RESUMO
We report here the derivation of two new human embryonic stem cell lines, Man-1 and Man-2, and their full characterization as novel pluripotent stem cell lines. Man-1 was derived from an embryo surplus to requirement from routine IVF, while Man-2 was obtained from an oocyte classified as failed to fertilise and subsequently chemically activated. We report the characterisation of pluripotency and the differentiation potential of these lines. Work is in progress to establish novel methods of stem cell derivation and culture, which will avoid the use of xenobiotics and be relevant to clinical production of human embryonic stem cell lines. Both newly derived human embryonic stem cell lines will be available for the research community from the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk).
Assuntos
Técnicas de Cultura de Células/métodos , Linhagem Celular/citologia , Pesquisas com Embriões , Células-Tronco Embrionárias/citologia , Adaptação Fisiológica/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Bandeamento Cromossômico , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Imunofluorescência , Humanos , CamundongosRESUMO
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 ß1 integrin-dependent attachment of hES cells to fibronectin was at least partially via the α5 subunit but independent of integrin αV.