行染色以证实有效的定形内胚层诱导(超过40-50%的细胞应该是Cxcr4 + sup/cKit sup>)。
      2. 收集EB并在IMDM中清洗。
      3. 旋转5分钟,300×g <4℃,4℃
      4. 重悬在10-15毫升前化培养基和板悬浮在新的P100培养皿
    4. 时间点:144小时(第6天)
      1. 收集EB。
      2. 在IMDM中清洗。
      3. 旋转5分钟,300×g <4℃,4℃
      4. 通过胰蛋白酶消化1ml等份的EB以获得单个细胞并计算总细胞数当量来获得细胞计数。
      5. 重悬在腹侧培养基中
      6. 在明胶包被的平板或皿(例如24孔板的100,000 /孔)上涂布50,000个细胞/cm 2的EB当量。 涂层程序参见下面的步骤2
      7. 每隔一天更换一次媒体。
      8. GFP + 将在第8-9天开始出现
    5. 时间点:第15天
      1. 取出腹腔介质并加入适当体积的胰蛋白酶(例如,每孔6ml板1ml/P100皿3-4ml)。
      2. 转移细胞从50毫升锥形的几个井,通过温和研磨形成单细胞悬浮液,并与等体积的FBS灭活。
      3. 重悬于PBS + (PBS + 2%FBS)中
      4. 排序GFP + cells。
      5. 旋转7分钟,500 x g ,4°C(注意离心机设置的变化)。
      6. 在补充有FGF2(250ng/ml),FGF10(100ng/ml)的cSFDM中复制25,000个细胞/cm 2(例如,24-孔板的50,000个细胞/ml)和肝素盐(100ng/ml)
      7. 每隔7天更换一次媒体。
    6. 时间点:第22天
      1. 取出介质并用PBS冲洗。
      2. 切换到DCI + K媒体
      3. 在第25天收获细胞(程序与第15天相同)

  2. 明胶涂层
    在室温下(例如在6孔板的孔中1ml)施加0.1%明胶(溶解在超纯水中)25-30分钟。 吸出明胶,用PBS冲洗,并再次吸出。 该板现在可以使用了。

  3. Cxcr4/cKit的FACS
    1. 细胞计数
      1. 取出1毫升EB,旋转和胰蛋白酶消化(0.5毫升胰蛋白酶),计数细胞
      2. 根据以前的细胞计数,去除对应于2-2.5×10 6 细胞的培养体积。
      3. 对未分化ES细胞重复步骤。
    2. 制备用于染色的细胞(在冰上的所有步骤)
      1. 离心EB,重悬在1ml胰蛋白酶(60秒在37℃),用P1000移液管单分散。
      2. 用1ml血清灭活,离心(5分钟,300×g,4℃),并用5ml IMDM洗涤一次。
      3. 重悬于500μlPBS中(PBS + 2%FBS),细胞浓度应为0.4-0.5×10 6细胞/100μl。
      4. 准备2×5 Eppendorf管(未染色,同种型,Cxcr4,cKit,Cxcr4/cKit(双)),标记每个管系列(D5内胚层或未分化的ES细胞),并将100μl每种培养物转移到每个管。
    3. 细胞染色
      1. 每管加入合适的抗体(例如,在"未染色"管中没有抗体,"双"管中的两种抗体)。
        用于此协议的抗体为08/15/12:
        同种型:APC大鼠IgG 2b,κ
        同种型:PE大鼠IgC 2b,κ
        Cxcr4:APC大鼠α - 小鼠CD184
        cKit:PE鼠α小鼠CD117
      2. 短暂涡旋并在冰上转移30分钟(用铝箔覆盖,在15分钟再次涡旋)
      3. 每个管中加入1ml PBS ,在台式离心机中以300×g旋转5分钟,小心地吸出上清液。
      4. 在500μlPBS +中重悬沉淀。
      5. 转移到带有细胞过滤器帽的FACS聚苯乙烯管
      6. 将细胞送到LSRII进行分析。

食谱

  1. IMDM
    1包IMDM粉末
    3.02g NaHCO 3/v/v 10ml Pen/Strep
    1 L Cellgro水
    检查pH(可接受范围6.9≤pH≤7.3)
  2. 无血清分化培养基(SFD)
    IMDM-375 ml
    火腿的F-1-125毫升
    B-27补充RA-5 ml
    N-2补充-2.5毫升
    BSA 7.5%的PBS-3.3ml
  3. 完全无血清分化培养基(cSFDM)
    SFD -100 ml
    MTG300μl储备液(库存:26μlMTG至2ml IMDM)
    200 mM L-Glut -1 ml
    抗坏血酸-1ml原液(库存:5mg/ml蒸馏水,新鲜制备)
  4. DCI + K的BASE介质
    Ham's F-12-243.7 ml
    1.0 M HEPES(pH 7.4)-3.75 ml
    1.0M CaCl 2-200μl
    BSA-0.625g
    100x ITS补充-2.5毫升
  5. 前置媒介
    cSFDM-10 ml
    mNoggin-100ng ml(储备:10μg/ml) SB431542-10μM(储备液:10mM,在DMSO中)
  6. 中和介质
    cSFDM - 10ml
    mWnt3a-100ng/ml(储备:100μg/ml) hBMP4-10ng/ml (储备:10μg/ml) hEGF-20ng/ml(储备:20μg/ml) mFGF2-250ng/ml(储备:100μg/ml) mFGF7-10ng/ml(储备:10μg/ml) hFGF10-10ng/ml(储备:10μg/ml) 肝素钠盐-100ng/ml血浆(储备液:1mg/ml)
  7. DCI + K介质
    BASE培养基 - 25 ml
    地塞米松-50nM(储备液:250μM,在乙醇中)
    KGF(FGF7)-10ng/ml(储备:10μg/ml) cAMP + IBMX-0.1mM(储备液:1mM cAMP + 1mM IBMX)
  8. 制备10x cAMP + IBMX公司
    将22.22mg IBMX溶于1ml DMSO(0.1M IBMX储备液,-20℃储存)中
    为了制备1mM cAMP + 1mM IBMX(10x)原液,将21.5mg 8BrcAMP溶解在49.5ml BASE培养基中,并加入0.5ml IBMX贮液器
    0.22μm过滤器
    在4°C下存储最多4周

致谢

该协议最初作为以下文件的一部分发布:Longmire等人(2012)。作者希望感谢Kotton实验室的所有成员进行有益的讨论和编辑。 DNK由NIH PO1 HL047049-16A1,1RC2HL101535-01,1R01 HL095993-01,1R01 HL108678,USAMRRA奖,Alpha-1基金奖和波士顿大学Evans交叉研究中心ARC奖提供支持。 TAL由NIH培训补助T32 HL007035支持。 LI由R01 HL111574和ATS/ChILD基金会奖励。

参考文献

  1. Gonzales,L.W.,Guttentag,S.H.,Wade,K.C.,Postle,A.D.and Ballard,P.L。(2002)。 通过糖皮质激素加cAMP在体外分化人类肺II型细胞。 Am J Physiol Lung Cell Mol Physiol 283(5):L940-951.
  2. 长期以来,我们的研究结果表明,该方法可以有效地降低血液中的葡萄糖浓度, Serra,M.,Weiss,DJ,Green,MD,Snoeck,HW,Ramirez,MI和Kotton,DN(2012)。 从胚胎干细胞高效地推导纯化的肺和甲状腺祖细胞。细胞 干细胞 10(4):398-411。
  3. Wray,J.,Kalkan,T.,and Smith,A.G。(2010)。 用干细胞技术革新药物发现。 Biochem Soc Trans。38,1027-1032。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Longmire, T. A., Ikonomou, L. and Kotton, D. N. (2012). Mouse ESC Differentiation to Nkx2.1+ Lung and Thyroid Progenitors. Bio-protocol 2(22): e295. DOI: 10.21769/BioProtoc.295.
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