20 October 2008

Research of the Week

Viruses no longer required to generate stem cells

In this colony of human embryonic stem cells, the cell nuclei, which contain DNA, are stained green; the cell surfaces appear red.

A major breakthrough in stem cell technology could allow scientists to transform adult human cells into stem cells without the use, and accompanying risks, of using a virus to carry genetic material.

In November 2007 Shinya Yamanaka and colleagues published a method to transform adult human skin cells into stem cells. Mature cells, such as skin cells, are normally locked into their identities — skin cells do not transform into brain cells, much like middle-aged scientists do not have the ability to leave the lab and become professional athletes.

Yamanaka’s breakthrough was the identification of four factors, four genes, that when added to and turned on in mature cells could reprogram them into stem cells. These stem cells could, under the right conditions, develop into heart cells, brain cells, or a number of other different types of cells. In the jargon of science, such stem cells are called induced pluripotent stem cells: “induced” because their creation was not spontaneous, “pluripotent” because they can become any cell type found in an embryo, fetus or mature organism.

The problem with Yamanaka’s original method was that he used a virus to insert the genes into a mature cell and generate a stem cell. Using a virus to generate stem cells poses huge safety concerns for therapeutic applications in humans. The virus inserts itself into the cell’s genome permanently, which could disrupt the way genes normally function.

Now Yamanaka has generated stem cells without using a virus. An article published October 9 in Science magazine reports his laboratory generated induced pluripotent stem cells from mouse embryonic fibroblasts (cells that make up connective tissue). Instead of using a virus, Yamanaka applied a well-known chemical cocktail to allow the critical genes to slip into the fibroblasts.

Scientists still have work to do. Yamanaka’s nonviral technique is much less efficient at generating stem cells than using a virus. Future studies need to apply this method to cells from adult humans — Yamanaka used cells from a mouse embryo — and confirm that there are no adverse effects. However, this breakthrough brings researchers one step closer to a nontoxic method for generating human stem cells from adult tissue.

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Gladstone Institutes of Health
University of California, San Francisco
October 10, 2008

Shinya Yamanaka MD, PhD, of Kyoto University and the Gladstone Institute of Cardiovascular Disease (GICD), has taken another step forward in improving the possibilities for the practical application of induced pluripotent stem (iPS) cell technology.

Previously, Dr. Yamanaka had shown that adult cells can be reprogrammed to become embryonic stem cell-like using a cancer-causing oncogene as one of the four genes required to reprogram the cells, and a virus to transfer the genes into the cells. In the last year, Dr. Yamanaka and other labs showed that the oncogene, c-Myc, is not needed. However the use of viruses that integrate into the genome prohibit use of iPS cells for regenerative medicine because of safety concerns: its integration into the cell's genome might activate or inactivate critical host genes.

Now Dr. Yamanaka's laboratory in Kyoto has eliminated the need for the virus. In a report published this week in Science, they showed that the critical genes can be effectively introduced without using a virus. The ability to reprogram adult cells into iPS 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 iPS field and stem cell research in general is progressing rapidly," said GICD Director Deepak Srivastava, MD. "But, as Shinya has shown, each step forward reveals a new set of challenges."

Dr. Yamanka's 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 with the injected cells contributing to almost all cell types.

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.

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Okita K, Nakagawa M, Hyenjong H, Ichisada T, Yamanaka S. Generation of mouse induces pluripotent stem cells with viral vectors. Science, In press.

About the Gladstone Institutes

The J. David Gladstone Institutes, affiliated with the University of California, San Francisco (UCSF), is dedicated to the health and welfare of humankind through research into the causes and prevention of some of the world's most devastating diseases. Gladstone is comprised of the Gladstone Institute of Cardiovascular Disease, the Gladstone Institute of Virology and Immunology and the Gladstone Institute of Neurological Disease. More information can be found at www.gladstone.ucsf.edu.

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