That recently enacted trillion-dollar, decade-long tax break breaks even with a different trillion-dollar levy by the year 2010. It hits 2 million Americans with end-stage kidney disease. Moreover, "as many as 3,000 Americans die every day from diseases that in the future may be treatable with tissues derived from embryonic stem cells."

Those exacting projections are cited in Nature Biotechnology for July 2002, released online June 3. They appear in a cover story titled: "Generation of histocompatible tissues using nuclear transplantation." Its lead author is tissue engineer Robert Lanza, vice president of medical and scientific development at Advanced Cell Technology Inc., of Worcester, Mass. The paper's senior author is clinical immunologist Anthony Atala, director of tissue engineering at Children's Hospital in Boston.

"The study is proof of principle," Atala observed, "that therapeutic cloning can create tissues without the threat of rejection." He pointed out, "Approximately 100,000 individuals in the U.S. are awaiting an organ for transplantation and approximately 250,000 are on kidney dialysis."

ACT's Lanza told BioWorld Today: "The goal of nuclear transplantation is to clone genetically matched cells and donor organs for patients suffering from a wide range of disorders that result from tissue loss or dysfunction. Besides heart, lung, liver and/or kidney ailments, millions more suffer from diabetes, arthritis, AIDS, strokes, cancer and other diseases that may one day be treatable using this technology.

"Its use in medicine to generate immune-compatible cells by cloning," Lanza continued, "would overcome two major scientific challenges in transplantation - immune rejection and organ shortage. Our results in this paper suggest that it may be possible to overcome both of those hurdles. So, in addition to demonstrating that we can generate immunologically compatible tissues," he added, "we showed that we could also bioengineer these cloned cells into complex structures such as functioning mini-kidneys."

As well as creating skeletal muscle and heart "patches," the co-authors employed nuclear transplantation to generate immune-compatible, 2-inch kidney units. These miniature organs not only survived and functioned as kidneys, but also evinced no rejection response to the cloned tissues.

The team's point of departure was an 1,800-pound domestic cow as the donor.

For Lack Of Stem Cells, A Bovine Embryo

"We wanted to use a large animal with a sophisticated immune system similar to a human's," Lanza recounted. "Unfortunately, all the animals we could clone - livestock species such as cows, pigs, goats - have no embryonic stem cells [ESCs]. Stem cells have not been isolated from cows or any of the livestock species that could be differentiated into replacement cells of any sort. So for this study we created a bovine embryo as a source of donor tissue. Now in humans this step is unnecessary, because ESCs have already been isolated. So what we had to do was the same procedure one would follow for nuclear transfer."

The researchers obtained donor eggs (oocytes) from cow ovaries and removed and discarded their nuclei - which contain the cells' genetic material - leaving behind just the shell. A skin cell punched from the cow's ear was placed inside the egg shell, and burst with an electrical jolt to expand the single cell into several. The resulting blastocysts (6-day-old embryonic cell masses) were transferred into the uteruses of surrogate-mother cows for a four- to six-week incubation period.

"That would not affect the proof of principle," Lanza pointed out, "because right from the early onset - be it a blastocyst, an embryo, a fetus or an entire animal - the nuclear genome was the same, and the cellular mitochondrion was the same. Our main concern was whether we were going to run into rejection problems because of the mitochondrial genome.

"We knew that cloned animals inherit the DNA in their mitochondria from the egg, and not from the cell that's being cloned," he noted. "So the presence of that DNA raised questions whether the foreign proteins in the cloned cells would lead to rejection after transplantation and defeat the main objective of the procedure.

"The acid test," Lanza said, "is always the clinical outcome: Does it actually work? And the answer in our study was yes. The cloned cells all survived, despite the presence of the foreign DNA. We did sequence the entire mitochondrial genome of both the clones and the nuclear donor animals, and confirmed that the DNA was indeed allogeneic - foreign."

Clones Defy Organ-Graft Rejection

"For organ transplant, it's the most sensitive way of actually picking up those minor compatibility antigens, which would be the problem in this particular case. For our results they were pretty spectacular. The kidney not only survived, but also produced yellow urine just as any human would. We also did a skin test for delayed hypersensitivity. That's considered the gold standard for picking up an immunological rejection response to transplanted tissue. We were unable to discern an immune response directed against cloned tissue. Our concern was whether or not the mitochondrial peptides would be recognized by the immune system as foreign. And the answer in these studies was no.

"The reason it worked for us," Lanza summed up, "is that the cloned tissue is the identical copy of the cow's nuclear genome. At that level, which is 99 percent of the DNA in the cell, it is absolutely identical to the clone from which it's derived. So the objective, in terms of the genome, was the animal's own cells."

Lanza commented: "Considering all the cloning debate that's going on in the House, the Senate and overseas, you would have thought that at this point, someone would at least have tested cloned tissue out in the mouse or some other animal. Ours is the first experimental data to address that question - whether the immune system would accept or reject the cloned tissue. This proof-of-principle study is not an example of therapeutic cloning, and could not be undertaken in humans," he added. "We think it's ethically unacceptable to transfer a cloned embryo into a woman's uterus to generate a fetus for any purpose.

"But we have a long way to go," he concluded, "before we can actually apply this technology in the clinic. We need to have an implantable kidney that can carry on sufficient function to replace a patient's own organ."