Cloning gains in race to make human stem cells

Two new studies revive interest in cloning as a means to replace diseased organs. Daniel Cossins and Elizabeth Finkel report.

These embryonic stem cells were generated from the cloned embryo of a 32 year-old diabetic woman. As a “proof of concept”, the cells were used to make insulin-producing pancreatic cells, a perfectly matched tissue graft that might cure her diabetes. – Bjarki Johannesson, NYSCF

In 1962 John Gurdon cloned a frog and fired the imagination of a generation. The medical goal was not to produce a cloned human being, but cloned body parts for people with diabetes or failing hearts, liver disease or ailing kidneys – perfectly matched tissue for a second lease on life.

In recent times that quest has become something of a horse race. Gurdon’s technique required creating and destroying an embryo. That approach seemed to be left in the dust by a technique that required no embryo. Now, two reports have finally shown that the embryo-based technique can be made to work to generate perfectly matched replacement cells for patients.

As reported in Cell Stem Cell, an international team comprising labs from South Korea and the US used skin cells of two healthy men, aged 35 and 75, to clone matching embryos and derive human embryonic stem cells capable of generating any tissue. Ten days later, a team of US and Israeli scientists achieved the same feat using skin cells from a 35-year-old diabetic woman. Furthermore her embryonic stem cells were coaxed into producing the functional pancreatic cells that she lacked. Their work was published in Nature.

“This progress gives us the basis to start moving toward patient-specific stem cell therapies,” says Dieter Egli of the New York Stem Cell Foundation Research Institute, who led the US and Israeli team. “It also gives us a chance to compare cells produced in this way with cells made using different methods.”

Cloning human embryos also carries ethical baggage. Harvesting
a woman’s eggs is a risky medical procedure.

John Gurdon’s method with the frog worked by taking advantage of the remarkable properties of an egg cell. When you insert the DNA of a mature cell, the egg performs something like a reboot. The DNA rewinds its clock and restarts frog development from scratch. So when Gurdon took a frog egg, removed the DNA-filled nucleus and replaced it with the nucleus from a tadpole’s intestinal cell, that egg developed into a frog that was a clone of the DNA donor.

Some said this would be impossible with mammals. Dolly the sheep disproved that in 1996 and mice, cats, dogs and cows followed. But researchers could not get human cloning to work. They could get an embryo started using "somatic cell nuclear transfer" – the same technique that worked for Dolly the sheep. But the embryo would stop developing before it was possible to cultivate embryonic stem cells – the crucial point from which tissues of any type might be generated. For some, the frustration was too much: after trying with some 2,000 human eggs, South Korean researcher Woo Suk Hwang claimed success in 2005, only to have his work proven fraudulent.

Cloning human embryos also carries ethical baggage. Harvesting a woman’s eggs is a risky medical procedure. The woman either sells or donates those eggs, and ethicists are concerned that the economically disadvantaged could be exploited and their health compromised. Then there are ethical concerns about destroying the embryo when embryonic stem cells are “harvested” around five days into development and alarm at the prospect of cloning babies if the technique were perfected.

A cloned embryo formed by replacing the egg’s DNA with that of a skin cell. The green fluorescence shows DNA that originated in the skin cell is active in the multiplying cells. – Dieter Egli, NYSCF

Many scientists speculated there might be another way to reprogram mature cells back to an embryonic state using whatever “magic” factors the egg carried to reboot DNA. In 2006, Shinya Yamanaka fired the imagination of the world by doing just that. He showed that just four genes could reprogram a mature mouse cell, making it behave like an embryonic stem cell. Cells made this way are termed induced pluripotent stem cells, or iPS cells. Yamanaka and Gurdon shared the Nobel Prize in 2012 for their bookend discoveries on reprogramming cells made 44 years apart. Yamanaka’s method however, unlike Gurdon’s, was extremely easy to apply to human cells. So much so that laboratories all around the world have made human iPS cells and trained them to produce various types of tissue. Japan is planning the first clinical trial later this year using retinal-pigmented epithelial cells for patients with macular degeneration.

While most scientists gave up on the nuclear transfer approach, some persevered. In May last year, Shoukhrat Mitalipov and colleagues at Oregon Health and Science University announced success with reprogramming human cells. It wasn’t entirely a surprise. They had been working at the method using rhesus monkeys, and in 2007 reported a triumph in cloning embryos and harvesting embryonic stem cells. Mitalipov used cells that were still very young – in one case cells from a foetus, and in another from an eight-month-old baby. It was a similar approach to Gurdon, who had reprogrammed the cells of a tadpole before stepping up to the skin cells of an adult frog.

Mitalipov’s success with human cells came down to a few key experimental tricks. He’d found, as had others, that the cloned embryos tended to stop growing after a few cell divisions. To keep them going, he added so-called HDAC inhibitors, chemicals known to help activate the mature donor DNA. Yet although the embryos developed, they never yielded embryonic stem cells. The critical factor turned out to be caffeine added to the culture medium, which seemed to help buy time for the new DNA to finish reprogramming. The method has the great advantage of not requiring a vast number of human eggs. Out of eight embryos, four yielded embryonic stem cells, and at least one embryonic stem cell line was produced for every harvest of eggs.

With cloning now well and truly back in the race, the question
now becomes: which cells do you back?

The two new groups have not only corroborated Mitalipov’s method. Using slight tweaks, they have shown that it will also reprogram the DNA of cells from fully mature individuals as old as 75. Both Egli, and Young Chung, from CHA Health Systems in Los Angeles and lead author of the Cell Stem Cell paper, say that much of the success depended on the eggs themselves. Some donors provided better eggs than others, a finding hinted at by Mitalipov.

“Three separate labs have now independently confirmed you can do this,” says Alan Trounson, president of the California Institute for Regenerative Medicine in San Francisco. “That’s important because it doesn’t always work out that way,” he says, referring to the recent inability to confirm a report that stem cells could be made by simply dipping mature cells in acid.

With cloning now well and truly back in the race, the question now becomes: which cells do you back?

There’s no doubt cloned cells are still technically much harder and more costly to make, carry hefty ethical baggage, and require women to donate or sell their eggs.

“The problem of getting access to large numbers of human eggs is a bottleneck,” warns Trounson. “It’s hard to imagine you could ever apply this to the large scale needs of patients for matched tissue,” adds Martin Pera, Program leader of Stem Cells Australia. (The cloning method is legal under licence in Australia and the UK. In the US, laws vary from state to state, and in the rest of the world, country to country.)

On the other hand, iPS cells may be easy to make and carry little ethical baggage, but they are susceptible to DNA programming glitches. The reboot triggered by the four genes doesn’t always clear the slate, so iPS cells made from a skin cell may still retain a predilection for forming skin. As a result, iPS cells have garnered a reputation for being highly variable when it comes to their ability to form specific tissues.

By contrast cloned stem cells are reprogrammed in a more normal way because they derive from the embryo. “It’s true, these cells are a bit cleaner,” says Jose Polo, a cell-reprogramming expert at Monash University in Melbourne. Nevertheless, he points out that, even if iPS cells have a bias to their cell of origin, it might be used to advantage. If you need more pancreas cells, for example, then reprogram a cell from the pancreas and smooth the way.

Of more concern is that iPS cells might be predisposed to forming cancers. Early methods of making iPS cells were particularly concerning because the four genes used to reprogram the cells became permanent fixtures of the iPS cell DNA, and one of them (cMyc) was a known cancer-causing gene. Modern techniques jettison the four genes after the iPS cells have formed; yet it remains possible that they are still more inclined to form cancers.

“I think if people were given the choice today of which they would prefer to have transplanted back into humans, they would choose the cells made by nuclear transfer,” says Egli. But that might change, says Pera. “It all depends on whether the wrinkles in the iPS method can be ironed out or become fatal flaws,” he counters.

But as far as placing bets on nuclear transfer cells versus iPS cells, it’s still early days. There are more than 2,000 iPS cell lines, and just a handful of those made via nuclear transfer. Putting them through their paces lies in the future.

“The field is open,” says Polo.

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