Cloning is in the news again. Dolly, above, was cloned in 1996. Now, cloning has generated immunologically compatible embryonic stem cells for older humans: two men aged 35 and 75. (Source: Wikimedia/Toni Barros, Hello, Dolly!)Cloning has at last generated immunologically compatible embryonic stem (ES) cells for older humans: two men aged 35 and 75. It may also generate “significantly” fewer tumor-causing mutations than the popular “induced pluripotent stem cell” (iPSC) method of making ES cells.

All this is according to recent papers in Cell Stem Cell and Stem Cell Reports. Together, they indicate that cloning— as a method to dedifferentiate older cells, and create potent, immunologically matched ES cells from them— may stage a comeback for study, if never for regular clinic use.

“(Cloning) is the most physiologically relevant condition to reprogram the somatic (mature adult cell) nucleus into a pluripotent state,” emailed University of California stem cell researcher Yang Xu, senior author on the mutation paper.

The papers are timely. Work published in Nature this January claimed that ordinary mature cells can be dedifferentiated to a stem cell state via a simple “acid bath.”  But that work was found by some of the co-authors’ own institute to be rife with errors and fabrication. No one has repeated the work— including most of the co-authors.

Dedifferentiation has been under fire generally, as a result.

But cloning, or SCNT (somatic cell nuclear transfer), is an older route to the same place, and has been pulled off repeatedly (if spottily) in animals since Dolly the Sheep was cloned in 1996. It lets Mother Nature do some--even much--of the labor. The DNA from the nucleus of an old donor cell is fused with an enucleated egg cell. The egg's cytoplasm then works its magic on the older DNA in much the same way it works magic on DNA in older sperm, resulting in cellular dedifferentiation to an earlier, more potent state: an embryonic state. Until last year, the feat only worked in animals.

Lanza’s Human Cell Cloning Paper

But this week’s Cell Stem Cell study, led by ACT chief scientific officer Robert Lanza and Dong Lee of Cha Health Systems, backs up the work of the Oregon Stem Cell Center’s Shoukhrat Mitalopov. Mitalipov reported last June he created the first cloned human ES cells— if the donor cells were from fetuses and babies, so closer to the pluripotent stem cell state than older cells. Lanza produced cloned ES cells from two 35- and 75-year-old men. Mitalipov said in an email that a key to his June success was the addition of caffeine— which Lanza also added.

“Lanza and others tried, unsuccessfully, human SCNT for more than a decade, until we showed them our approach last year,” says Mitalipov. “Now they used our protocols and produced the cells. We are not sure of the exact mechanism, but as we described last year, caffeine helps to preserve oocyte's cytoplasm in metaphase. We believe that is the key for success with human SCNT.”  

In the new paper, Lanza also incubated the egg for two hours after fusion with the older DNA— instead of Mitalipov’s 30 minutes. Lanza said in an email that may have eased the dedifferentiation of the older cells. Mitalipov says his monkey work found no difference between 30 minutes and two hours. More work is needed, but some think Lanza may have broken new ground. The team derived no ES cell lines from fused eggs incubated for 30 minutes. But it derived two lines out of fused eggs incubated for two hours. In cloning, that yield is high.

Xu’s Cloning Mutations Paper

Lanza also found karyotyping results, even after 30 cell divisions, were clean. There were no notable cancer-causing chromosomal abnormalities. This complements data from Xu’s above paper comparing ES cells made via both the iPSC and SCNT methods (from genetically identical mice). Xu found ES cells made via SCNT were “significantly” — or twice as -- free of genetic mutations. SCNT also did not generate unique mutations with each round, as the iPSC method did.

Xu believes his less mutagenic SCNT ES cells owe much to the dedifferentiation skills of that egg. In cloning there is less wheel-reinventing than in iPSC-making, which often involves altering three or four genes. “SCNT does not involve the overexpression of oncogenic proteins such as Oct4, Sox2, and Klf4 used in iPSC reprogramming— which can promote genetic instability,” Xu emailed. “The iPSC technology needs to be optimized to better mimic the SCNT condition.”

Says Mitalipov: “We know for sure that SCNT allows correction of mutations in mitochondrial DNA.”

Xu says gene expression should be analyzed next. “The epigenetics of SCNT ESCs and iPSCs should be compared to determine whether the epigenetics of SCNT ESCs are more similar to ESCs than iPSCs.”

The Future

Lanza believes nuclear transfer can offer the stem cell field practical help. If his work is repeated, it could provide an additional supply of cells for ES cell banks someday, he says. Standard ES cells have been called controversial because they come from spare IVF clinic embryos— and are not immunologically identical to adult patients.

“We at ACT currently have three clinical trials using (standard IVF clinic) human ES cell-derived retinal pigmentosa epithelial cells,” he said. “We could almost certainly eliminate the need for immunosuppression in such patients in the future by using SCNT (or iPSC generation) to create HLA cell banks.” Such banks could match ES cells with patients possessing near-identical DNA sequencing in key immunological sites.

“Although there’s a shortage of human eggs,” Lanza said, “it’s important to realize the future of stem cell technology isn’t making hundreds-of-millions of patient-specific stem cell lines, but banks of matching HLA lines. In the U.S., just 100 human ES cell lines would generate a complete HLA haplotype match for over half of the population. It doesn’t matter how the initial lines are made, since both human iPSCs and human ES cells are immortal and grow forever.”

Some Skepticism

Besides noting that no one has established perfect methods for keeping any ES cells mutation-free in very long-term culture, some are skeptical the two-hour incubation is key. They note 30 minutes was sufficient for monkeys, and work by mouse cloner Teru Wakayama of Yamanashi University found 30 minutes sufficient in mice. Emailed one cloning expert about the Lanza paper: “Does this advance anything? Do we have a better understanding of mechanisms? The field doesn’t seem to be doing bread-and-butter characterizations. What recent (human cell) papers boil down to is, `Who has access to good quality eggs?’ Trouble is, there is no indication as to what 'good quality' means apart from whether the eggs support development.”

Both human cell cloning studies have, “a 'champion donor' from whose eggs a disproportionately high number of nuclear transfer blastocysts are derived. But no meaningful curiosity has been expressed as to what in molecular terms makes these eggs special,” said the researcher.

Lanza says “evidence says otherwise” about the need for two-hour incubation. “Please see the table in the paper. We did a side-by-side comparison with the same egg donors and the same nuclear donors. Every species cloned has a unique set of reproductive and physiological differences.”

He believes the work advances knowledge. It offers proof-of-principle for Mitalipov. And the clean karyotyping he saw in 30 cell divisions shows cloning may not involve massive chromosome damage.

“Everyone thought the problem with human cloning was aneuploidy. We clearly show this is not the case as many scientists had thought in the past.”

He does note human eggs need more study.

Regarding the Xu mutation paper, MIT nuclear transfer expert Rudolf Jaenisch tells Bioscience the significance of Xu's iPSC mutations must be established. If they are not functional--not oncogenic, not dominant--do they matter?

Xu says one study tried to address “the functional outcome of somatic mutations in reprogramming by studying the impact of individual mutations, and found that very few have an impact.” But, he says, the issue is complex. “It is possible a panel of these mutations must work together to have a significant impact on reprogramming. Or the mutation has to work with a specific epigenetic state in order to have a significant impact on reprogramming. Studies have shown an enrichment of mutations in cancer-related genes. In any case, the accumulation of somatic mutations indicates the overall state of genomic instability, which could also include chromosomal translocations and aneuploidy—a bigger problem for inducing cancer.”

Jaenisch also notes that, by definition, cells in nuclear transfer are clones, whereas iPSC starting cells are heterogenous. But Xu responds that “the individual iPSC clones we analyzed were derived from a single reprogrammed cell, the same as the SCNT ESCs.”

For years, cloning produced mutant animals. It is still wildly inefficient. But recent work offers tantalizing hints science can start taking key stem-cell-making tips from the unfertilized human egg.

Even Jaenisch agrees that while it is unlikely nuclear transfer will be used in the clinic, as it is “too complicated and too much trouble to get human eggs, the technology is interesting. Why didn’t it work on human? That was a scientifically interesting question which this last paper seems to have confirmed. There is more." SCNT definitely rejuvenates cells, elongating their telomeres, for example, he says. (Indeed, only weeks ago, Cell Stem Cell published a paper concluding SCNT's superior ability to rejuvenate telomeres should be capitalized to help iPSC creation. Last year, a similar paper was published.) Cloning may be able to answer "some very important scientific questions," Jaenisch says. "Much remains unsettled.”