RESUMO
Clonal cell culture is crucial for experimental protocols that require growth or selection of pure populations of cells. High-density derivation of neural progenitors from human embryonic stem cells (hESCs) can lead to incomplete differentiation, and transplantation of resulting heterogeneous cell mixtures can cause proliferation of tumorigenic clusters in vivo. We have identified the neural precursor that resides among normal hESC colonies as a TRA-1-60(-)/SSEA4(-)/SOX1(+) cell and developed a method that allows for the clonal expansion of these FACS-selected progenitors to neural stem cells (NSCs) in serum-free conditions. Single TRA-1-60(-)/SSEA4(-)/SOX1(+) cells grown in serum-free media give rise to multipotent NSCs with an efficiency of 0.7%. The fate of the TRA-1-60(-)/SSEA4(-)/SOX1(+) neural precursor becomes specified in maintenance conditions by inhibition of BMP signaling. This clonal culture method can be scaled up to produce NSCs for differentiation and use in cell therapies.
Assuntos
Células Clonais/fisiologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Neurais/fisiologia , Proteínas Morfogenéticas Ósseas/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Diferenciação Celular/fisiologia , Linhagem da Célula , Sobrevivência Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Meios de Cultura Livres de Soro , DNA Complementar/biossíntese , DNA Complementar/genética , Fatores de Crescimento de Fibroblastos/fisiologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Neostriado/citologia , Neostriado/fisiologia , Reação em Cadeia da Polimerase , RNA/biossíntese , RNA/isolamento & purificação , Transplante de Células-Tronco , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
There are two types of neural stem cells (NSCs). Primitive NSCs [leukemia inhibitory factor (LIF) dependent but exogenous fibroblast growth factor (FGF) 2 independent] can be derived from mouse embryonic stem (ES) cells in vitro and from embryonic day 5.5 (E5.5) to E7.5 epiblast and E7.5-E8.5 neuroectoderm in vivo. Definitive NSCs (LIF independent but FGF2 dependent) first appear in the E8.5 neural plate and persist throughout life. Primitive NSCs give rise to definitive NSCs. Loss and gain of functions were used to study the role of vascular endothelial growth factor (VEGF)-A and its receptor, Flk1, in NSCs. The numbers of Flk1 knock-out mice embryo-derived and ES cell-derived primitive NSCs were increased because of the enhanced survival of primitive NSCs. In contrast, neural precursor-specific, Flk1 conditional knock-out mice-derived, definitive NSCs numbers were decreased because of the enhanced cell death of definitive NSCs. These effects were not observed in cells lacking Flt1, another VEGF receptor. In addition, the cell death stimulated by VEGF-A of primitive NSC and the cell survival stimulated by VEGF-A of definitive NSC were blocked by Flk1/Fc-soluble receptors and VEGF-A function-blocking antibodies. These VEGF-A phenotypes also were blocked by inhibition of the downstream effector nuclear factor kappaB (NF-kappaB). Thus, both the cell death of primitive NSC and the cell survival of definitive NSC induced by VEGF-A stimulation are mediated by bifunctional NF-kappaB effects. In conclusion, VEGF-A function through Flk1 mediates survival (and not proliferative or fate change) effects on NSCs, specifically.