Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines.
The role of individual supplements necessary for the self-renewal of human embryonic stem (hES) cells is poorly characterized, and furthermore we have found that previously reported feeder cell- and serum-free culture systems used for individual hES cell lines are unable to maintain HUES7 cells for more than one passage. We have therefore derived a feeder/serum-free culture system that can support the long-term (at least 10 passages) self-renewal of several euploid hES cell lines including MAN1, HUES7, and HUES1 with minimal spontaneous differentiation and without the need for manual propagation. This system contains fibroblast growth factor 2, activin A, neurotrophin 4, and the N2, B27 supplements together with a human fibronectin substrate. We demonstrate that these components exert distinct functions: both FGF2 and activin A were necessary to prevent differentiation of hES cells while NT4 promoted cell survival, FGF2 could not be substituted by IGFII, and the fibronectin substrate supported a rapid rate of hES culture expansion. Inhibition studies showed that β1 integrin-dependent attachment of hES cells to fibronectin was at least partially via the α5 subunit but independent of integrin αV.
Faculty of Life Sciences, The University of Manchester, 2nd Floor CTF Building, Oxford Road, Manchester, UK. Melissa.baxter@manchester.ac.uk
Melissa A
MA
Baxter
Maria V
MV
Camarasa
Nicola
N
Bates
Fiona
F
Small
Patricia
P
Murray
David
D
Edgar
Susan J
SJ
Kimber
D028341:Activins / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005353:Fibronectins / Q000494:pharmacology; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism; D009414:Nerve Growth Factors / Q000494:pharmacology
D016895:Culture Media, Serum-Free; D005353:Fibronectins; D019012:Integrin beta1; D009414:Nerve Growth Factors; C422405:activin A; D016222:Fibroblast Growth Factor 2; D028341:Activins; C073922:neurotrophin 4