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Stem cells are often difficult to transfect, and the methods used for stem cell transfection vary depending on cell type, species, the molecule being delivered, and the intended downstream application. Transfection methods include electroporation lipid-mediated delivery and gene gun delivery, as well as cell transduction via virus-mediated gene delivery.
Electroporation uses an electric shock to temporarily disrupt the lipid bilayer of the plasma membrane, allowing gene molecules to enter the cell. The electroporation protocol is optimized for various cell types, including optimized electrical parameters and improved buffer solutions to allow DNA to enter the nucleus and thus enhance gene expression. A variety of nuclear transfection methods based on electroporation have been proven to be effective in transfecting mouse embryonic stem cells and human embryonic stem cells.
The commonly used lipid reagent is a mixture of cationic lipid and co-lipid DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine), which has been widely used in neural stem cells, embryonic stem cells, and other stem cells. In lipid-mediated transfection, lipids are mixed with nucleic acids and then added directly to cells. Lipid-coated molecules are thought to enter the cell through endocytosis. Transfection efficiency varies with reagent and cell type. Inevitably, lipid toxicity is a problem when transfecting cells.
Nanostraws are hollow alumina tubes (typically 100 to 200 nm in diameter and 1 to 3 μm in length) embedded in a cell culture-compatible polymer membrane. The nanostraws form a direct fluidic path from the cargo-containing compartment below the nanostraw membrane into the cytoplasm of the cultured cell at the top of the membrane. Adherent cells cultured on nanostraws pull themselves onto the nanostraws, causing them to pierce through the cell membrane. The cargo is delivered electrically by passive diffusion or by a weak pulsed electric field. Nanostraws have been shown to target small molecules, RNA, DNA, and proteins to different adherent cells (e.g., human hematopoietic stem and Primary stem cells) in a tunable, non-toxic and efficient manner. Nanostraws have been used to efficiently deliver molecular cargo to cells with minimal impact on cell viability.
Biolistic transfection is a mechanical method, which has potential applications in a wide variety of cells (including stem cells) and tissue types. Biolistic particle delivery works by coating small compounds, proteins, or nucleic acids onto inert micron-scale particles, which are injected into cells using high-pressure helium gas as microbombs. The biggest advantages of biolistic are the ability to overcome physical barriers such as the epidermis, the possibility of multiple uses in the same sample, transfecting a large number of cells each time as well as the possibility of transfecting two or more DNA simultaneously in a single use, what makes it time-saving and easy to use.
Adenovirus is a double-stranded DNA virus that has been used for gene delivery and can infect a wide range of dividing and non-dividing cells. Adenovirus vectors can contain up to 35 kb of exogenous DNA. They exhibit much lower host immunogenicity and achieve long-term expression of multiple transgenes in a single vector. Adenoviral infection begins with the attachment to the cell surface receptors and the interaction of the pontoons with αvβ3 and αvβ5 integrins. Subsequently, by receptor-mediated endocytosis, adenovirus escapes from the endosome and heads toward the nucleus, where viral transcription and replication take place. Completion of the infection cycle induces cell death and the release of progeny viruses.
Currently, adenovirus vector-based gene delivery strategies have been developed to directly eliminate tumorigenic human pluripotent stem cells for safer regenerative medicine.
Although viruses are the preferred gene delivery systems in clinical trials due to their high in vivo transfection efficiency and sustained gene expression after incorporation into the host genome, they have several shortcomings, including immunogenicity and cytotoxicity, the technical challenges and cumbersome production process of the vectors, the high cost of biosafety requirements, and the low packaging capacity (most viral vectors are about 10 kb, rather than the non-viral vector about 100 kb), and the variability of infectivity of the viral vector preparation.
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