The connections existing between vessels and nerves go beyond the structural architecture of vascular and nervous systems to comprise cell fate determination. The analysis of functional/molecular links that interconnect endothelial and neural commitments requires a model in which the two differentiation programs take place at the same time in an artificial controllable environment. To this regard, this work presents an in vitro model to differentiate embryonic stem (ES) cells simultaneously into mature neurons and endothelial cells. Murine ES cells are differentiated within an artificial environment composed of PA6 stromal cells and a serum-free medium. Upon these basal culture conditions ES cells preferentially differentiate into neurons. The addition of basic fibroblast growth factor (FGF2) to the medium allows the simultaneous maturation of neurons and endothelial cells, whereas BMP4 drives endothelial differentiation to the disadvantage of neural commitment. The responsiveness of the system to exogenous cytokines was confirmed by genes expression analysis that revealed a significant upregulation of endothelial genes in presence of FGF2 and a massive downregulation of the neural markers in response to BMP4. Furthermore, the role played by single genes in determining endothelial and neural fate can be easily explored by knocking down the expression of the target gene with lentiviruses carrying the corresponding shRNA sequence. The possibility to address the neural and the endothelial fate separately or simultaneously by exogenous stimuli combined with an efficient gene silencing strategy make this model an optimal tool to identify environmental signals and genes pathways involved in both endothelial and neural specification.
Mature endothelium and neurons are simultaneously derived from embryonic stem cells by 2D in vitro culture system
NOGHERO, ALESSIO;ARESE, Marco;BUSSOLINO, Federico;GUALANDRIS, Anna
2011-01-01
Abstract
The connections existing between vessels and nerves go beyond the structural architecture of vascular and nervous systems to comprise cell fate determination. The analysis of functional/molecular links that interconnect endothelial and neural commitments requires a model in which the two differentiation programs take place at the same time in an artificial controllable environment. To this regard, this work presents an in vitro model to differentiate embryonic stem (ES) cells simultaneously into mature neurons and endothelial cells. Murine ES cells are differentiated within an artificial environment composed of PA6 stromal cells and a serum-free medium. Upon these basal culture conditions ES cells preferentially differentiate into neurons. The addition of basic fibroblast growth factor (FGF2) to the medium allows the simultaneous maturation of neurons and endothelial cells, whereas BMP4 drives endothelial differentiation to the disadvantage of neural commitment. The responsiveness of the system to exogenous cytokines was confirmed by genes expression analysis that revealed a significant upregulation of endothelial genes in presence of FGF2 and a massive downregulation of the neural markers in response to BMP4. Furthermore, the role played by single genes in determining endothelial and neural fate can be easily explored by knocking down the expression of the target gene with lentiviruses carrying the corresponding shRNA sequence. The possibility to address the neural and the endothelial fate separately or simultaneously by exogenous stimuli combined with an efficient gene silencing strategy make this model an optimal tool to identify environmental signals and genes pathways involved in both endothelial and neural specification.
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