Gene targeting technique extended to stem cells

Feb. 10, 2003

by Terry Devitt

The technique that helped revolutionize modern biology by making the mouse a crucible of genetic manipulation and a window to human disease has been extended to human embryonic stem (ES) cells.

In a study published today (Feb. 10) in the online editions of the journal Nature Biotechnology, a team of scientists from UW-Madison reports that it has developed methods for recombining segments of DNA within stem cells.

By bringing to bear the technique, known in scientific parlance as homologous recombination, on DNA in human embryonic stem cells, it is now possible to manipulate any part of the human genome to study gene function and mimic human disease in the laboratory dish.

"Indeed, homologous recombination is one of the essential techniques necessary for human ES cells to fulfill their promise as a basic research tool and has important implications for ES cell-based transplantation and gene therapies," write Wisconsin researchers Thomas P. Zwaka and James A, Thomson, the authors of the new study.

The technique has long been used in the mouse and is best known in recent years for its use to generate mice whose genomes have been modified by eliminating one or more genes. Known as 'knockouts,' genetically altered mice have become tremendously important for the study of gene function in mammals, and have been used to explore everything from the underlying mechanisms of obesity and other conditions to the pinpointing of genes that underpin many different diseases.

Significant differences between mouse and human embryonic stem cells have, until now, hampered the application of the technique to human ES cells, according to Zwaka, the lead author of the Nature Biotechnology report and a research scientist working in the laboratory of James Thomson. Thomson was the first to isolate and culture human embryonic stem cells nearly five years ago.

"This is a big benefit for the human ES cell field," Zwaka said. "It means we can simulate all kinds of gene-based diseases in the lab - almost all of them."

To demonstrate, the team led by Zwaka and Thomson were able to remove from the human genome the single gene that causes a rare genetic syndrome known as Lesch-Nyhan, a condition that causes an enzyme deficiency and manifests itself in its victims through self-mutilating behavior such as lip and finger biting and head banging.

The study of genes derived from human ES cells, as opposed to those found in mice, is important because, while there are many genetic similarities between mice and humans, they are not identical. There are human genes that differ in clinically significant ways from the corresponding mouse genes, said Zwaka. The gene that codes for Lesch-Nyhan is such a gene, as mice that do not have the enzyme do not exhibit the dramatic symptoms of the disease found in humans whose genes do not make the enzyme.

Another key aspect of the new work is that it may speed the effort to produce cells that can be used therapeutically. Much of the hype and promise of stem cells has centered on their potential to differentiate into all of the 220 kinds of cells found in the human body. If scientists can guide stem cells - which begin life as blank slates - down developmental pathways to become neurons, heart cells, blood cells or any other kind of cell, medicine may have access to an unlimited supply of tissues and cells that can be used to treat cell-based diseases like Parkinson's, diabetes, or heart disease. Through genetic manipulation, 'marker' genes can now be inserted into the DNA of stem cells destined for a particular developmental fate. The presence or absence of the gene would help clinicians sort cells for therapy.

"Such 'knock-ins' will be useful to purify a specific ES-cell derived cell type from a mixed population," Zwaka said. "It's all about cell lineages. You'll want dopamine neurons. You'll want heart cells. We think this technique will be important for getting us to that point."

Genetic manipulation of stem cells destined for therapeutic use may also be a route to avoiding transplant medicine's biggest pitfall: overcoming the immune system's reaction to foreign cells or tissues. When tissues or organs are transplanted into humans now, drugs are administered to suppress the immune system and patients often need lifelong treatment to prevent the tissue from being rejected.

Through genetic manipulation, it may be possible to mask cells in such a way that the immune system does not recognize them as foreign tissue.