EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs

Stem Cell Res. 2013 May;10(3):417-427. doi: 10.1016/j.scr.2013.01.009. Epub 2013 Feb 4.


We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation / drug effects
  • Cells, Cultured
  • Culture Media / chemistry
  • Epidermal Growth Factor / pharmacology
  • Fibroblast Growth Factor 2 / pharmacology
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Intermediate Filament Proteins / metabolism
  • Multipotent Stem Cells / cytology*
  • Nerve Tissue Proteins / metabolism
  • Nestin
  • Neural Stem Cells / cytology*
  • Neural Stem Cells / metabolism
  • Octamer Transcription Factor-3 / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / pharmacology
  • SOXB1 Transcription Factors / metabolism
  • Up-Regulation


  • Culture Media
  • Intermediate Filament Proteins
  • NES protein, human
  • Nerve Tissue Proteins
  • Nestin
  • Octamer Transcription Factor-3
  • Recombinant Proteins
  • SOXB1 Transcription Factors
  • Fibroblast Growth Factor 2
  • Epidermal Growth Factor