iPS Cell Generation Using a Single Lentiviral Stem Cell Cassette

By Cesar Sommer, M. Stadtfeld, George J. Murphy, K. Hochedlinger, Darrell N. Kotton, G. Mostoslavsky. Stem Cells, 2009

697 citations recorded by the source corpus.

Induced pluripotent stem (iPS) cells can be generated using retroviral vectors expressing Oct4, Klf4, Sox2, and cMyc. Most prior studies have required multiple retroviral vectors for reprogramming, resulting in high numbers of genomic integrations in iPS cells and limiting their use for therapeutic applications. Here we describe the use of a single lentiviral vector expressing a “stem cell cassette” composed of the four transcription factors and a combination of 2A peptide and internal ribosome entry site technology, generating iPS cells from postnatal fibroblasts. iPS cells generated in this manner display embryonic stem cell‐like morphology, express stem cell markers, and exhibit in vivo pluripotency, as evidenced by their ability to differentiate in teratoma assays and their robust contribution to mouse chimeras. Combining all factors into a single transcript achieves the most efficient reprogramming system to date and allows derivation of iPS cells with a single viral integration. The use of a single lentiviral vector for reprogramming represents a powerful laboratory tool and a significant step toward the application of iPS technology for clinical purposes. STEM CELLS 2009;27:543–549

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/822d8eea4414e770c93abb3809482f5586f7f438

How researchers see this paper

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In Genetics

1. Michel Sadelain
2. M. Stadtfeld
3. K. Hochedlinger

In Molecular Biology

1. M. Stadtfeld
2. K. Hochedlinger
3. Michel Sadelain
4. L. Studer
5. S. Yamanaka

Citations in the corpus

• Induced pluripotency: history, mechanisms, and applications. (2010, background): “Lentiviral vectors are also more efficient than retroviral vectors at infecting different somatic cell types and can be used to express polycistronic cassettes encoding all four reprogramming factors, thus increasing reprogramming efficiency (Carey et al. 2009; Sommer et al. 2009).”
• Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site (2012, neutral): “3% (5) K111M 59 Top10 ,200 (n51) 10 50% (5) T239D 63 Top10 ,200 (n51) 10 60% (6) T239V 63 Top10 19 (n51) 10 90% (9)”
• Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation (2009, background): “The stoichiometric requirements presented here will prove useful in guiding the implementation of optimized protocols for hiPSC induction and the rational design of improved reprogramming vectors, such as polycistronic and non-integrating vectors (9, 31, 32).”
• Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors (2011, methodology): “Toward this end, several techniques have been used for obtaining integration and/or transgene-free iPSCs, including the use of plasmids (3, 4), the Cre/ loxP system (5, 6), adenoviruses (7), piggyBac (8, 9), minicircle vector (10), and proteins (11, 12).”