Cracking Cloning

Nuclear transfer research encompasses some of the most compelling biological and ethical puzzles of our time. In an online publishing experiment, we asked you, The Scientist readers, to help us create the article. Here's how you would solve the mysteries of the egg, fertilization, and cloning.

By The Readers and Editors of The Scientist

The thunder of a late spring rain rumbles through Philadelphia. Inside a Temple University lab, an ovulated mouse egg rests in metaphase II. Devoid of a nuclear envelope, its chromosomes lie naked, held near the edge of the cell, suspended mid division by a spindle, and just barely visible as a bright hump in the near perfect sphere.

If a sperm were to enter this cell, it would set off a remarkable chain of events: The completion of meiosis and extrusion of a second polar body containing 20 maternal chromosomes; the stripping of protamines from the sperm's DNA and replacement with histones; the unification and reprogramming of the two haploid genomes; and eventually a rapid succession of divisions that transform the cell from egg to embryo with some potential to become a full grown mouse. What scientists have discovered about the process could fill several books.

Such books, however, provide only snapshots, lightning flashpoints in a dark developmental world. Sitting at a row of microscopes at Temple's Fels Institute for Cancer Research, Zhiming Han hopes to crack the secrets of this ovulated mouse egg. To do so, she's going to replace a key feature, the maternal DNA, with that from an adult cell.

Her tool, a piezoelectric micropipette makes infinitesimal jabs when Han steps on a foot switch. The thunder outside doesn't distract her as she uses this micron sized jackhammer to punch through the dense zona pelucida surrounding the egg. She locates the spindle chromosome complex and draws it into the pipette. With a fluid motion, she retracts it, dumps it, and pushes the egg out of the field of view - ready to move on to the next.

For researchers studying nuclear transfer, what Han has done is mere routine - the equivalent of chopping an onion for a prep cook - something she can do upward of 300 times in a day. But the unknown biological consequences of this deft nuclear theft are no less unsettling. The myriad roles that discarded material might have played are unclear. The spindle-depleted cell will be put aside to rest for an hour before Han comes back and injects a nucleus from a mouse cumulus cell. Han has been working to elucidate why a viable mouse embryo develops only two percent of the time.

"The egg is round and fairly simple in appearance but it's entrusted with executing the essential, remarkable events for starting a new life," says Keith Latham, the Temple professor in whose lab Han works. Because it's in mice, his group's research is fairly uncontroversial. And, Latham jokes, not exactly lucrative: "There isn't a tremendous market for cloned mice."

Nevertheless, the work here represents part of a field of research with largely unprecedented challenges, both in its approach and its intersection with public policy: nuclear transplantation for the derivation of human embryonic stem cell lines, or therapeutic cloning, as it has been called in the media.

Throughout the month of April, we posed three related questions to the readers of The Scientist online: How can science overcome the technical hurdles to somatic cell nuclear transfer (SCNT) with human cells? Is it time to reevaluate the ethics in doing so? And is there a better way to communicate to the public exactly what such studies mean and what promise they hold?

Responses to our queries epitomized the frustration of those trying to study a phenomenon about which we know little, and the polarization of the ethical debate. Still, in the online discussion and separate discussions with researchers in the field, there was a sense that success, depending on how you define it, is inevitable. On the following pages, we discuss four issues that will define this success: The egg, the nucleus, the animal models, and the embryo.

Eggs aren't just enigmatic. They're also the most precious material in this line of research. The process for donors is intense and poses uncertain risk. "Getting the egg donors is not a trivial matter;" writes Deepak Srivistava, director of the Gladstone Institute of Cardiovascular Disease, in our forum. "I don't think [there are] going to be that many people who come forward for egg donation purely for research purposes."

In an effort to protect donors, guidelines for stem cell research by the National Academy of Sciences (NAS), in 2005, and by the International Society for Stem Cell Research (ISSCR), earlier this year, addressed egg donors specifically¹. The guidelines were similar, but differed in how research should be overseen. Still, say Leonard Zon and colleagues at the Children's Hospital Boston Stem Cell Program, in a post to our Web discussion, "Debate continues around the world about when monetary incentives may become an undue factor in a woman's decision to donate."

Kevin Eggan, from Harvard, says he's aggravated with what he perceives as a double standard. In the Spring of last year, he and Doug Melton received approval for human nuclear transfer experiments. But while fertility clinics routinely provide financial incentive for donors Eggan's team was barred from doing so. Expenses such as child care and travel can be covered, but protocols are scrutinized for anything that might be considered "undue inducement." "Doug and I have spent more than $50,000 on advertising in the last year and many hundreds of women have responded to our ads ? but when they can go and do the exact same thing, and be paid to help another woman get pregnant, they choose to do that." They have yet to see a single human egg and Eggan predicts the same fate will stall the work planned by the California Institute for Regenerative Medicine.

Evan Snyder, director of the Stem Cells and Regeneration program at Burnham Institute in La Jolla, Calif. affirms in our forum: "As to the rights of egg donors, they should be addressed, as we do all organ donations-with respect and without direct compensation." In a phone interview, Snyder says that in vitro fertilization (IVF) clinic-associated compensation appears an outlier in tissue donation, but says, "I'm a little reluctant to compromise the ideal position that we've all been assuming."

Snyder says he worked on developing the laws in place in California, and is comfortable that a nuclear reprogramming study now underway can abide by the laws, in part due to partnerships with clinical research institutes that may be able to assist in providing unwanted eggs. This is similar to a stance that Srivastava presented in our forum. "I've advocated a scenario where people who don't get in vitro fertilization done for financial reasons, even though they have the fertility problems, get the procedure partially or fully paid for in exchange for donating a portion of their eggs to research. In many cases of IVF, some of the eggs get frozen and never used, and that doesn't do anybody any good."

While laws in Massachusetts and California clearly prohibit compensation, the ISSCR placed the review of reimbursement practices in the hands of an unspecified form of stem cell research oversight. This was seen by some as a step back from established principles. Marcy Darnovsky of the Center for Genetics and Society, in Oakland, Calif., wrote in our Web forum that the laws passed in California were enacted to protect women. "In order to head off the emergence of a market in which predominantly poor women are the ones who wind up selling their eggs, it limits payment to reimbursement for direct expenses." In her comments she urges more federal regulatory measures to oversee nuclear transfer research and embryonic stem cell research.

When asked about the perceived double standard in fertility clinics, Darnovsky says, "The fact that we're doing something wrong in assisted reproduction sphere doesn't justify doing it that same wrong way in the research sphere."

While human eggs are scarce, those of non-primate mammals, are abundant. For this reason and perceived incentives in agriculture - such as cloning a top milk-producing cow for use in breeding - nuclear transfer in non-primate mammals has proceeded apace. This could hasten success with human cells. "Use of animal models may help identify the type of adult human cell that is most amenable to reprogramming and successful nuclear transfer," writes Zon. And research groups have taken another step - transplanting human nuclei into rabbit or cow eggs to investigate the reprogramming interaction between egg and genome, albeit, with limited success. Snyder says this may be a direction for research he and colleagues have planned at Burnham.

Embryologist Davor Solter at the Max Planck Institute for Immunobiology wrote us asking "Why are people trying to use rabbit or cow eggs to reprogram human cells but not mouse? It seems to me that one could use WI38 or other diploid human cell lines and mouse eggs without needing anybody's permission and maybe one could learn a lot." Indeed, consent would not be needed from egg or nucleus donor in this case, but public reaction to chimera experiments such as these has not been entirely positive.

"Avignus," who commented on our Web site offered this imperative: "The techniques employed must be centered around our increasing understanding of phenomena such as DNA remodeling and epigenetic reprogramming that naturally occur in fertilized zygotes."

Understanding nuclear reprogramming can be seen both as a step in furthering the success of nuclear transplant, but would be a pretty impressive result in its own right. Our online discussion contained the word "reprogram" more than 20 times in 60 posts. The remarkable chemistry of the ooplasm contains necessary ingredients to, in a sense, turn back the genomic clock allowing an adult nucleus to shed the expression profile of its differentiated state and become part of a totipotent embryonic cell. But it's no secret that this reprogramming is incomplete. The low efficiency of nuclear transfer isn't the only indication that this is true.

A recent study from Latham's lab looked at gene expression between mouse embryos created via fertilization, SCNT, and parthenogenesis. They found a large number of genes misexpressed in SCNT constructs, particularly those of transcription factors likely involved in the long-term fate determination of cells.² A normally fertilized egg needs to terminate the gene expression program that led to the creation of both gametes and turn on the genes needed by an embryo. With a transferred nucleus, scientists are giving the egg something very different to work with, "like putting a Russian-speaking person in a room full of people who only speak French," Latham says. These cloned constructs appear to "behave" more like the adult cells from which the nucleus was taken. They even seem to grow better in media designed for adult cells, according to Latham. Eggan, however, says his group hasn't had to use different media for cloned mouse embryos.

Solter, under whom Latham once worked at the Wistar Institute, writes: "We are sort of in agreement that ovulated oocyte can reprogram but zygote cannot. Why? Are the 'reprogramming factors' used up following fertilization by reprogramming sperm? Or are the 'reprogramming factors' normally in the nucleus so they are released to the cytoplasm in MII oocyte but removed with the zygote pronuclei? Or both?" The consensus seems to be that when it comes to nuclear reprogramming, there are more questions than answers.

In our online discussion, a great deal of debate revolved around the moral status of the embryo. One of the very first commenters, under the moniker "blahboy," expresses the frustration felt by many who argue that embryos have rights and feel their arguments are being marginalized: "You seem to want to lump me in with moonbats that believe aliens are hiding in comets tails." This individual came back to our comment board to ask that "rather than labeling things 'ethical concerns,'" we list them "for all to see."

Leonard Zon and colleagues pointed to two: "a) human egg donation and b) the moral status of pre-implantation embryos (or the embryo-like entities created by nuclear transfer)." By doing so, they imply a distinction between embryos created by the union of sperm and egg and the entities created by nuclear transfer. In a 2004 issue of the Connecticut Law Review, Harvard Medical School professor Ann Kiessling suggested terminology such as "pre-embryo" or "ovasome" for embryos created by nuclear transfer because of differences in their potential to develop (for more discussion on terminology see "A Stem Cell Makeover").³

In a response to Kiessling, in the same journal, Latham and Carmen Sapienza, also at the Fels Institute at Temple, rejected efforts to rename nuclear-transfer derived embryos on the basis of their developmental potential. Such "distortion of simple, well-defined, broadly accepted, and widely-understood terms such as 'embryo,'" they wrote, "serves political, not scientific ends."⁴ As politicization of stem cell research has become front and center, scientists have often debated whether "framing" an issue to make it more publicly palatable detracts from the science.

A recent Science article on framing science sparked wide debate among science bloggers on the Web⁵. Matthew Nisbet, a social scientist at American University and Chris Mooney, a science writer based in Washington, DC, demanded in a short policy forum that scientists work to actively frame their arguments. In our web discussion, Nisbet offered that a small self-selecting public will actually access news about stem cell research and nuclear transfer and those that do will generally use preconceived frames of reference such as political stance or religious ideology to make decisions about it. "The challenge then is to use a combination of new media platforms such as entertainment TV and film; targeted documentaries; YouTube; (non-science) blogs, and other outlets to engage the wider public," he writes in our forum. Several commenters called for more educational outreach to the public.

But education, it would seem won't change every mind, even among scientists. Comments on our website included a few entreaties from scientists that nuclear transfer and embryonic stem cell research be abandoned or at least postponed. Shanthi Raam, a retired cancer researcher from Tufts University School of Medicine, wrote: "Pluripotent cells are available in adults, placentas that we throw out at birth, neonatal cord blood; let us focus on these first and exhaust all possible uses." Other comments were more adamant. "Human lives are not just cells to be manipulated. Each of us, and each of our children, deserves to be treated with more dignity than that," wrote Margie Roach.

Bernard Siegel of the Genetics Policy Institute wrote that positively framed messages about stem cell science and cloning were being understood by the public: "People understand there's a difference between cloning a baby and cloning a stem cell line." Yet, other commenters still expressed fear that a slippery slope would lead to live births of human clones. One commenter even wrote that he looks forward to reproductive cloning, citing a recent Nature editorial that called the event inevitable.⁶ But most researchers commenting on the website agreed to the consensus widely reported that an absolute proscription on reproductive cloning should be in place.

This may be one of the only points on which much consensus will take hold. Although many predict that progress in therapeutics may change perceptions, for now, people can only make predictions about stem cell lines that have never existed, and will require a great deal of work in both research and public policy to create.

The editors of The Scientist chose to spark this discussion on the web because it is an interesting scientific puzzle with ethical concerns that are worth talking about. We hope that readers will continue to talk.