New Life for Human (Therapeutic) Cloning? Whence Come the Eggs?

by Kenneth W. Krause.

Kenneth W. Krause is a contributing editor and “Science Watch” columnist for the Skeptical Inquirer.  Formerly a contributing editor and books columnist for the Humanist, Kenneth contributes regularly to Skeptic as well.  He may be contacted at krausekc@msn.com.

Physicians have long fantasized over the promises of regenerative medicine—the ability to replace a suffering patient’s diseased or damaged body cells instead of her organs, for example.  And to that auspicious end, medical researchers continue to search for ways to procure stem cells with at least two critical attributes.  First, they should be pluripotent, or capable of forming all three germ layers and any of the body’s tissue types.  Second, in order to avoid immune system rejection, they should be patient-specific, or derived from the ailing person’s own genome.

In 2006, Shinya Yamanaka revolutionized the field with the development of induced pluripotent stem cells (iPSCs).  He was the first to successfully reprogram adult fibroblasts to a pluripotent, embryonic-like state through the forced expression of four transcription factors (SOX2, KLF4, MYC, and OCT4).  Shortly thereafter, staggering progress was made in differentiating human iPSCs into neurons and heart, liver, pancreas, and eye tissues.

In 2010, researchers from Ohio reported success in coaxing human iPSCs (and separately cultured human embryonic stem cells (hESCs)) to form a three-dimensional organ resembling an intestine.  They were also able to recapitulate smooth muscle tissue, nutrient absorbing cells, and mucous, hormone, and enzyme secreting cells.  Others have used iPSCs to cure diabetes in mice.  Many experts now claim that iPSCs are poised to have a major impact on disease modeling, drug screening, and cell-based therapies.

But iPSCs pose serious problems too.  First, the insertion of external genes into reprogrammed cells can cause any number of expression anomalies.  Second, one of Yamanaka’s transcription factors is known to cause tumors.  Third, the reprogramming process can be very inefficient.  Indeed, one recent study found that iPSCs were thousands of times less likely to proliferate and suffered greatly increased rates of early senescence (aging) and apoptosis (cell death) when compared to their embryonic counterparts.

The evidence also indicates that iPSCs tend to retain traits from their tissue of origin—residual DNA methylation signatures that could seriously compromise these cells’ suitability for medical use.  In one study describing the phenomenon of “epigenetic memory,” researchers compared iPSCs reprogrammed through the Yamanaka method to ESCs generated via somatic cell nuclear transfer.  They learned that iPSCs were less likely to achieve “ground state pluripotency,” and, disappointingly, that they tended to differentiate into their original cell types.

Even so, scientists have addressed and, in exceptional cases, overcome some of these obstacles.  Derrick Rossi, for one, used synthetic RNA molecules corresponding to the standard Yamanaka factors to produce RNA-induced pluripotent stem cells, or “RiPS.” His method was 100 times more efficient than its viral counterparts and took roughly half the time.  Sheng Ding, for another, effectively reprogrammed human skin cells by treating them with drugs and only one virus-delivered gene.  Nevertheless, no leading researcher believes that iPSCs can provide a solution to every challenge.

The epitome of pluripotency, of course, is represented by the ESC.  But unfortunately these cells pose major problems too.  First, the destruction of any embryo remains controversial for many religious enthusiasts.  Second, fertilized embryos generally do not yield patient-specific stem cells.

But what if scientists could instead use human oocytes to reprogram soma, or body cells, back to pluripotency?  This “cloning” process, referred to in professional circles as somatic cell nuclear transfer (SCNT), has a checkered history.  In 1996, Dolly the sheep proved that it was possible to clone an animal by transferring adult DNA into an oocyte, or egg cell.  But since then, only a few experiments—involving mice and rhesus macaques, for example—have reportedly produced cloned ESC lines for research or tissue therapy.  And despite a fraudulent report in 2004 from biologist Woo Suk Hwang, no human cell has ever been successfully cloned.

Nevertheless, George Q. Daley—stem cell guru at Harvard Medical School and the Children’s Hospital Boston—recently argued that “Although it is premature to conclude that … iPS cells pose insurmountable risks, comparative studies of mouse stem cells suggest that SCNT may be more effective” (Daley 2011).

Indeed, on October 6, the possibility of deriving pluripotent, patient-specific stem cells via SCNT came one colossal step closer to becoming reality.  Scott Noggle and Dieter Egli from the New York Stem Cell Foundation Laboratory reported the reprogramming of human somatic cell genomes to a pluripotent state, thus demonstrating for the first time that human oocytes can transform differentiated soma—skin cells, in this case— into stem cells (Noggle, S., Fung, H., et. al. 2011).

Yet their triumph was incomplete.  In order for researchers to derive stem cells, reconstructed eggs must be brought to the blastocyst, or seventy- to 100-cell stage of development.  This can occur only after about five days of development.  To achieve that result, Noggle and Egli were forced to create cells that were abnormal and unfit for practical use.

More than a decade of previous research involving primates and strict nuclear transfer—where the egg genome is removed, á la Dolly the sheep—had ended mostly in cell developmental arrest at about three days.  And, unsurprisingly, the results obtained by Noggle and Egli were consistent with that history.

Their study involved 270 mature, high-quality human oocytes from sixteen donors along with somatic cell genomes gathered from male adults, both diabetic and healthy.  The latter were marked with a green fluorescent protein (GFP).  In the end, genome exchange consistently led to developmental arrest and death without expression of the GFP.

But something exciting and largely unexpected happened as part of the team’s control experiment.  Therein, the somatic cell genomes were merely added, and the oocyte genomes were left in place.  On the fourth day of development the embryos began to express the GFP, and in thirteen out of 64 attempts, Noggle and Egli were able to develop the reconstructed eggs to the blastocyst stage.  From the inner cell masses of those blastocysts they derived two pluripotent stem cell lines.

So what’s the hitch?  Two’s company and three’s a crowd, mostly.  When the diploid genome of the somatic cell was added to the haploid genome of the oocyte, each of the resulting stem cells was rendered triploid, containing three copies of every chromosome instead of the familiar two.  That made the stems abnormal, unstable, and thus currently irrelevant for medical purposes.

But in terms of continuing research, this discovery’s value remains inestimable.  One wonders, for example, whether their method can assist scientists in surmounting the aforementioned obstacles related to iPSCs.  Importantly, the cells produced by Noggle and Egli displayed no evidence of epigenetic memory.  So further investigation might well reveal transcription factors—thus far unidentified by Dr. Yamanaka or his successors—capable of enhancing or even perfecting the reprogramming process.

And perhaps the team’s method can be adapted directly to therapeutic uses, to one day generate usable patient-specific blastocysts.  In a nutshell, we now realize two things.  First, if the oocyte’s genome is removed too early, it fails to provide a very special something capable of reprogramming the somatic cell nucleus.  But, second, if it is left in the embryo too long, it fuses with its genomic complement and cannot be extracted.  Can researchers find a way to remove it at the last possible moment?  Might the use of a different kind of somatic cell—from blood or neural tissue, for example—solve the problem?

Noggle and Egli are now convinced that somatic cell reprogramming using human eggs is feasible.  “With a reliable source of human oocytes,” they predict, “it should be possible to overcome the requirement of the oocyte genome for somatic cell reprogramming, allowing the generation of diploid pluripotent stem cells.”

That scientific possibility forces us to confront yet another set of predicaments, this time socio-political in nature.  Many will find Noggle’s and Egli’s results troubling for at least three reasons.  First, continued research will result in the creation and destruction of embryos.  Second, successful human SCNT might someday be employed to clone people.  Third, and most significantly for researchers at this critical juncture, renewed enthusiasm for the process will likely produce increased demand for human oocytes.

The New York Stem Cell Foundation enjoyed abundant access to oocytes because it collaborated with a Columbia University program that paid $8000 to each eligible woman who expressed an interest in donation for research.  The Empire State has allowed compensation for that purpose since 2009.  The rules vary from state to state, however.  Such payment is banned in Massachusetts, for example, and guidelines recently issued by the U.S. National Academy of Sciences allow no more than reimbursement for expenses like travel and lost wages.

In the United Kingdom, direct payment is also prohibited.  Instead, pursuant to an “egg sharing” program, women who donate voluntarily are given a discount on the cost of in vitro fertilization treatments.  A new report compiled by the Nuffield Council on Bioethics, however, recommends reasonable payment balanced by ethical oversight and a registry designed to limit repeated donation.

Such bans and regulations are purportedly intended to prevent women from feeling unduly coerced.  But, as Noggle and Egli observe, “very few women agree to donate their oocytes for research without payment.”  One might judge such oversight to be unnecessary, of course, or even paternalistic.  Indeed, each of the team’s sixteen donors was gainfully employed.

Jan Helge Solbakk, medical ethicist at the University of Oslo, heartily applauds the team’s decision to not only pay their donors, but to acknowledged them as study participants as well (Sobakk 2011).  Such recognition, he contends, focuses attention on the women instead of on the oocytes, avoids reducing the latter to mere things to be sold on the open market, and appreciates the donors’ contributions as valuable services for which they should be modestly paid.

Noggle’s and Egli’s novel approach this sensitive issue, Solbakk reverently concludes, “represents the first step towards acknowledging women as genuine participants—co-producers even—in the generation of new knowledge.”  What could be more laudable than that?  Now I almost wish I had eggs!

References:

Daley, George Q. 2011. Imperfect yet striking. Nature 478:40-41.

Noggle, S., Fung, H. et. al. 2011. Human oocytes reprogram somatic cells to a pluripotent state. Nature 478:70.

Solbakk, Jan H. 2011. Persons versus things. Nature 478:41.

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