Human embryonic stem (hES) cells have the unique capability of differentiating into all cell types, leading to the development of an entire organism. As the integrity of ES cells is critical for the developing embryo, these cells have likely evolved mechanisms that detect and respond rapidly to adverse stimuli.
Indeed, hES cells have been shown to be highly sensitive to DNA damage, but the molecular mechanisms underlying this rapid death remain unclear. Caspases are critical mediators of apoptosis in mammalian cells, and a key protein that controls their activation is Bax, a proapoptotic member of the Bcl-2 family. While the main components of the apoptotic pathway have been identified, exactly how this pathway is regulated in various primary cells remains unclear.
Here, we examined the apoptotic pathway in hES cells and report a unique mechanism engaged by hES cells that can prime them to undergo rapid apoptosis inresponse to genotoxic damage.To visualize GFP-tagged Bax, 3-day colonies of hES cells were transfected with 2 mg of hBaxC3-EGFP (Addgene) with FuGENE HD transfection reagent.
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The process of introducing nucleic acids into eukaryotic cells by nonviral methods is defined as transfection. Using various chemical, lipid or physical methods, this gene transfer technology is a powerful tool to study gene function and protein expression in the context of a cell. Development of reporter gene systems and selection methods for stable maintenance and expression of transferred DNA have greatly expanded the applications for transfection.
Assay-based reporter technology, together with the availability of transfection reagents, provides the foundation to study mammalian promoter and enhancer sequences, trans-acting proteins such as transcription factors, mRNA processing, protein:protein interactions, translation and recombination events (Groskreutz and Schenborn, 1997).
Transfection is a method that neutralizes or obviates the issue of introducing negatively charged molecules (e.g., phosphate backbones of DNA and RNA) into cells with a negatively charged membrane. Chemicals like calcium phosphate and DEAE-dextran or cationic lipid-based reagents coat the DNA, neutralizing or even creating an overall positive charge to the molecule.
This makes it easier for the DNA:transfection reagent complex to cross the membrane, especially for lipids that have a "fusogenic" component, which enhances fusion with the lipid bilayer. Physical methods like microinjection or electroporation simply punch through the membrane and introduce DNA directly into the cytoplasm. Here we describe the striking observation that healthy undifferentiated hES cells maintain Bax in its preactivated state at the Golgi.
This is in contrast to other cell types in which Bax is typically present in an inactive form in the cytosol. Our results also highlight the fact that the apoptotic machinery undergoes dynamic changes even at early stages of differentiation.While undifferentiated hES cells have constitutively active Bax and undergo rapid apoptosis in response to DNA damage, just 2 days of differentiation induced significant changes suchthat Bax was no longer active, and the cells were no longer highly sensitive to DNA damage.
This could be manifested with even greater complexity in vivo as cells during early embryogenesis undergo rapid proliferation and differentiation.
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