Alex Philippidis Senior News Editor Genetic Engineering & Biotechnology News

“Eureka Moment” Helped Researchers Make History 20 Years Ago

The greatest catalyst for the cloning of Dolly the sheep, according to the research team leader, was a conversation in a Dublin bar.

Sir Ian Wilmut was attending the 1987 annual conference of the International Embryo Transfer Society when he went out with scientists that included Geoff Mahon, whom he knew from their days at University of Cambridge. Dr. Mahon shared how veterinarian Steen Willadsen, Ph.D., obtained calves through the transfer of nuclei from embryos at the blastocyst stage.

“Steen's success suggested to me in that revelatory moment that if we could derive embryo stem cells from livestock species, nuclear transfer was feasible. Taken together, these two techniques would make it possible to introduce precise genetic modifications into livestock species for the first time,” Sir Ian wrote in 2010 in Times Higher Education. “It was this eureka moment that led me to change the emphasis of my own research completely, and ultimately led to the birth of Dolly.”

Dolly was born July 5, 1996, healthy and weighing 6.6 kg (14.55 pounds), though her birth wasn’t announced until February 22, 1997. The research leading to that event began years earlier, when scientists doubted that cloning adults was possible, based on amphibian research that appeared to be confirmed by early mammal studies that James McGrath, M.D., Ph.D., and Davor Solter, M.D., Ph.D., declared in a 1984 Science paper: “The cloning of mammals by simple nuclear transfer is biologically impossible.”

After hearing about Dr. Willadsen’s work, then visiting him, Sir Ian sought and received commercial funding for new research. That funding enabled Sir Ian to recruit Keith Campbell, Ph.D., to the Roslin Institute at the University of Edinburgh. While Sir Ian studied the reproductive biology of livestock, Dr. Campbell focused on mechanisms that regulate the growth and division of cells.


Starving Nuclei

A series of key experiments yielded the conclusion that nuclei that were induced to quiescence by being starved of some nutrients had greater potential to support normal development. The first experiment saw the birth of two sheep, Megan and Morag in 1995, following the transfer of nuclei from differentiated cells derived from late blastocyst-stage sheep embryos by Jim McWhir, Ph.D., of the Roslin Institute.

Because the procedure showed low efficiency, researchers next sought to transfer nuclei from later stages of development and introduce genetic modifications into cells before nuclear transfer. When government funding proved insufficient, the researchers turned to a Roslin Institute spinout PPL Therapeutics, which set out to derive similar cell lines from late blastocysts, while the researchers agreed to assess their developmental potential. They then won government funds to transfer nuclei from fetuses in the first sheep season and from adult cells the following season.

PPL’s Angelika Schnieke, Ph.D. (now chair, livestock biotechnology at Technical University of Munich), had cultures derived from the mammary gland of pregnant sheep, since the company’s main business was producing clinically useful human proteins in sheep milk. “It was she who suggested that they be used because they were a well-characterized, stable cell line. So it was that a few months later it was appropriate to christen our first clone from an adult ewe ‘Dolly,’ after Dolly Parton,” Sir Ian wrote in 2013 in Cellular Reprogramming, published by GEN publisher Mary Ann Liebert Inc.

To create Dolly, Sir Ian and colleagues removed mammary gland cells from a 6-year-old Finn Dorset sheep, then preserved the cells in test tubes so the researchers would have genetic material for DNA fingerprinting, he told The New York Times in 1997. Dolly’s progenitor died by the time the clone was born.

Dolly herself died at age 6. The Roslin Institute confirmed on February 14, 2003, that she was euthanized after developing a progressive lung disease, a year after she was diagnosed with premature arthritis.


Inflection Point

“Historically, it will be viewed as an inflection point in the history of mankind,” Bernard Siegel, J.D., executive director of the Regenerative Medicine Foundation, told GEN. “The struggle that society has had is a tug-of-war between vast scientific advances wrought by convergence of technologies on one side, and on the opposite side a rising specter and fear of a dystopian future where science has no boundaries, and we’re facing species-altering events that can’t be reversed.

“The cloning of Dolly was the starting gun of this deep societal debate that goes on till this day,” Siegel added.

A month after Dolly’s birth was announced, President Bill Clinton barred use of federal funds for human cloning. President George W. Bush went further, limiting federal funding for research on human embryonic stem cells (hESCs) derived by means that destroy human embryos and to just stem cell lines already available—an action reversed in 2009 by President Barack Obama.

Since Dolly, animal cloning from cultured cell populations derived from adult animals has been repeated in species that include sheep, pigs, cows, rats, mice, dogs, cats, horses, and deer. Those successes have also raised fears that someday humans will join the list of successfully cloned animals.

“There’s nobody running around saying, ‘I can make a fortune by cloning humans.’ Nevertheless, it’s where a lot of the public perceives there to be still a good deal of danger,” Arthur Caplan, Ph.D., the Drs. William F and Virginia Connolly Mitty Professor and founding head of the Division of Bioethics at New York University Langone Medical Center, told GEN.

That trepidation, he said, can be seen in the debates over GMOs, the synthetic genome, and three-person in vitro fertilization. Fears of cloning misuse also resurfaced in April last year, when Junjiu Huang, Ph.D., and colleagues at China’s Sun Yat-sen University published results in the journal Protein & Cell from their use of CRISPR/Cas9 to replace the HBB gene, mutations of which have been implicated in β-thalassemia. Out of 86 human embryos subjected to genetic manipulation, 71 survived. Of the surviving embryos, 54 were genetically tested. Just 28 were successfully spliced. An even smaller number contained the desired genetic material. Off-target effects were seen.

“We did learn a little bit from Dolly in that we’re at least trying to surface the ethical issues before anybody does anything,” Dr. Caplan added.

The first announcement of a human cloning more than a decade ago sparked calls by politicians to ban the practice, as well as allegations of fraud that remain as unproven as the claims that sparked them. In 2002, Clonaid—the project name of a Bahamas-registered company—claimed to have successfully performed the first reproductive cloning of a human baby, called “Eve.” Siegel, a lawyer, petitioned the Broward County Circuit Court to appoint a special guardian for Eve and sued Clonaid, arguing that he was seeking to protect the rights of the allegedly cloned child. No proof confirming the alleged cloning of Eve and subsequent babies has surfaced.  Four years later, Hwang Woo-suk, Ph.D., admitted that he misrepresented research results in which he claimed the first-ever cloning and extracting of patient-specific hESCs, as well as the first cloned canine.


Breakthroughs

No similar taint followed the 2006 disclosure by Shinya Yamanaka, M.D., Ph.D., and Kazutoshi Takahashi, Ph.D., that they generated induced pluripotent stem cells (iPSCs) capable of germline transmission from mouse somatic cells by transduction of four transcription factors, Oct3/4, Sox2, Klf4, and c-Myc. The researchers and colleagues later used those factors to generate iPSCs from adult human dermal fibroblasts that were similar to hESCs in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Dr. Yamanaka co-won the 2012 Nobel Prize in Physiology or Medicine for the discovery, building on the work of co-winner Sir John B. Gurdon, D.Phil., D.Sc.

Those breakthroughs made iPSCs an alternative to somatic cell nuclear transfer (SCNT), which required large quantities of eggs derived from embryos created through fertility treatments—raising moral objections about the resulting destruction of human life. But iPSC technology  hasn’t been able to create some cell types, such as cartilage, hence the interest by some researchers in improving SCNT.

In 2013, a team led by Shoukhrat Mitalipov, Ph.D., of Oregon Health & Science University successfully carried out SCNT on human oocytes, reprogramming human skin cells to become hESCs. Dr. Mitalipov earlier led the successful replacement of the mitochondrial genome in mature non-human primate oocytes (Macaca mulatta) by spindle–chromosomal complex transfer from one egg to an enucleated, mitochondrial-replete egg, a key technique in development of three-parent embryos.

The following year, Dieter Egli, Ph.D., of the New York Stem Cell Foundation (NYSCF) Research Institute and Mark V. Sauer, M.D., of Columbia University Medical Center, created the first disease-specific diploid-state hESC line from one adult donor with type 1 diabetes and a healthy control. The researchers used SCNT to add the nuclei of adult skin cells to unfertilized donor oocytes. “By reprograming cells to a pluripotent state and making beta cells, we are now one step closer to being able to treat diabetic patients with their own insulin-producing cells,”  the authors stated.


Valuable Role

Those studies showed human SCNT is not only possible, but can play a valuable role in regenerative medicine, Geoff Lomax, Dr.P.H., senior officer for medical affairs and strategic centers with the California Institute for Regenerative Medicine, told GEN.

“Value may be found in comparing how iPSCs, SCNT cells, and hESC cells behave, such as differentiation of the pluripotent cells to a neuron,” Dr. Lomax said. “If and where one sees differences, one can generate hypotheses about what characteristics are best for regenerative medicine applications.”

Also in 2014, Dong Ryul Lee, Ph.D., of South Korea’s CHA Stem Cell Institute and Robert Lanza, M.D., now CSO at the Astellas Institute for Regenerative Medicine, replicated the cloning process of Dr. Mitalipov’s team, but derived hESCs using dermal fibroblasts from two adult cell donors. Since then, Dr. Lanza told GEN, “the team has improved the efficiency of SCNT enormously,” although some of the research has yet to be published.

Virtually all scientists consider reproductive cloning unsafe and unethical. However, SCNT could yet play a role in regenerative medicine someday, Dr. Lanza said. Cloning could also have value in conservation, where efforts are underway to use the Dolly technique, and variations, to help save several critically endangered animals. 

In 2001, Dr. Lanza and colleagues cloned the first endangered species, a gaur, followed two years later by an endangered wild ox (a Banteng) from the frozen cells of an animal that died at the San Diego Zoo a quarter-century earlier. More recently, CRISPR and other genome-editing techniques have been applied toward reviving a number of extinct species. Those efforts will require cloning to be successful, Dr. Lanza said.

“Whether you agree or disagree, we’re likely to see de-extinction become a reality in our lifetime,” Dr. Lanza said. “We may even see the resurrection of the mammoth and passenger pigeon, and all scientifically possible because of the Dolly experiment 20 years ago.”






































 

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