Inducing Cellular Senescence in Mouse Embryonic Fibroblasts (MEFs).

Inducing cellular senescence in mouse embryonic fibroblasts (MEFs) is a robust tool to study the molecular mechanisms underlying senescence establishment and their heterogeneity. This protocol provides a detailed guide to generate MEFs and routinely induce senescence in MEFs using several DNA damage-dependent and DNA damage-independent induction methods.

Identifying molecular tags selectively retained on the surface of brain endothelial cells to generate artificial targets for therapy delivery.

Current strategies to identify ligands for brain delivery select candidates based on preferential binding to cell-membrane components (CMC) on brain endothelial cells (EC). However, such strategies generate ligands with inherent brain specificity limitations, as the CMC (e.g., the transferrin receptor TfR1) are also significantly expressed on peripheral EC. Therefore, novel strategies are required to identify molecules allowing increased specificity of therapy brain delivery. Here, we demonstrate that, while individual CMC are shared between brain EC and peripheral EC, their endocytic internalization rate is markedly different. Such differential endocytic rate may be harnessed to identify molecular tags for brain targeting based on their selective retention on the surface of brain EC, thereby generating 'artificial' targets specifically on the brain vasculature. By quantifying the retention of labelled proteins on the cell membrane, we measured the general endocytic rate of primary brain EC to be less than half that of primary peripheral (liver and lung) EC. In addition, through bio-panning of phage-displayed peptide libraries, we unbiasedly probed the endocytic rate of individual CMC of liver, lung and brain endothelial cells. We identified phage-displayed peptides which bind to CMC common to all three endothelia phenotypes, but which are preferentially endocytosed into peripheral EC, resulting in selective retention on the surface of brain EC. Furthermore, we demonstrate that the synthesized free-form peptides are capable of generating artificial cell-surface targets for the intracellular delivery of model proteins into brain EC with increasing specificity over time. The developed identification paradigm, therefore, demonstrates that the lower endocytic rate of individual CMC on brain EC can be harnessed to identify peptides capable of generating 'artificial' targets for the selective delivery of proteins into the brain vasculature. In addition, our approach identifies brain-targeting peptides which would have been overlooked by conventional identification strategies, thereby increasing the repertoire of candidates to achieve specific therapy brain delivery.