RESUMO
We develop biodegradable polymeric nanoparticles to facilitate nonviral gene transfer to human embryonic stem cells (hESCs). Small (approximately 200 nm), positively charged (approximately 10 mV) particles are formed by the self assembly of cationic, hydrolytically degradable poly(beta-amino esters) and plasmid DNA. By varying the end group of the polymer, we can tune the biophysical properties of the resulting nanoparticles and their gene-delivery efficacy. We created an OCT4-driven GFP hES cell line to allow the rapid identification of nanoparticles that facilitate gene transfer while maintaining an hESC undifferentiated state. Using this cell system, we synthesized nanoparticles that have gene delivery efficacy that is up to 4 times higher than that of the leading commercially available transfection agent, Lipofectamine 2000. Importantly, these materials have minimal toxicity and do not adversely affect hESC colony morphology or cause nonspecific differentiation.
Assuntos
Células-Tronco Embrionárias/citologia , Técnicas de Transferência de Genes , Vetores Genéticos/química , Animais , Materiais Biocompatíveis/química , Cátions , Diferenciação Celular , Citometria de Fluxo , Técnicas Genéticas , Proteínas de Fluorescência Verde/metabolismo , Hidrólise , Camundongos , Nanotecnologia/métodos , Fator 3 de Transcrição de Octâmero/metabolismo , Polímeros/químicaAssuntos
Embrião de Mamíferos/citologia , Células-Tronco/citologia , Aneuploidia , Técnicas de Cultura de Células/métodos , Divisão Celular , Linhagem Celular , Células Cultivadas , Aberrações Cromossômicas , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Manejo de Espécimes/métodos , Fatores de TempoRESUMO
BACKGROUND: We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs) to neural precursors and neurons.HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII). RESULTS: A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7-10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons. CONCLUSION: This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4+ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.
