An Efficient and Reversible Transposable System for Gene Delivery and Lineage-Specific Differentiation in Human Embryonic Stem Cells

• Published in: Cell stem cell Vol. 5; no. 3; pp. 332 - 342
• Main Authors: Lacoste, Arnaud, Berenshteyn, Frada, Brivanlou, Ali H.
• Format: Journal Article
• Language: English
• Published: Cambridge, MA Elsevier Inc 04.09.2009; Cell Press
• Subjects: Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Animals; Applied cell therapy and gene therapy; Base Sequence; Biological and medical sciences; Biotechnology; Cell Differentiation - drug effects; Cell Lineage - drug effects; DNA Transposable Elements - genetics; Doxycycline - pharmacology; Embryonic Stem Cells - cytology; Embryonic Stem Cells - drug effects; Embryonic Stem Cells - metabolism; Fundamental and applied biological sciences. Psychology; Gene therapy; Gene Transfer Techniques; Genetic Engineering; Genome, Human - genetics; Health. Pharmaceutical industry; Humans; Industrial applications and implications. Economical aspects; Macaca; Medical sciences; Molecular Sequence Data; Mutagenesis, Insertional - drug effects; Neurons - cytology; Neurons - drug effects; Neurons - metabolism; Organ Specificity - drug effects; Phenotype; RNA, Small Interfering - metabolism; STEMCELL; Transfusions. Complications. Transfusion reactions. Cell and gene therapy; Transgenes; STEMCELL; Human; Differentiation; Gene therapy; Embryonic cell; Stem cell
• ISSN: 1934-5909; 1875-9777
• DOI: 10.1016/j.stem.2009.07.011

Unraveling the therapeutic potential of human embryonic stem cells (hESC) requires tools to modify their genome. We have engineered the PiggyBac transposable element to create an efficient system for gene delivery in hESCs. This redesigned system, named “ePiggyBac,” can deliver up to 18 Kb inserts, and transgene expression is observed in almost 90% of hES cells. ePiggyBac transposons can also carry insulators, inducible expression cassettes, and short hairpin RNAs for gain- and loss-of-function approaches. In hES cells, ePiggyBac's efficiency is superior to that of viral vectors and previously described transposons, including other PiggyBac-based systems. In addition, ePiggyBac transgenes can be removed from the hESC genome without leaving any mutation. We used this system to direct hESC differentiation toward a neuronal phenotype. We then removed the transposons to obtain transgene-free neuronal precursors and neurons. The ability to create fully reversible genetic modifications represents an important step toward clinical applications of hESCs.