Biologists have demonstrated two new techniques for deriving embryonic stem cells in mice, both designed to sidestep the main objection of opponents of stem cell research: the need to destroy embryos.

In separate papers published online Monday in the journal Nature, researchers proved that two techniques previously proposed to derive embryonic stem cells without destroying an embryo can work – at least in mice. In one paper, researchers avoided breaking up the embryo outright, while in the second, the team’s goal was to avoid producing a viable embryo.

Embryonic stem cells are “pluripotent,” meaning they have the ability to become any type of cell in the body. That offers potential cures to diseases such as ALS (Lou Gehrig’s disease), diabetes, cancer, and Parkinson’s. But isolating the cells requires the destruction of three- to five-day-old embryos. By order of President George W. Bush, federal funding for stem cell researchers is available only for projects using stem cell lines that existed before August 2001.

Scientists say the restriction has slowed research, and have long been hunting for ways to increase funding, such as state-level initiatives and rules changes that would fund experiments on any stem cells derived from embryos left over from in-vitro fertilization procedures and earmarked for disposal.

The new techniques may offer an alternative solution to the funding problem – but researchers and ethicists say neither approach is likely to provide a clean way out of the ethical debate over embryonic stem cell research, since each endangers embryos in its own way.

In one of the Nature papers, Robert Lanza, vice president of medical and scientific development at Advanced Cell Technology in Worcester, MA, and colleagues report the creation of embryonic stem cell lines using a technique similar to preimplantation genetic diagnosis, which is used to detect genetic abnormalities in IVF embryos before they are implanted in the mother’s uterus.

In preimplantation genetic diagnosis, or PGD, doctors allow the embryos to divide until they consist of eight cells, then remove a single cell and test it for genetic abnormalities. Lanza and his colleagues did the same in mice – but rather than testing the separated cell, they put it in a petri dish with previously derived embryonic stem cells, which provided factors that pushed it to divide and develop characteristics of stem cells.

Once enough cells had grown, they separated the two different cell types into fully competent embryonic stem cell lines. The remaining seven cells of the embryos were implanted into female mice and developed into normal mice at the same rate as undisturbed IVF embryos.

It could take years to see whether the technique works in humans; but if it does, Lanza envisions that prospective parents already electing PGD might agree to let the single cell removed for diagnosis divide overnight. Then one cell would be used for diagnosis and the other to create a stem cell line for research. “You would not change the clinical outcome or add any additional risk to that embryo in any way,” says Lanza.

The eventual goal is to create the first method for deriving human embryonic stem cells that might be eligible for federal funding. “We think since this does not involve destruction of embryos, we should be able to create lines within the existing framework of laws and regulations,” Lanza says.

But to some opponents of embryonic stem cell research, particularly Catholic ethicists, Lanza’s method isn’t much better than current techniques for deriving the cells – and may not sidestep proscriptions against endangering embryos.

The Roman Catholic Church opposes in vitro fertilization itself and considers preimplantation genetic diagnosis a further affront to the sanctity of life. “PGD applied to humans is unethical,” says Richard Doerflinger, deputy director of pro-life activities for the U.S. Conference of Catholic Bishops. “It poses a risk of death to the embryo, is done chiefly to select out genetically imperfect embryos for discarding, and poses unknown risks of future harm even to the child allowed to be born.”  

In fact, PGD experts believe the procedure holds little additional risk beyond that inherent in IVF, since removing a single cell from the embryo does not significantly decrease the chances it will implant. However, they do doubt it will be easy to make Lanza’s technique work in humans.

“A human single cell is much more difficult to grow in culture” than mouse cells, says Santiago Munne, director of Reprogenetics, a private genetics lab that specializes in preimplantation genetic diagnosis. Currently, only 10 percent to 20 percent of attempts to induce a single cell from a human embryo to divide are successful, Munne says. If the cell does not divide overnight, it may die or undergo alterations that close the window of opportunity to do PGD analysis.

“If you are paying for PGD, you want every embryo diagnosed,” he says. “I would never recommend this to a PGD patient of mine.” And removing more than one cell from the embryo isn’t an option either, Munne says, since that reduces the embryo’s viability by half.

Lanza says the co-culture technique should induce the separated cells to divide more reliably, obviating Munne’s concerns and creating a new source of embryonic stem cells. His group has already begun studies using donated IVF embryos and existing embryonic stem cell lines; Lanza says it will take a year or two to complete these studies. Still, he acknowledges that it’s not a sure bet: “We hope our approach can be perfected in humans,” he says. “But a lot of people don’t have time for us to work this out. I think it would be tragic not to pursue all of the options and methods that are available to get this technology to the bedside.”

One of those other options – reported in the second Nature paper – is a technique demonstrated by MIT biologist and Whitehead Institute member Rudolph Jaenisch and Alexander Meissner, a graduate student in his lab. Called altered nuclear transfer, the method adapts a cloning technique researchers in Korea have already used to produce stem cell lines tailored to specific patients.

In the original technique, called somatic cell nuclear transfer, researchers transfer the genetic material from an adult cell into an egg stripped of its own DNA. The egg “reprograms” the adult DNA, creating an embryo with genetic characteristics identical to those of the donor. (The same technique was used to create Dolly, the world’s first cloned mammal, and many subsequent clones.)

In 2002, William Hurlbut, a medical ethicist at Stanford University and member of the President’s Council on Bioethics, proposed altering that process to produce pluripotent stem cells – stem cells with the same potential as embryonic stem cells – without creating human embryos. Hurlbut’s idea involved altering either the nucleus of the adult cell, the cytoplasm of the egg, or both before they are joined in order to prevent the formation of an embryo. If researchers can succeed, says Hurlbut, “[it] could allow a true win-win solution to this – not a compromise, but a solution.”

In 2004, Hurlbut suggested that turning off a gene called CDX2, which is involved in the earliest stages of embryonic development and is required for the embryo to form a placenta and implant in the uterus, might be one way to prevent clones from developing into viable embryos. In their Nature paper, Meissner and Jaenisch showed that by turning off CDX2 in the nucleus of the donor cell before transferring it into the stripped-down egg cell, they could create an embryo that failed to develop properly – but which could still be used create a normal embryonic stem cell line. “What one gets is a clone which has no chance to ever develop into a fetus,” says Jaenisch. “But it still can make an embryonic stem cell.”

9”This is a really significant thing that he has done,” Hurlbut says of Jaenisch’s work. “It establishes the scientific feasibility of the idea.” Hurlbut believes that CDX2 acts so early in development – at the eight-cell stage in mice – that the entities created by Meissner and Jaenisch should not be called embryos. But  many ethicists disagree.

“It doesn’t solve the ethical problem,” says Nigel Cameron, chairman of the Center for Bioethics and Culture. “If you create a deformed embryo, it’s still an embryo.” Still, that doesn’t negate the entire concept of altered nuclear transfer, he says; scientists could look for different or additional genes to alter that might yield stem cells without creating embryos.

Rev. Tadeusz Pacholczyk, a Roman Catholic priest, PhD in neurobiology, and director of education for the National Catholic Bioethics Center, agrees. “I’m glad to see Jaenisch’s work,” he says. “I’m not convinced this resolves anything, but I think it points in a hopeful direction to the way science might be used in the future.”

The next step is to consider altering some combination of genes rather than a single gene, Pacholczyk says, “so it really doesn’t make an embryo.” One proposal he finds promising is that of Markus Grompe, a stem cell researcher at Oregon Health Sciences University. Rather than turning off one or more genes to disable embryonic development, Grompe plans to force the donor cells to produce one or more transcription factors typically found only in embryonic stem cells. These proteins direct the activity of hundreds of genes that keep the cells from developing into specific cell types. The eggs might also be encouraged to make these proteins. Grompe would then follow typical nuclear transfer techniques, injecting the nucleus of the donor cell into a nucleus-stripped egg. The end result would be a pluripotent stem cell that had never passed through an embryonic stage.

Hurlbut also thinks Grompe’s ideas have potential. The CDX2 work, he says, is just a starting point. “This is the beginning of a constructive conversation,” he says. “We’re going to get in there and have a cooperative dialog and see if we can arrive at an acceptable definition of embryo, organism, human. If we don’t do this, we’re going to have just one battle after another.”

Evan Snyder, director of Burnham Institute’s Program in Stem Cells and Regeneration in La Jolla, CA, has worked with Hurlbut in the past and shares his hope for a compromise. “Our hypothesis is that in a pluralistic society, efforts to reach a consensus are worthwhile,” he says. Even if the two new techniques fail to work in human cells or fail to satisfy critics, “no experiment is useless, and in trying to derive cells in alternate ways, we may learn some new things.” 

But other researchers and ethicists say there is probably no way to stop the moral merry-go-round to which both opponents and supporters of stem cell research find themselves strapped. Neither Lanza’s nor Jaenisch’s approach really solves the moral dilemma, says Sean Philpott, a research microbiologist with the New York State Department of Public Health and executive managing editor of the American Journal of Bioethics. “This is scientific hand-waving,” he says. “We are putting so much time and effort into trying to come up with solutions that are acceptable to all sides, when really there are no acceptable solutions.”

Ultimately, Philpott believes, “We’re going to have to accept that there are going to be a considerable number of Americans who are morally opposed to embryonic stem cell research. That doesn’t mean that we shouldn’t pursue it.”