Simple generation of neurons from human embryonic stem cells using agarose multiwell dishes

Ronit Birenboim, Amos Markus, Ronald S. Goldstein

Research output: Contribution to journalArticlepeer-review

14 Scopus citations


Human embryonic stem cells (hESC) are potentially an unlimited source of neurons for study and therapy for human disease. Directed differentiation of hESC has been performed using many different methods, often via neural precursor intermediates generated from aggregates of hESC. We describe here a protocol based on commercially available reusable silicone micromolds and two small molecule growth factor inhibitors to simply and reproducibly generate human neurons from hESC. Hundreds of neurospheres were generated with a single pipettation of hESC into agarose multiwell plates made with the micromolds. This was followed by suspension culture with two medium changes, and plating of clumps cut from the neurospheres on laminin-coated coverslips. After two weeks of terminal differentiation, 90%+ of cells expressed neuronal proteins, and many of the neurons expressed markers of peripheral sensory neurons. The neurons made with this method underwent productive infection with the human-specific pathogenic virus varicella zoster, demonstrating the utility of the neurons for addressing clinically relevant research questions. This simple method should allow laboratories experienced in growing human pluripotent cells to easily generate neurons for studies of nerve cell biology and pathology.

Original languageEnglish
Pages (from-to)9-14
Number of pages6
JournalJournal of Neuroscience Methods
Issue number1
StatePublished - 30 Mar 2013

Bibliographical note

Funding Information:
This study was supported by an Israel Science Foundation individual (ISF) grant #238/11 to RSG, and an ISF Center of Excellence grant #1803/10 to A. Klar (Hebrew University, Jerusalem, Israel, M. Fainzilber, Weizmann Inst. Science, Rehovot, Israel, and RSG).The recombinant VZV was generously provided by H. Zhu of the New Jersey Medical School. Our thanks to Chaya Morgenstern for logistic help. The monoclonal antibody 2H3 developed by Jessell, T.M.and Dodd, J. was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.


  • Embryoid bodies
  • Human embryonic stem cells
  • Neuronal differentiation
  • PNS
  • Varicella zoster virus


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