The Scientist

Researchers have used altered nuclear transfer (ANT) to generate pluripotent murine embryonic stem (ES) cells from blastocysts inherently unable to implant in the uterus. Scientists hope the findings, published in the October 16 issue of Nature, will provide ES cell researchers with a technique that sidesteps the destruction of embryos, triggering ethical controversies.

"This is just one possible approach using ANT, but this study provides the scientific basis for the conversation that could lead us to resolve our nation's impasse over ES cell research," William Hurlbut, from Stanford University, Ca., told The Scientist. Hurlbut was the first to conceive of the idea of ANT, but did not participate in the study.

In another study, published in the same issue of Nature, researchers report using a cell biopsy technique for diagnosing genetic defects to extract ES cells without diminishing the embryo's viability, another technique they hope will help overcome the ethical barriers to ES cell research.

Working with mice, Rudolph Jaenisch and Alexander Meissner from the Massachusetts Institute of Technology in Cambridge, Mass. created blastocysts unable to express the gene Cdx2. Cdx2 is known for its crucial role in the formation of the trophectoderm, which gives rise to the placenta.

"By silencing the expression of Cdx2 before nuclear transfer, our goal was to create a cellular system unable to establish the basic body pattern of a human embryo, but able to generate fully functional ES cells," said Meissner.

To do so, the researchers infected murine fibroblasts with a conditional lentiviral RNA interference (RNAi) construct targeting Cdx2. After nuclear transfer, the resulting blastocysts generated ES cells in vitro, but were unable to implant when transferred in the uterus.

"This procedure isn't only interesting for its potential applications in humans," Meissner told The Scientist. "It's a confirmation that the RNAi technique – which is a much faster, easier and cheaper technique than a knock-out – can be used to study gene function in animals."

Because the modified ES cells' developmental potency was diminished compared to control cells, the team restored normal Cdx2 expression levels and pluripotency by transfecting a Cre plasmid targeting the RNAi construct.

"We've proved (ANT) works in mice, now there's a long road before anything can be tested with human cells," said Meissner. "We've first got to learn more about Cdx2 in humans."

Some scientists, however, oppose ANT, arguing that the technique could create additional technical hurdles and might not work in humans. "Practically, nuclear transfer is already a challenge in itself. Add genetic modifications, gene transduction - it's getting pretty heavy just to reach the same results," Bruno Peault, from the University of Pittsburg Medical Center, Pa., who did not participate in the study, told The Scientist. "The more mending you accumulate, the more unsuspected consequences you might get," he said, adding that he still supports the use of ANT.

While ANT received the endorsement of groups traditionally opposed to NT for ethical reasons, others remain unsatisfied. "I consider it's an abuse of science to use cloning to deliberately create crippled human embryos," Robert Lanza from Advanced Cell Technology in Worchester, Mass., who did not participate in the ANT study, told The Scientist. "Blocking Cdx2's action prevents the embryo from attaching to the uterine wall, therefore it no longer receives oxygen and nutrients and simply starves to death. I don't see how that solves any ethical problems."

In the same issue of Nature, Lanza and his team published another report in which they removed single cells to produce ES lines, without affecting the viability of embryos. Using the cell biopsy technique used for IVF pre-implantation genetic diagnosis, the team extracted single blastomeres to grow pluripotent ES cells. Outside observer Irving Weissman, from Stanford University, Ca., told The Scientist this method could be used by couples undergoing IVF, but would not be useful for therapeutic cloning, given that it involved creating an embryo that would never be implanted. "Each method has its own limitations and both would have different fields of applications," he said.