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Investigators Convert Human Fibroblast into Embryonic Stem Cells

Valeo, Tom

doi: 10.1097/01.NT.0000431947.77890.bc
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Investigators successfully did a somatic cell nuclear transfer that involved inserting the nucleus from a patient's fibroblast into an enucleated oocyte from a healthy donor. The cytoplasm, which contains transcriptional and epigenetic factors, causes the transplanted nuclear DNA to revert to an embryonic state.

Creating embryonic stem cells with a patient's own DNA — the “Holy Grail” of stem cell engineering, according to one researcher — has finally been achieved, conjuring visions of effective treatments for an array of disorders including brain injury, neurodegeneration, genetic diseases, and spinal cord damage.

The cloning technique, known as somatic cell nuclear transfer (SCNT), involves inserting the nucleus from a patient's fibroblast into an enucleated oocyte from a healthy donor. The cytoplasm, which contains transcriptional and epigenetic factors, causes the transplanted nuclear DNA to revert to an embryonic state.

“The cytoplasm has this magic ability to reset epigenetic memory,” said Shoukhrat Mitalipov, PhD, lead author of the study appearing in the May 15 online edition of Cell. “The oocyte is the only cell in the body that has this powerful cytoplasmic power to control the nucleus. The nucleus controls everything else, but at an early stage the cytoplasm is in charge. How is this possible? No one knows for sure.”

The work emerges from similar research involving rhesus monkeys, conducted over several years by a team led by Dr. Mitalipov, a senior scientist at the Oregon National Primate Research Center, affiliated with the Oregon Health & Science University (OHSU) in Beaverton, OR.

In a 2007 paper in Nature, a team led by Dr. Mitalipov reported using SCNT to produce two embryonic stem cell lines from adult skin fibroblasts taken from rhesus macaques. This achievement, previously accomplished only in mice, demonstrated that therapeutic cloning in primates was possible.

However, achieving the same results in humans proved more difficult, in part because of regulations restricting the use of federal funds for embryonic stem cell research. To abide by those regulations, Dr. Mitalipov set up a separate lab that mirrored his own, which receives funds from the National Institutes of Health.

Then, procuring human eggs proved difficult since reimbursing egg donors is prohibited in some states, and regarded by some as unethical. “We found that if you don't reimburse, you don't get eggs,” said Dr. Mitalipov. “Our institution understood this, so they gave us approval to reimburse donors.” Egg donors received $3,000-$7,000.

Previous attempts to create SCNT cell lines have foundered because the nucleus of the cytoplasm of the oocyte hovers in metaphase, with the chromosomes lined up, ready to separate as soon as a sperm cell triggers cell division. Inserting a needle to remove the cell's own nucleus and deposit another tends to trigger cell division, disrupting the cytoplasm's ability to reprogram the new nucleus back into an embryonic, pluripotent state. “Human eggs appear to be very sensitive to poking with needles,” said Dr. Mitalipov. “During microsurgical manipulation to replace nuclear DNA, we would accidently trigger egg cytoplasm exit from metaphase, and if they exit, the reprogramming doesn't work.”

Dr. Mitalipov and colleagues tried a number of agents capable of holding the cytoplasm of the oocyte in metaphase during the removal of its own nuclear material and the insertion of the donor's nucleus, but most of the agents proved toxic. They finally settled on caffeine, which appears to block the degradation of kinases that maintain the metaphase cell cycle. The caffeine allows egg cytoplasm to remain in metaphase during the nuclear transfer procedure. Once the nucleus has been successfully transplanted, an electrical current is used to stimulate the oocyte to start dividing.

The resulting cell lines contain the donor's DNA, and no DNA from the oocyte except for mitochondrial DNA, which enables the cells to circumvent a problem encountered when induced pluripotent stem cells are used to treat diseases that involve mitochondrial dysfunction, such as Leigh disease and perhaps Parkinson's disease. Transplanting only the nucleus from a patient's fibroblast into a donated egg leaves the problematic mitochondrial DNA behind, enabling the oocyte's mitochondrial DNA to take over.

“The mitochondrial genome is highly resistant to genetic manipulation,” said Dr. Mitalipov. “If you insert a patient's cell nucleus into the cytoplasm of an oocyte, which contains its own mitochondrial DNA, cytoplasmic factors can reprogram the nucleus into an embryonic stem cell. Because the nucleus comes from the patient, you have a personalized stem cell line without the patient's mutated mitochondrial DNA. The egg cytoplasm provides the new mitochondrial genome.”

The SCNT technique also effectively eliminates the danger of rejection, since the stem cells contain the patient's own nuclear DNA, unlike conventional embryonic stem cells derived from unrelated fertilized embryos.

Since the technique creates a human embryo, and involves methods that produced the first cloned animal, Dolly the sheep, it is expected to revive ethical concerns about human stem cell research and human cloning. For example, Daniel Sulmasy, MD, PhD, Kilbride-Clinton professor of medicine and ethics at the University of Chicago Divinity School, told National Public Radio that the technique involves creating a human being “for the sole purpose of destroying that human being,” which he considers morally wrong, “no matter how much good could come of it.”

In addition, Dr. Sulmasy warned that this procedure could lead to the first cloning of a human being. “We already know there are people out there who are itching to be able to be the first to bring a cloned human being to birth,” he said, “and I think it's going to happen.”

Dr. Mitalipov pointed out that SCNT cells are not fertilized, and are probably incapable of developing into a healthy baby. “We tried it with monkeys many times, and we couldn't do it, so we believe it won't work for human reproduction,” he said. “No primates have ever been cloned with somatic cells.”

The Cell paper has aroused widespread enthusiasm in the stem cell community.

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“This paper describes excellent new methods,” said Lawrence S.B. Goldstein, PhD, professor in the department of cellular & molecular medicine at the University of California, San Diego (UCSD) School of Medicine, and director of the UCSD Stem Cell Program. “It makes an improvement on existing methodology, and it demonstrates that the efficiency of this process could be far better than we originally thought, which is very good news. Now the work needs to be replicated to see how reproducible it is, especially that high efficiency.”

The use of SCNT cells faces significant obstacles, however. The Cell paper probably would have caused a greater stir eight years ago before the fraudulent work published by South Korean researcher Hwang Woo-suk, according to Arnold Kriegstein, MD, PhD, John G. Bowes distinguished professor in stem cell and tissue biology, and director of Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at the University of California, San Francisco School of Medicine.

“Then it caused a great deal excitement because it represented the only way to create patient-specific stem cell lines,” he said. “Since then it has become possible to do that with induced pluripotent stem cells (iPSCs), which don't involve embryonic cells.”

[The work by Dr. Hwang Woo-suk, of Seoul National University in South Korea, claimed to have created a pluripotent human embryonic stem line using SCNT, but his results were later discredited.]

Dr. Kriegstein noted that the technique developed by Dr. Mitalipov requires donated oocytes, “which creates a much higher barrier for entry for most labs. It's much easier to take skin or blood cells,” he said.

However, now it will be possible to compare iPSCs and SCNT cells, Dr. Kriegstein added.

“It allows head-to-head comparison, and that may teach us how these cell lines differ,” he said. “If they do differ, that may provide a benchmark for improving the iPS technology — for making it perhaps a better model of what the oocyte knows how to do.”

Although stem cells have long been regarded as a potential source of new therapies, that won't happen anytime soon, according to Evan Y. Snyder, MD, PhD, director of the stem cells and regenerative biology program, and a professor with the Del E. Webb Center for Neuroscience, Aging, and Stem Cell Research, at the Sanford/Burnham Medical Research Institute.

“This is an exciting technological advance, but as to whether it's a therapeutic advance, I don't think it is,” Dr. Snyder said. “How do you use cell therapy to treat neurologic disease? With most diseases, we don't even know what we want to fix.”

Stem cells may show more promise, at least in the short term, as a way to detoxify a diseased brain environment, he said. He and his colleagues recently published a meta-analysis in Science Translational Medicine of 11 studies in which neural stem cells were transplanted into the spinal cord of a mouse model of ALS, slowing onset and progression of the disease, and increasing survival — not by replacing the malfunctioning neurons, but by creating colonies of cells that performed various housekeeping functions, such as producing trophic factors and reducing astrogliosis and inflammation.

“With a neurodegenerative disease like amyotrophic lateral sclerosis you think the cell you want to replace is a motor neuron, but the key may be to replace the astrocytes — the support cells — which don't seem to be doing their job in these patients, and may even be making molecules that are toxic to motor neurons,” Dr. Snyder said. “What we demonstrated was that by transplanting better support cells, the animal stops cranking out toxic astrocytes. That's a form of cell replacement, but not the kind everybody talks about where you literally rewire the brain.”

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Shoukhrat Mitalipov, PhD, and his colleagues briefly fell under suspicion after their paper appeared in Cell when anonymous reviewers on an Internet website,, noted that one cell microscopy image was a cropped version of another, and some of the cells were mislabeled.

After assessing the errors, Emilie Marcus, editor-in-chief of Cell, posted a response stating, “We do not believe these errors impact the scientific findings of the paper in any way.”

Dr. Mitalipov said he and his colleagues are working with Cell editors on an erratum that will appear in an upcoming issue. He believes the errors triggered so much concern because suspicions about the Hwang data began with inconsistencies in published images.

“That is fresh in everyone's memory,” Dr. Mitalipov said. “We are doing the same sort of work and we made a similar mistake in assembling the data, and that's what brought all this attention. The mistakes involving the images will be corrected.”

—Tom Valeo

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•. Tachibana M, Amato P, Mitalipov S Human embryonic stem cells derived by somatic cell nuclear transfer. Cell 2013; E-pub 2013 May 15.
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