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Peer-reviewed

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

TL Tyler A. Longmire
LI Laertis Ikonomou
Darrell N. Kotton Darrell N. Kotton

发布: 2012年11月20日第2卷第22期 DOI: 10.21769/BioProtoc.295 浏览次数: 11477

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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) search Directed Differentiation (定向分化) search Lung Progenitors (肺细胞) search Thyroid Progenitors (甲状腺细胞) search Nkx2-1 (NKX2-1) search

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

English
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文章信息

版权信息

© 2012 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

干细胞 > 胚胎干细胞 > 维持和分化

细胞生物学 > 细胞分离和培养 > 细胞分化

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