"How can either 'nuclear transfer' or 'twinning' produce Hwang's 'patient-specific' stem cells?"

Dianne N. Irving
Copyright December 17, 2005
Reproduced with Permission

The driving force behind the frantic hope for curing so many debilitating diseases has been the claim by most stem cell researchers that it is possible to prepare "patient-specific" stem cells derived from their cloned embryos (their "genetic twins") that will not cause serious immune rejection reactions in the patient-donors. But for "patient-specific" stem cells to be a "genetic match" with the patient, both the nuclear and the mitochondrial DNA of those stem cells must match the nuclear and mitochondrial DNA of the patient. If either the nuclear or the mitochondrial DNA  or both  don't match the genetic makeup of the patient, then they would cause serious immune rejection reactions in the patient.

Nicholas Wade notes two different kinds of cloning techniques in his latest excellent article on the Hwang cloning controversy (Nicholas Wade, "Cell Researcher's Retraction Leaves Vexing Questions", New York Times, Dec. 17, 2005, at: http://www.nytimes.com/2005/12/17/science/17research.html?pagewanted=1; copied below). He observes that the possibility of Hwang's having used either or both of these cloning techniques must obviously be resolved. But given the scientific facts, how can either type of cloning be used to produce "patient-specific" stem cells? Aside from the issue of scientific fraud, wouldn't stem cells produced either way cause serious immune rejection reactions in the patient?

Two different kinds of cloning (asexual reproduction) are noted in the article: (1) "nuclear transfer" (if using somatic cells, SCNT); and, (2) "twinning" (blastomere division, blastomere separation, or blastocyst splitting). Neither would produce an embryo with "patient-specific" stem cells.

Why? In SCNT, the nucleus of a patient-donor is removed from one of the patient's adult body cells (somatic cells) and placed into an oocyte whose own nucleus has been removed. However, the oocyte's mitochondrial DNA remains in the cytoplasm of the oocyte, and becomes incorporated into the new embryo. Thus the nuclear DNA of the cloned embryo and that of the patient-donor are the same, but the mitochondrial DNA are different. Thus the embryo's stem cells would not be a genetic match for the patient-donor, and would cause immune rejection reactions if injected.

In monozygotic "twinning", an embryo that is sexually reproduced (fertilization) begins to grow and multiply its cells (blastomeres; all of which are totipotent). If one or more of these totipotent blastomeres are separated from the whole embryo, then the natural biological process of regulation could set in and if successful could revert the isolated totipotent cell or cells to new embryos (organisms). The first twin would be produced sexually; the second twin would be produced asexually. This is what happens in naturally occurring human monozygotic twinning, as well as artificially in in vitro cloning (as is done in IVF clinics for "infertility treatments).

In "twinning", the twins are genetically identical  they have the same nuclear and mitochondrial DNA. But neither have the same nuclear or mitochondrial DNA as the patient, because half of the original sexually reproduced embryo's nuclear DNA is derived from the male sperm of the father. Thus the second twin will also carry the father's DNA. So one twin is genetically identical to the other twin, but neither are genetically identical to the patient -- thus also causing immune rejection reactions if these stem cells are injected.

What is so baffling is that if Hwang's "patient-specific" stem cells are really derived from SCNT-cloned embryos, then the mitochondrial DNA of those stem cells should be different from the genetic makeup of the donor-patient. This genetic difference should be detected in the DNA fingerprints Hwang produced. But if this is so, then these cells are not "patient-specific", and if injected into the patient-donor would cause immune rejection reactions.

If Hwang was really using "twinning" instead of "nuclear transfer", then the DNA fingerprints of the stem cells and the patient would be even more genetically different (because of the incorporation of male chromosomes during fertilization). In this case, the DNA fingerprints of the twins would be genetically identical, but these DNA fingerprints would obviously not be "patient-specific"  and if injected would cause immune rejection reactions in the patient.

The only way that the DNA fingerprints of the "stem cells" and the "patients" could be identical is if they are of twins.


New York Times
December 17, 2005

Cell Researcher's Retraction Leaves Vexing Questions

Now that Dr. Hwang Woo Suk has asked Science magazine to withdraw the June 17 article in which he reported what seemed a striking advance toward the goal of treating patients with their own regenerated tissues, scientists have two formidable tasks ahead of them.

The first is to decide how much of the work of Dr. Hwang, of Seoul National University in South Korea, can be relied on. The second is whether scientific journals' procedures for detecting spurious claims can and should be improved. The editor of Science, Dr. Donald Kennedy of Stanford University, said yesterday that he would allow the paper to be retracted if all the other authors agreed with the request. Dr. Gerald P. Schatten of the University of Pittsburgh, the senior author, asked to withdraw his name from the article on Monday.

Dr. Hwang had conceded that there were problems with some of the data in the article, Dr. Kennedy said, but that "there is certainly no basis right now for a charge of scientific misconduct."

Dr. Hwang claimed yesterday that he had succeeded in making embryonic cell colonies from some patients, but that the cells were killed by a fungus. His withdrawal of the paper and his acknowledgment of problems with some of its data have inevitably raised doubts about his previous work.

"It is clear now that every experiment published by Hwang will have to be confirmed in some way before being believed," said Rudolf Jaenisch, a biologist at the Whitehead Institute in Cambridge.

Scientists disagree as to whether Science's reviewers should have detected the problems with Dr. Hwang's article. "Should reviewers have caught some of this? Yeah, probably they should have," said John Gearhart, a stem cell expert at Johns Hopkins University and a member of Science's board of reviewers. "Obviously great claims require great proof, and maybe more people should review such a paper," he said. Dr. Arthur Levine, dean of the University of Pittsburgh medical school, where a committee is investigating the work in the Science article for possible misconduct, said he agreed that Science's referees "might have been more critical, but that is hindsight." He added, "Almost six months have elapsed since the paper was published, and it had been widely read by many fine scientists without challenge."

Unlike readers, a journal's reviewers can demand more data on points that do not persuade them. But reviewers are unpaid, and their main task is to judge whether the data presented to them support the claim being made. Dr. Kennedy said that the reviewers could not be expected to detect deliberate falsehoods and that he could not see any generic fault in the peer review system.

Dr. Levine said he saw "terribly important lessons" in the Hwang incident, chiefly that the senior author of an article is responsible for its integrity and must therefore be "intimately familiar" with the data. However, the true test of science is replication of a claim by others, Dr. Levine said.

Other scientists have expressed the fear that a replication - the success by a second laboratory in cloning human cells - might have seemed to vindicate Dr. Hwang's work, if the present criticisms had not come to light. Any Nobel Prize might then have gone to him, not to the scientists who had apparently come second. "If the procedure works indeed and other labs would have repeated it, the credit would have gone to Hwang," Dr. Jaenisch said.

In the wake of the problems with the June Science article, scientists are now looking more skeptically at some of Dr. Hwang's other work. In an article published in Science in March 2004, he claimed to have performed the first nuclear transfer with human cells, the cloning procedure in which a nucleus from a person's adult cell is inserted into a human egg, from which embryonic stem cells are obtained.

An unusual feature of the 2004 paper is that Dr. Hwang inserted a woman's nucleus into her own cell. Both Dr. Jaenisch and Dr. Gearhart said that Dr. Hwang had offered a reasonable explanation for this self-cloning procedure and should be given the benefit of the doubt.

But Dr. Robert Lanza, a rival cloner at Advanced Cell Technology in Worcester, Mass., said the design would protect Dr. Hwang against critics who asked for the donor and cell to be subjected to a DNA fingerprinting test. DNA fingerprint tests were indeed demanded by Science's reviewers for the 11 colonies allegedly derived from patients in the June 2005 paper, and Dr. Hwang provided them.

But critics have said that in some cases the fingerprints Dr. Hwang provided have the identical background noise, as if a single test were being presented twice, instead of two tests having been made independently.

Dr. Lanza also pointed to a similar article by Dr. Hwang in the August 2005 issue of the journal Molecular Reproduction and Development. The identical photograph, a test of pig embryonic cell colonies, seemed to have been presented twice but as representing different cells. The first photo shows embryonic cells said to have been cloned from an adult pig, and the second embryonic cells labeled as from a fertilized pig egg.

Dr. Lanza also expressed doubt about the cloned dog, Snuppy, which Dr. Hwang announced in Nature, Science's rival publication, in August 2005. Nature's editors should have required a test of the mitochondrial DNA of Snuppy and the dog it was cloned from, Dr. Lanza said. If a true clone, the mitochondrial DNA of Snuppy and the egg's donor would be different.

But Dr. Hwang, in a very brief report with little data, reported only that the nuclear DNA of Snuppy and the adult cell donor were identical. This would not rule out the possibility that the two dogs were in fact identical twins obtained by splitting an embryo and delaying the gestation of the second twin. "I think it's essential that he immediately allow independent testing of the original and cloned animals," Dr. Lanza said.

Dr. Gearhart agreed, saying, "That would be the very first thing that anyone would have asked."

Seoul National University is conducting an inquiry into Dr. Hwang's work, and its committee, with access to Dr. Hwang's colleagues and data, may be in the best position to decide which, if any, parts of his work are valid.

For the committee members' findings to have credibility, Dr. Gearhart said, it was "critical they bring in people from outside and who know the field."

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