Human Embryonic Stem (hES)

Human embryonic stem (hES) cell has a unique ability of differentiating into all cell types, which leads to the development of the whole organism. As the integrity of ES cells is crucial for the developing embryo, these cells have likely evolved a mechanism that will detect and respond to adverse stimuli. Indeed, hES cell has 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 the cells of mammals, and is considered a key protein that is responsible for controlling their activation is Bax, a Bcl-2 family proapoptotic member. While the main components of the apoptotic pathway have been known and identified, exactly how this pathway is functioning and regulated in various primary cells is still unclear. The study examined the apoptotic pathway in the hES cells and also reported a unique mechanism of hES cells that can help them to undergo apoptosis in response to the genotoxic damage. To visualize GFP-tagged Bax, the 3-day colonies of hES cells were transfected with 2 mg of hBaxC3-EGFP from Addgene with FuGENE HD transfection reagent from Roche. Transfection is the process of by which nucleic acids is introduce to the eukaryotic cells by methods that are nonviral. With the help of some various methods such as chemical, physical, lipid methods, this technology of gene transfer technology is a very powerful tool to investigate the gene function and the expression of protein of a cell. Assay-based reporter technology, and with the availability of transfection reagents will actually give and provides the foundation necessary to study sequences of mammalian promoter and enhancer, the trans-acting proteins such as transcription factors, processing of mRNA, the interactions of different proteins, recombination, and translation events. In general, transfection is a method or protocol used to neutralize or obviate the issue of introducing the negatively charged molecules such as phosphate backbone of DNA and RNA into the negatively charged membrane of the cells. In addition, chemicals such as calcium phosphate and cationic lipid-based reagents that coat the DNA, neutralize or even creates an overall positive charge to the molecule is also used. This makes it easier for the DNA to transfection reagent complex to cross the said membrane, especially for lipids which has a fusogenic component that further enhances the fusion with the different lipid bilayer. Other methods such as physical methods like microinjection or electroporation have also been used that is simply punch through the membrane and will introduce DNA directly into the cytoplasm. In this study, they describe the striking feature of the healthy undifferentiated hES cells, which maintain Bax in its preactivated state at the Golgi that is in contrast to other cell types. The results also highlight the fact that the apoptotic machinery undergoes a dynamic change even if its an early stages of differentiation.