High-Content Imaging Phenotypic Screen for Neurogenesis Using Primary Neural Progenitor Cells

  • Li Sharon Wu
  • Jingjun Li
Part of the Methods in Molecular Biology book series (MIMB, volume 1787)


Neurogenesis phenotypic screen of small-molecule library enables the discovery of small-molecule inducers, and identification of associated biological targets and pathways that control neuronal formation from neural progenitor cells (NPCs). Here, we describe protocols for preparing mouse embryonic NPCs, setting up a high-content imaging assay that quantifies the production of Tuj1-labeled neurons, and analysis of high-throughput screens.

Key words

Neurogenesis High-content imaging Phenotypic screen Neural progenitor cells Neuronal differentiation Small-molecule chemical library 


  1. 1.
    Gage FH, Temple S (2013) Neural stem cells: generating and regenerating the brain. Neuron 80:588–601CrossRefGoogle Scholar
  2. 2.
    Goldman SA, Nedergaard M, Windrem MS (2012) Glial progenitor cell-based treatment and modeling of neurological disease. Science 338:491–495CrossRefPubMedGoogle Scholar
  3. 3.
    Lu P, Wang Y, Graham L, McHale K, Gao M, Wu D et al (2012) Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell 150:1264–1273CrossRefPubMedGoogle Scholar
  4. 4.
    Li J, Ma J, Meng G, Lin H, Wu S, Wang J et al (2016) BET bromodomain inhibition promotes neurogenesis while inhibiting gliogenesis in neural progenitor cells. Stem Cell Res 17:212–221CrossRefGoogle Scholar
  5. 5.
    Schneider JW, Gao Z, Li S, Farooqi M, Tang TS, Bezprozvanny I et al (2008) Small-molecule activation of neuronal cell fate. Nat Chem Biol 4:408–410CrossRefGoogle Scholar
  6. 6.
    Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O et al (2010) Selective inhibition of BET bromodomains. Nature 468:1067–1073CrossRefPubMedGoogle Scholar
  7. 7.
    Nicodeme E, Jeffrey KL, Schaefer U, Beinke S, Dewell S, Chung CW et al (2010) Suppression of inflammation by a synthetic histone mimic. Nature 468:1119–1123CrossRefPubMedGoogle Scholar
  8. 8.
    Kriegstein A, Alvarez-Buylla A (2009) The glial nature of embryonic and adult neural stem cells. Annu Rev Neurosci 32:149–184CrossRefPubMedGoogle Scholar
  9. 9.
    Danovi D, Folarin AA, Baranowski B, Pollard SM (2012) High content screening of defined chemical libraries using normal and glioma-derived neural stem cell lines. Methods Enzymol 506:311–329CrossRefGoogle Scholar
  10. 10.
    Kim KJ, Wang J, Xu X, Wu S, Zhang W, Qin Z et al (2012) A chemical genomics screen to discover genes that modulate neural stem cell differentiation. J Biomol Screen 17:129–139CrossRefGoogle Scholar
  11. 11.
    Azari H, Sharififar S, Rahman M, Ansari S, Reynolds BA (2011) Establishing embryonic mouse neural stem cell culture using the neurosphere assay. J Vis Exp 47:2457Google Scholar
  12. 12.
    Ricardo R, Phelan K (2008) Freezing, thawing, and packaging cells for transport. J Vis Exp 17:757Google Scholar
  13. 13.
    Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73CrossRefGoogle Scholar
  14. 14.
    Buchser W, Collins M, Garyantes T, Guha R, Haney S, Lemmon V et al (2004) Assay development guidelines for image-based high content screening, high content analysis and high content imaging. In: Sittampalam GS et al (eds) Assay guidance manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences, BethesdaGoogle Scholar
  15. 15.
    Schenone M, Dancik V, Wagner BK, Clemons PA (2013) Target identification and mechanism of action in chemical biology and drug discovery. Nat Chem Biol 9:232–240CrossRefPubMedGoogle Scholar
  16. 16.
    Chen Y, Stevens B, Chang J, Milbrandt J, Barres BA, Hell JW (2008) NS21: re-defined and modified supplement B27 for neuronal cultures. J Neurosci Methods 171:239–247CrossRefPubMedGoogle Scholar
  17. 17.
    Dandapani S, Rosse G, Southall N, Salvino JM, Thomas CJ (2012) Selecting, acquiring, and using small molecule libraries for high-throughput screening. Curr Protoc Chem Biol 4:177–191PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Li Sharon Wu
    • 1
  • Jingjun Li
    • 2
  1. 1.School of Life SciencesNanchang UniversityNanchangChina
  2. 2.Lilly China Research and Development Center, Eli Lilly and CompanyShanghaiChina

Personalised recommendations