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Mouse ESC Differentiation to Nkx2.1+ Lung and Thyroid Progenitors

Stem Cell > Embryonic stem cell > Maintenance and differentiation
Authors: Tyler A. Longmire, Laertis Ikonomou and Darrell N. Kotton
Vol 2, Iss 22, 11/20/2012, 2336 views, 0 Q&A

[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.

Materials and Reagents

  1. Mouse ESCs or iPSCs carrying a GFP reporter knocked in to the Nkx2.1 locus (Nkx2.1-GFP ESCs; see reference 1).
  2. 0.05% Trypsin-EDTA (1X) (Gibco, 25300-054)
  3. Defined Fetal Bovine Serum (Hyclone, SH30070.03)
  4. IMDM powder (Invitrogen, 12200-036)
  5. NaHCO3 (Sigma, S-5761)
  6. Pen/Strep (Invitrogen, 15140-148 10,000 U Penicillin and 10 mg Streptomycin per ml))
  7. Cellgro water (VWR, #45000-672)
  8. Ham’s F-12 (Cellgro, 10-080-CV)
  9. B-27 supplement with RA (Invitrogen, 17504-044)
  10. N-2 supplement (Invitrogen, 17502-048)
  11. BSA Fraction V 7.5% in PBS (Invitrogen, 15260-037)
  12. 1-thioglycerol (MTG) (Sigma, M6145-25ml)
  13. L-Glutamine (200 mM, Invitrogen, 25030-081)
  14. Ascorbic Acid (Sigma, A4544-25G)
  15. 1M HEPES (Gibco, 15630-080)
  16. CaCl2 (Sigma, C4901)
  17. BSA (Sigma, A9418-10G)
  18. ITS supplement, 100X (BD, 354352)
  19. mNoggin (R&D Systems, 1967-NG-025)
  20. SB431542 (Sigma, S4317)
  21. mWnt3a (R&D Systems, 1324-WN-010)
  22. hBMP4 (R&D Systems, 314-BP-050)
  23. hEGF (R&D Systems, 236-EG-01M)
  24. mFGF2 (R&D Systems, 3139-FB-025)
  25. mFGF7 (R&D Systems, 5028-KG-025)
  26. hFGF10 (R&D Systems, 345-FG-025)
  27. Heparin sodium salt (Sigma, H4784-250mg)
  28. Dexamethasone (Sigma, D4902)
  29. 8‐Br‐cAMP (Sigma, B7880)
  30. IBMX (Sigma, I5879)
  31. DMSO, Hybri-Max (Sigma, D2650)
  32. Ethanol (Sigma, E7023)
  33. Activin A (R&D Systems, 338-AC) )
  34. PBS (Gibco, 14190-250)
  35. 0.1% Gelatin in ultrapure water (Millipore, ES-006-B)
  36. Cxcr4 Antibody: APC Rat anti-mouse CD184 (Cxcr4) (BD-Pharmigen Cat#: 558644)
  37. cKit Antibody: PE Rat anti-mouse CD117 (cKit) (BD-Pharmigen Cat#: 553355)
  38. APC Isotype: APC Rat IgG2b, κ (BD-Pharmigen Cat #: 553991)
  39. PE Isotype: PE Rat IgG2b, κ (BD-Pharmigen Cat#: 553989)
  40. 1.5-ml Eppendorf Snap-Cap microcentrifuge tubes (Fisher Scientific, 05-402-25)
  41. 12x75 mm, 5 ml polystyrene round bottom test tube with a cell strainer cap (BD, 352235)


  1. P100 Petri dish (100 mm x 15 mm Bacteriological Petri Dish, nontreated polystyrene, BD Falcon, 351029)
  2. P150 Petri dish (150 mm x 15 mm Bacteriological Petri Dish, nontreated polystyrene, BD Falcon, 351058)
  3. Centrifuge
  4. LSRII flow cytometer
  5. Filter 0.22μm


  1. Timeline
    1. Timepoint: 0 hours
      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 (100 mm x 15 mm Bacteriological Petri Dish, nontreated polystyrene, BD Falcon, 351029) 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
    1. Timepoint: 60 hours
      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 minutes, 300xg, 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 minutes, 300xg, 4°C
      9. resuspend the cells in 5 ml cSFDM
      10. count cells
      11. plate 0.5-1×106 cells (in suspension) per P100 or P150 Petri dish (150 mm x 15 mm Bacteriological Petri Dish, nontreated polystyrene, BD Falcon, 351058) in 25 ml cSFDM supplemented with 50 ng/ml Activin A for definitive endoderm induction and EB formation
    1. Timepoint: 120 hours (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 minutes, 300xg, 4°C
      4. resuspend in 10-15 ml of Anteriorization media and plate in suspension in new P100 Petri dish
    1. Timepoint: 144 hours (Day 6)
      1. collect EBs
      2. wash in IMDM
      3. spin 5 minutes, 300xg, 4°C
      4. get cell count by trypsinizing an 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 Day 8-9
    1. 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 minutes, 500xg, 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
    1. 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
  1. 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.
  2. 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 2‐2.5x106 cells
      3. repeat procedure for undifferentiated ES cells
    1. 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 minutes, 300xg, 4°C ) and wash once with 5 ml IMDM
      3. resuspend in 500 μl PBS+ (PBS+2% FBS), cell concentration should be 0.4--‐0.5x106 cells/100 μl
      4. prepare 2x5 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
    1. 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 BD-Pharmigen Cat #: 553991
        Isotype: PE Rat IgC2b, κ BD-Pharmigen Cat#: 553989
        Cxcr4: APC Rat α-mouse CD184 BD-Pharmigen Cat#: 558644
        cKit: PE Rat α-mouse CD117 BD-Pharmigen Cat#: 553355
      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 300xg 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


  1. IMDM
    1 packet IMDM powder (Invitrogen, 12200-036)
    3.02 g NaHCO3 (Sigma, S-5761)
    10 ml Pen/Strep (Invitrogen, 15140-148(10,000 U Penicillin and 10 mg Streptomycin per ml))
    1L Cellgro water (VWR, #45000-672)
    check pH (acceptable range 6.9≤pH≤7.3)
  2. Serum Free Differentiation Medium (SFD)
    IMDM–375 ml
    Ham’s F-12 (Cellgro, 10-080-CV)–125 ml
    B-27 supplement withRA (Invitrogen, 17504-044)–5 ml
    N-2 supplement (Invitrogen, 17502-048)–2.5 ml
    BSA 7.5% in PBS (Invitrogen, 15260-037)–3.3 ml
  3. Complete Serum Free Differentiation Medium (cSFDM)
    SFD –100ml
    MTG (Sigma M6145-25ml) 300 μl of stock (Stock: 26 μL MTG to 2 ml IMDM)
    L-Glut –1 ml (200 mM, Invitrogen, 25030-081)
    Ascorbic Acid –1 mL of stock (Stock: 5 mg/ml distilled water, prepare fresh!)
  4. BASE medium for DCI+K
    Ham’s F-12 (Cellgro, 10-080-CV)–243.7 ml
    HEPES (1.0 M, pH 7.4)–3.75 ml
    1.0 M CaCl2 –200μl
    BSA (Sigma, A9418-10G) –0.625 g
    ITS supplement, 100X (BD, 354352) –2.5 ml
  5. Anteriorization medium
    cSFDM – 10 mL
    mNoggin -100 ng/mL (Stock: 10 μg/ml, R&D Systems 1967-NG-025)
    SB431542 – 10 μM (Stock: 10 mM in DMSO, Sigma, S4317)
  6. Ventralization medium
    cSFDM – 10mL
    mWnt3a – 100 ng/ml (Stock: 100 μg/ml, R&D Systems, 1324-WN-010)
    hBMP4 – 10 ng/ml (Stock: 10 μg/ml, R&D Systems, 314-BP-050)
    hEGF – 20 ng/ml (Stock: 20 μg/ml, R&D Systems, 236-EG-01 M)
    mFGF2 – 250 ng/ml (Stock: 100 μg/ml, R&D Systems, 3139-FB-025)
    mFGF7 – 10 ng/ml (Stock: 10 μg/ml, R&D Systems, 5028-KG-025)
    hFGF10 – 10 ng/ml (Stock: 10 μg/ml, R&D Systems, 345‐FG-025)
    Heparin sodium salt - 100 ng/ml (Stock: 1 mg/ml, Sigma, H4784-250mg)
  7. DCI+K medium
    BASE media – 25 ml
    Dexamethasone – 50 nM (Stock: 250 μM in ethanol, Sigma, D4902)
    KGF (FGF7) – 10 ng/ml (Stock: 10 μg/ml, R&D Systems, 5028-KG-025)
    cAMP+IBMX – 0.1 mM (Stock: 1 mM cAMP+1mM IBMX, see recipe at the end of protocol)
  8. Preparation of 10X cAMP+IBMX stock:
    Dissolve 22.22 mg IBMX (Sigma, I5879) 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 (Sigma, B7880) in 49.5 ml of BASE media and add 0.5 ml of IBMX stock
    Filter (0.22 μm filter)
    Store at 4°C for up to 4 weeks


  1. Longmire, T.A., Ikonomou, L., Hawkins, F., Christodoulou, C., Cao, Y., Jean, J.C., Kwok, L.W., Mou, H., Rajagopal, J., Shen, S.S., et al. (2012). Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell 10, 398-411.
  2. 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, L940-L951.
  3. Wray, J., Kalkan, T., and Smith, A.G. (2010). Revolutionizing Drug Discovery with Stem Cell Technology. Biochem Soc Trans 38, 1027-1032.

How to cite this protocol: Longmire, T., Ikonomou, L. and Kotton, D. (2012). Mouse ESC Differentiation to Nkx2.1+ Lung and Thyroid Progenitors. Bio-protocol 2(22): e295. http://www.bio-protocol.org/e295

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