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Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation

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Author

  • Catharina Ellerström
  • Raimund Strehl
  • Karin Noaksson
  • Johan Hyllner
  • Henrik Semb

Summary, in English

Traditionally, human embryonic stem cells (hESCs) are propagated by mechanical dissection or enzymatic dissociation into clusters of cells. To facilitate up-scaling and the use of hESC in various experimental manipulations, such as fluorescence-activated cell sorting, electroporation, and clonal selection, it is important to develop new, stable culture systems based on single-cell enzymatic propagation. Here, we show that hESCs, which were derived and passaged by mechanical dissection, can be rapidly adjusted to propagation by enzymatic dissociation to single cells. As an indication of the stability of this culture system, we demonstrate that hESCs can be maintained in an undifferentiated, pluripotent, and genetically normal state for up to 40 enzymatic passages. We also demonstrate that a recombinant trypsin preparation increases clonal survival compared with porcine trypsin. Finally, we show that human foreskin fibroblast feeders are superior to the commonly used mouse embryonic fibroblast feeders in terms of their ability to prevent spontaneous differentiation after single-cell passaging. Importantly, the culture system is widely applicable and should therefore be of general use to facilitate reliable large-scale cultivation of hESCs, as well as their use in various experimental manipulations.

Department/s

Publishing year

2007

Language

English

Pages

1690-1696

Publication/Series

Stem Cells

Volume

25

Issue

7

Document type

Journal article

Publisher

AlphaMed Press

Topic

  • Cell and Molecular Biology

Keywords

  • enzymatic
  • single cells
  • human embryonic stem cell
  • human feeders
  • dissociation
  • expansion

Status

Published

ISBN/ISSN/Other

  • ISSN: 1549-4918