- Regenerative Medicine at Gladstone -

Another Step Forward for iPS Cells

Shinya Yamanaka

Major scientific breakthroughs represent more of a beginning of experiments than an end. Often many practical problems must be solved before the new technology can be brought into practice.

The remarkable discovery by Shinya Yamanaka that adult cells can be reprogrammed to become stem cell-like cells is just such a beginning. His induced pluripotent stem (iPS) cell technology has revolutionized stem cell research and brought the promise of stem cell-based therapies much closer. But many challenges remain. In the case of iPS cells, these challenges include the need to use an oncogene as one of the four genes required to reprogram the cells, a virus to transfer the genes into the cells, and the poor efficiency of the reprogramming process. In the last year, Dr. Yamanaka and other labs showed that the oncogene, c-Myc, is not needed.

Now Dr. Yamanaka's laboratory in Kyoto has eliminated the need for the virus. In a recent paper in Science, they showed that the transfection can be accomplished with a plasmid instead.

Use of the retrovirus was a major safety concern for iPS cells in regenerative medicine: its integration into the cell's genome might activate or inactivate critical host genes. The ability to reprogram cells without viral integration into the genome also lays to rest concerns that the reprogramming event might be dependent upon viral integration into specific genomic loci that could mediate the genetic switch.

The Yamanaka team began this series of experiments by replacing the retrovirus with an adenoviral vector. While transfections with the genes on separate vectors didn't work, they did work when the genes were arranged in a specific order on a single vector. The same arrangement worked when the genes were incorporated into a plasmid. To determine if the plasmid-mediated reprogrammed cells were pluripotent, the scientists transplanted the cells under the skin of immunocompromised mice. The resulting tumors contained a wide variety of cell types from all three germ layers. iPS cells injected into embryos resulted in chimeric mice.

Major scientific breakthroughs represent more of a beginning of experiments than an end. Often many practical problems must be solved before the new technology can be brought into practice.

Still, other problems remain to be solved. The efficiency of the gene transfer with the plasmid was lower than with the retrovirus. Nevertheless, this significant step moves us closer to realizing the promise of stem cells in the understanding and eventual cure of diseases.

Generating virus-free iPS cells. (A) Expression plasmids for iPS cell generation. The three cDNAs encoding Oct3/4, Klf4, and Sox2 were connected in this order with the 2A-peptide and inserted into a plasmid. In addition, the c-Myc cDNA was inserted into another plasmid. Thin lines (O-1, O-2, K, K-S, 1 to 11, and M) indicate amplified regions used to detect plasmid integration into genome. The locations of the CAG promoter, the ampicillin-resistant gene (Amp), and the polyadenylation signal (pA) are also shown. (B) Time schedules for induction of iPS cells with plasmids. Open arrowheads indicate the timing of cell seed, passage, and colony pickup. Solid arrowheads indicate the timing of transfection.

Okita K, Nakagawa M, Hyenjong H, Ichisada T, Yamanaka S (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science 322:949-953.