After years of failed attempts, researchers have finally generated stem cells from adults using the same cloning technique that produced Dolly the sheep in 1996.

A previous claim that Korean investigators had succeeded in the feat turned out to be fraudulent. Then last year, a group at Oregon Health & Science University generated stem cells using the Dolly technique, but with cells from fetuses and infants.

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In this case, cells from a 35-year-old man and a 75-year-old man were used to generate two separate lines of stem cells. The process, known as nuclear transfer, involves taking the DNA from a donor and inserting it into an egg that has been stripped of its DNA. The resulting hybrid is stimulated to fuse and start dividing; after a few days the “embryo” creates a lining of stem cells that are destined to develop into all of the cells and tissues in the human body. Researchers extract these cells and grow them in the lab, where they are treated with the appropriate growth factors and other agents to develop into specific types of cells, like neurons, muscle, or insulin-producing cells.

Reporting in the journal Cell Stem Cell, Dr. Robert Lanza, chief scientific officer at biotechnology company Advanced Cell Technology, and his colleagues found that tweaking the Oregon team’s process was the key to success with reprogramming the older cells. Like the earlier team, Lanza’s group used caffeine to prevent the fused egg from dividing prematurely. Rather than leaving the egg with its newly introduced DNA for 30 minutes before activating the dividing stage, they let the eggs rest for about two hours. This gave the DNA enough time to acclimate to its new environment and interact with the egg’s development factors, which erased each of the donor cell’s existing history and reprogrammed it to act like a brand new cell in an embryo.

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The team, which included an international group of stem cell scientists, used 77 eggs from four different donors. They tested their new method by waiting for 30 minutes before activating 38 of the resulting embryos, and waiting two hours before triggering 39 of them. None of the 38 developed into the next stage, while two of the embryos getting extended time did. “There is a massive molecular change occurring. You are taking a fully differentiated cell, and you need to have the egg do its magic,” says Lanza. “You need to extend the reprogramming time before you can force the cell to divide.”

While a 5% efficiency may not seem laudable, Lanza says that it’s not so bad given that the stem cells appear to have had their genetic history completely erased and returned to that of a blank slate. “This procedure works well, and works with adult cells,” says Lanza.

The results also teach stem cell scientists some important lessons. First, that the nuclear transfer method that the Oregon team used is valid, and that with some changes it can be replicated using older adult cells. “It looks like the protocols we described are real, they are universal, they work in different hands, in different labs and with different cells,” says Shoukhrat Mitalopov, director of the center for embryonic cell and gene therapy at Oregon Health & Science University, and lead investigator of that study.

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Second, the findings confirm that the key factor in making nuclear transfer work with human cells is not the age of the donor cell, as some experts have argued, but the quality of the donor egg. “No matter how much you tweak the protocols or optimize them, it looks like the major player in efficiency is the individual egg quality,” says Mitalipov. He notes that all of his stem cell lines came from the same egg donor. The two cell lines described by Lanza’s group also came from one egg donor.

This latest success should reignite the debate over which reprogramming method generates the most reliable, and potentially useful, stem cells for eventually treating patients. The nuclear transfer method may join two other ways of making stem cells: one, developed by James Thomson in 1998, relied on extracting them from days-old embryos left over from IVF, and another, developed by Japanese scientist Shinya Yamanaka in 2006 (and for which he was awarded the Nobel Prize), bypassed the egg and embryo completely, allowing researchers to make stem cells by modifying an adult’s cells using a mixture of just four genes.

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Each method has it advantages and risks, however. IVF embryos are difficult to come by, since they require permission from couples to be used for stem cells research, and they may not be genetically matched to patients who might benefit from cells they generate.

While so-called induced pluripotent stem cells, or iPS cells, avoid the need for embryos and could be matched to patients, some studies suggest that the process may not completely reprogram cells, leaving populations of some partially reprogrammed ones in the mix. In addition, iPS cells aren’t useful for treating mitochondrial diseases, which result from mutations in the cell’s energy factories, which have their own DNA outside of the cell’s DNA in the nucleus. If a cell with a mitochondrial mutation is reprogrammed using the iPS technique, any mutations would be reprogrammed as well.

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Nuclear transfer, however, could treat these disorders since it involves an egg that provides its own, healthy mitochondria. But the process requires a good supply of eggs, which have to be donated by healthy volunteers. That raises ethical concerns since the technique could produce human clones. That’s why research on nuclear transfer is not funded by the federal government, and scientists know less about these cells and their potential than they do about iPS cells. “They have become kind of like cursed cells,” says Mitalipov of the stem cells generated through nuclear transfer. “But we clearly need to understand more about them.”

For patients who might one day benefit from stem cell-based therapies, that understanding could mean the difference between life and death, which is why the latest findings are potentially significant. “We have another way to skin the cat,” Lanza says. “The hope is that iPS cells work out, but for the future application of stem cell therapies to treating disease, it’s good knowing there is another way to make stem cells should we need to.”

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