By David N. Leff

Half a dozen frisky Holstein heifers are living disproof of the theory propounded when Dolly the sheep became the first mammal cloned from an adult cell. Dolly's Scottish creators, and other cloners, cautioned that cloned cells may have prematurely shortened life spans, and that their cloned animals would face too-early old age.

Today's Science, dated April 28, 2000, carries a photograph of the Holstein female sextet, named Crocus, Daffodil, Forsythia, Lily, Persephone and Rose. The article reporting their birth is titled: "Extension of cell life span and telomere length in animals cloned from senescent somatic cells." Its senior author is cell biologist Michael West, president and CEO of privately held Advanced Cell Technology (ACT) in Worcester, Mass., and a telomere research pioneer.

The paper's lead author, Robert Lanza, ACT's vice president of medical and scientific development, told BioWorld Today: "The suggestion that cloned animals might reflect the age of their donor nucleus would mean - unfortunately for medical therapeutics - that you would have an animal prematurely old. So its cells and tissues, even for therapeutic cloning applications, would be of minimal value. Because," he added, "you wouldn't want to be transplanting tissues and organs into a patient with a disease process, only to have him fail in a few months.

"So we thought," Lanza continued, "seeing as that was the prevailing view at that time in the scientific community, it was extremely important for ACT to undertake a rigorous, stringent test. If in fact the age of the donor cell is reflected in the animal, if we were to take cells totally to senescence - to the end of their life span - where they had only one or two population doublings left, in theory it would be an impossibility to get a fetus, much less a term animal."

"Now in Science," Lanza observed, "not only were we able to show that cloning could take the old cells backward in time and restore them to a youthful state, but in fact we were able to get healthy cows that appeared to have cells even younger than their chronological age."

ACT's Michael West described the co-authors' cloning and nuclear transfer rationale: "It's really a repeating theme here that there is an immortal substratum to life - the immortal germ line. If we took an old somatic cell and dunked it into an oocyte - a germ-line cell - we wondered whether it would really work. That came to a head when Ian Wilmut, who cloned Dolly in 1997, published since that it didn't work - that Dolly's telomeres [the DNA chromosomal caps that measure cell senescence] were not regenerated."

Sheep In Lamb's Clothing?

"Gene targeting is highly inefficient," West mused, "but if you could clone 1 in 100,000 cells, and expand it out, you could make an animal. And if you can grow cells in a dish, the way we grow and transform bacteria, then isolate colonies and grow them up with the ability to rejuvenate the cells, then you could do multiple rounds of gene targeting, and edit the DNA the way you edit your word-processing document. That has always been our dream."

But dreaming aside, West went on, "For whatever purpose we'd ever want to clone an animal - to make a pharmaceutical or a xenograft or for agriculture - we'd like to have those animals healthy. So if we were making the world's best dairy cow, but geriatric from day one - or a sheep in lamb's clothing as with Dolly - that would not be good."

West pointed out that whereas Dolly's Scottish creators had patented their approach using relatively young, quiescent or resting cells, ACT's patent claims actively proliferating cells.

"Essentially," he recounted, "we ran two sets of nuclear transfer experiments, one using fetal cells, the other, adult-derived ones. In both cases, we used 80 percent or more senescent or near-senescent cells. In the first case, we extracted 40-day-old bovine fetuses, from which enzymes digested the fibroblasts. The adult tissues used skin fibroblasts.

"Both fetal and adult were somatic bovine cells," West pointed out. "Then we took bovine oocytes from slaughterhouse ovaries and follicles. After maturing these eggs in the lab, we held the zona pellucida with a little suction micropipette, then went in with a much smaller micropipette and removed the nuclear DNA.

"Then we took the fibroblast used as somatic cell donor," West went on, "and placed that cell just inside the zona, into the space separating it from the oocyte. What's nice about this," he observed, "is that we did electrofusion to fuse the somatic cell and the egg, and help activate the oocyte. The rest of course is magic; it's a black box. We don't know the fundamental mechanisms of that embryonic reprogramming."

After delivery by Caesarean section, the six newborn calves had cells, Lanza said, "with a longer life span than those from normal animals. The cloned heifers, one of which is celebrating her first birthday this week, have telomeres that look like those of newborn calves, despite having been cloned from senescing cells. Our results," he pointed out, "show that cloning actually has the potential to reverse the aging of cells. This has profound implications for treating age-related disease."

Futuristic Therapeutic Cloning Case History

West proposed one scenario for such therapeutic cloning: "Let's take an elderly patient who has CML - chronic myelocytic leukemia - and needs chemotherapy. For a bone marrow transplant there are no relatives that can offer a compatible graft, so we're looking at the possibility of an autologous [self] graft.

"But the patient's blood-forming stem cells," West continued, "are too senescent to repopulate the bone marrow. So what we could do is therapeutic cloning. We could take a cue tip, swab a budding cell from inside of the cheek, do a nuclear transfer, and take the cells back to their totipotent embryonic state. These differentiate into blood-forming cells, which we put back into the patient, hopefully giving him or her cells that have robust proliferative capacity, similar to what's seen from umbilical cords. But they would be the patient's own cells.

"We're doing some animal studies trying to make this little time machine work," West concluded, "but they're not technically preclinical at this point."

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