Abstract
The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.
Highlights
Cell transplantation is a valid approach to treat some types of diseases; this treatment is limited by the low availability of many types of cells.Stem cell differentiation is a valuable tool to approach this problem, but several issues have to be overcome before it can be used on a regular basis for cell therapy
To confirm its ability to yield the desired fusion protein (VP22.eGFP of,70 kDa), this construct was transfected into 293T cells, and these cells were subjected to analysis by both Western blotting (Figure 1B) and confocal microscopy (Figure 1C), revealing the production of the desired fusion protein
To document the capacity of VP22 to penetrate and cross membranes, we first analyzed whether the VP22 protein and the VP22.eGFP fusion protein were secreted by the cells and could be detected in the culture medium
Summary
Stem cell differentiation is a valuable tool to approach this problem, but several issues have to be overcome before it can be used on a regular basis for cell therapy. Safety is an important issue, especially when cell differentiation is achieved by stable gene expression, due to the risk of mutagenesis, which may be associated with prolonged expression of the transgene [1]. An attractive alternative is proteins containing a protein transduction domain (PTD), which are characterized by their ability to cross the plasma membrane and translocate into living cells. PTD proteins contain a short arginine- or lysine-rich peptide sequence. These basic amino acids seem to be important for the contact with the negatively charged lipids present in the cell membrane, which is the first step of protein transduction [3]. Several theories have been proposed to explain how these PTD proteins enter the cell [4,5], but the actual mechanism is yet to be understood
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