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Isolation of Mouse Cardiac Neural Crest Cells and Their Differentiation into Smooth Muscle Cells

小鼠心脏神经嵴细胞的分离及向平滑肌细胞的分化

XW Xia Wang
SA Sophie Astrof

发布: 2017年09月05日第7卷第17期 DOI: 10.21769/BioProtoc.2530 浏览次数: 9681

评审: Anonymous reviewer(s)

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Abstract

Cardiac neural crest cells (CNCCs) originate at the dorsal edge of the neural tube between the otic pit and the caudal edge of the 3rd somite, and migrate into the pharyngeal arches and the heart. We have shown that fibronectin (Fn1) plays an important role in the development of the CNCC by regulating the differentiation of CNCCs into vascular smooth muscle cells around pharyngeal arch arteries (Wang and Astrof, 2016). This protocol describes the isolation of CNCCs from the neural tube and from the caudal pharyngeal arches, and the differentiation of neural crest-derived cells into smooth muscle cells. This protocol was adapted from (Newgreen and Murphy, 2000; Pfaltzgraff et al., 2012).

Keywords: Cardiac neural crest (心脏神经嵴) search Vascular smooth muscle cells (血管平滑肌细胞) search Neural tube (神经管) search Pharyngeal arch (咽弓) search Differentiation (分化) search

Background

Previous published protocols described the isolation of neural crest cells from the neural tube. However, neural crest cells in the region of the neural tube between the otic pit and the 3rd somite include neural crest cell populations that contribute to a number of different cell types; for example, vagal neural crest cells also originate from this region. In this protocol, we modified the conventional method for the isolation of cardiac neural crest cells. Instead of using the neural tube, we used the caudal pharyngeal arch region at embryonic day (E) 9.5 (22-25 somite stage). This is prior to differentiation of cardiac neural crest cells into vascular smooth muscle cells. It is common for neural crest cultures to contain contaminating mesenchymal cell types, which often express smooth muscle genes. To identify neural crest-derived cells, we isolated neural crest cells from embryos resulting from the following cross: Fn1flox/flox;ROSAmTmG/mTmG female mice x Fn1+/−;Tfap2αIRESCre/+ male mice. In 50% of the progeny from this cross, neural crest cells are lineage-labeled by the expression of GFP, so we could easily identify neural crest cells by their GFP expression without the need for cell sorting (Wang and Astrof, 2016). Additional Cre-expressing strains that can be used are Wnt1-Cre2 (Lewis et al., 2013) and P3ProCre (Li et al., 2000) transgenic strains, e.g., (Wang and Astrof, 2016). All experimental procedures were approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University and conducted in accordance with federal guidelines for humane care of animals.

Materials and Reagents

  1. 12 mm round glass coverslips (Electron Microscopy Sciences, catalog number: 72231-01 )
  2. 24-well plates (Corning, Falcon®, catalog number: 353047 )
  3. Nunc 4-well dishes untreated (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 144444 )
  4. 35 cm Petri dish (Corning, Falcon®, catalog number: 353001 )
  5. 1.5 ml centrifuge tube
  6. Sterile transfer pipet (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: PP89SB )
  7. Glass pipet (Fisher Scientific, model: 13-678-6A )
  8. Parafilm
  9. Glass slide
  10. 0.2 μm syringe filter unit
  11. Pregnant mice
  12. Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 ), for dissection of embryos and for cell culture
  13. Trypsin (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  14. 4% PFA prepared in 1x PBS
  15. 0.1% Triton X-100 in 1x PBS
  16. Phosphate-buffered saline (PBS)
  17. Donkey serum (Sigma-Aldrich, catalog number: D9663-10ML )
  18. Anti-GFP antibody (Aves Labs, catalog number: GFP-1020 )
  19. Anti-αSMA antibody (Sigma-Aldrich, catalog number: SAB2500963 )
  20. Anti-calponin antibody (Abcam, catalog number: ab46794 )
  21. Donkey anti-goat IgG (H+L) secondary antibody, Alexa Fluor® 555 conjugate (Thermo Fisher Scientific, catalog number: A-21432 )
  22. Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor® 647 conjugate (Thermo Fisher Scientific, catalog number: A-31573 )
  23. Alexa Fluor® 488 AffiniPure F(ab’)2 fragment donkey anti-chicken IgY (IgG) (H+L) (Jackson ImmunoResearch, catalog number: 703-546-155 )
  24. ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, InvitrogeTM, catalog number: P36931 )
  25. Collagenase and dispase (Roche Diagnostics, catalog number: 10269638001 )
  26. Glacial acetic acid
  27. Collagen I (Corning, catalog number: 354249 )
  28. DMEM-low glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11885076 )
  29. Neurobasal medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
  30. Chicken embryo extract (MP Biomedicals, catalog number: 092850145 )
  31. Penicillin/Streptomycin (Pen/Strep) (Mediatech, catalog number: 30-001-CI )
  32. N2-supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 17502048 )
  33. B27-supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  34. Retinoic acid (Sigma-Aldrich, catalog number: R2625 )
  35. Ethanol
  36. 2-Mercaptoethanol (Thermo Fisher Scientific, GibcoTM, catalog number: 21985023 )
  37. Fibroblast growth factor-basic (bFGF) (Sigma-Aldrich, catalog number: F0291 )
  38. Tris pH 7.6
  39. Insulin-like growth factor 1 (IGF-1) (R&D Systems, catalog number: 291-G1 )
  40. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 100-125 )
  41. DMEM-high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
  42. Collagenase/dispase stock solution (see Recipes)
  43. Collagen I working solution (see Recipes)
  44. Neural crest self-renewal medium (see Recipes)
  45. Differentiation medium (see Recipes)

Equipment

  1. Autoclave
  2. Biological safety hood (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series Class II, Type A2 )
  3. Scissors (Fisher Scientific, model: 13-804-6 )
  4. Forceps 9 cm (Fine Science Tool, model: 14060-09 )
  5. Forceps 0.1 x 0.06 mm (Fine Science Tool, model: 11251-23 )
  6. Forceps 11 cm (Fine Science Tool, model: 11254-20 )
  7. EdgeGARD® horizontal flow hood (The Baker Company, model: EdgeGARD® HF )
  8. Flat bench
  9. Dissection microscope and light source (Carl Zeiss, model: Stemi 2000-C )
  10. Humidified, 37 °C tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
  11. Fluorescence microscope

Procedure

English
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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Wang, X. and Astrof, S. (2017). Isolation of Mouse Cardiac Neural Crest Cells and Their Differentiation into Smooth Muscle Cells. Bio-protocol 7(17): e2530. DOI: 10.21769/BioProtoc.2530.

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分类

发育生物学 > 细胞生长和命运决定 > 肌纤维

细胞生物学 > 细胞分离和培养 > 细胞分化

细胞生物学 > 细胞分离和培养 > 细胞分离

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