Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

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: 9150

评审: Anonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Development, featuring study using the protocol.

Jan 2016

Advertisement

A protocol guide to micro milling for bio-microfluidics
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

基于 PKD2L1 启动子驱动的慢病毒系统在体外选择性富集与鉴定脑脊液接触神经元

基于 PKD2L1 启动子驱动的慢病毒系统在体外选择性富集与鉴定脑脊液接触神经元

Wei Tan [...] Qing Li

2025年11月20日 1324 阅读

重新优化原代小胶质细胞分离流程：改进的小胶质细胞提取方法

重新优化原代小胶质细胞分离流程：改进的小胶质细胞提取方法

Jianwei Li [...] Guohui Lu

2025年12月05日 1419 阅读

小鼠与人源细胞培养模型中脂肪生成分化方案的优化

小鼠与人源细胞培养模型中脂肪生成分化方案的优化

Junwan Fan [...] Wenyan He

2026年01月20日 61 阅读

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
中文翻译

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.

ris ris Download Citation in RIS Format

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

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件