Human Stem Cells Made by Cloning

Wouldn’t it be great if every time a patient needed a new kidney, a stronger heart or a batch of new brain cells scientists could simply grow the required spare part in a petri dish? While a few decades ago this idea would have sounded like a plot for a science fiction novel, it may very well become standard procedure within decades thanks to the discovery of stem cells.
There are about 300 different types of regular “somatic” cells in the body. (Somatic cells make up most of the body’s tissues—the term is used in contrast to cells, such as sperm and egg cells, that serve reproductive purposes.) Each type of somatic cell has unique properties that allow it to perform a particular function. For example, muscle cells contract and expand to allow us to move, while nerve cells transmit signals that let us experience the world around us, recognize familiar faces, memorize poems, and so forth.

While somatic cells are not interchangeable—you wouldn’t expect a photoreceptor cell from an eye to drive muscle contraction or a skin cell to transport oxygen—stem cells lack a specific identity and can develop into different types of tissue. The so-called embryonic stem cells make up the blastocyst, which forms when an egg cell is fertilized and eventually gives rise to all of the different tissues of the body. Like a blank tile in Scrabble that can turn into any letter you want, an embryonic stem cell has the potential to become any type of body cell. However, once it does, there’s no turning back: just like the wildcard Scrabble piece that can only be used once, a stem cell is locked into its “chosen” identity.

While embryonic stem cells are present only in the earliest stages of development, certain non-embryonic varieties stay with us through adulthood. Non-embryonic stem cells are more specialized and allow the body to repair itself. For example, bone marrow, blood, and even the brain have some natural capacity for regeneration.

The unique flexibility of stem cells makes them a godsend for regenerative medicine. Like a customized tissue factory, they can be used to produce a constant supply of replacement cells genetically identical to the recipient. However, even though scientists have been looking for ways to put stem cells to medical use for over a decade, the task has been difficult due to technical complications, legal issues and ethical dilemmas. In a recent issue of Cell, scientists from the Oregon Health and Science University (OHSU) describe how they were able to create human embryonic stem cells via cloning. Their results are a milestone in what scientist John Gearhart calls “a holy grail that we’ve been after for years.”
Crime or Cure

For the last few decades, doctors have been able to use certain types of stem cells to treat certain diseases. For example, since bone marrow contains stem cell precursors to different cells found in blood, bone marrow transplants can help treat a number of different blood-related disorders, such as leukemia. While treatments using embryonic stem cells are still experimental, one such approach recently restored sight to a man whose vision went from 20/400 to 20/40. Another team of scientists managed to use antibodies to “reprogram” bone marrow stem cells to give rise to brain cells.

And this is just the beginning: the potential benefits of using stem cells in other medical procedures are almost inexhaustible. Doctors may someday be able to use them to treat anything from Alzheimer’s or Parkinson’s disease to heart disease, cancer, or even baldness. It’s hard to think of a medical condition that wouldn’t offer some point of attack for this potentially revolutionary tool.

While the field of regenerative medicine is very promising, it is also one of the most controversial areas of research, marked by technical complications, ethical dilemmas, and even scientific fraud. The main sticking point is the use of human embryos to harvest stem cells—a task first accomplished by a University of Wisconsin team in 1998—and the fact that the embryos are destroyed in the process. Even though the embryos used in research are usually ones discarded from in vitro fertilization, some people feel, on religious and ethical grounds, that this is wrong, and consequently the research became a political football, with some arguing that embryonic stem cell research should be federally funded since it held such great promise, and others contending that federal funding of such research would be a grave mistake.

To bypass some of the ethical dilemmas, scientists began to look for alternative ways of harvesting stem cells. A major advance came a few years ago, when Gladstone Institute of Cardiovascular Disease scientist Shinya Yamanaka developed the first “induced pluripotent stem cells” (referred to as iPS). Unlike embryonic stem cells, iPS cells are derived from regular somatic cells that get “reprogrammed” back to their undifferentiated state.

However, while Yamanaka’s discovery earned him a Nobel Prize in 2012 and provided an option acceptable to both sides in the stem cell debate, it remained unclear whether or not iPS cells are really just as good as embryonic stem cells. Critics point out that iPS cells are created using retroviruses, which may cause potentially dangerous genetic abnormalities. Moreover, iPS cells tend to age quickly and don’t last very long.
Hello Again, Dolly

While some researchers continue trying to improve iPS, others have turned to a third method known as somatic cell nuclear transfer (SCNT). The technique, also referred to as “therapeutic cloning,” allows scientists to create embryos by swapping the genetic material of an egg cell for the DNA of another person—for example, that of a sick patient.

In nature, an embryo develops when an egg cell is fertilized by a sperm cell, both of which are “haploid” and have half the total number of chromosomes found in somatic cells. However, by replacing the modified egg cell’s genes with a full set of chromosomes from a body cell a researcher can “trick” the egg into acting as if it had been fertilized. As a result, it starts dividing and grows into an “embryo” genetically identical to the body cell’s donor.

The overall idea isn’t a new one: 17 years ago, Roslin Institute scientist Ian Wilmut used this method to create Dolly, the famous cloned sheep. Since then, enough species have been cloned to fill a whole zoo— for example, Dewey the deer at Texas A&M University; Snuppy, a South Korean Afghan Hound; two ferrets, Libby and Lilly; Ralph the rat; a mouse named Cumulina—among many others.[See Adult Mammal Cloned for First Time, April 1997; On the Heels of Dolly Come Polly and Molly, February 1998; 5 Cloned Piggies, All in a Row, May 2000; Doggy Double: Korean Scientists Create First Canine Clone, August 2005.]

However, the process didn’t seem to work for human egg cells, which are more fragile than those of other species. The search for ways around this obstacle turned out to be very messy. When Seoul National University veterinary scientist Hwang Woo Suk published reports of successful SCNT trials with human cells in 2004 and 2005, it seemed that finally a solution had been found. However, what looked like a groundbreaking discovery turned out to be one of the most infamous cases of scientific fraud, with falsified data underlying the claims. [See Hwang Woo Suk and Fraud in Science, January 2006; Hwang Woo Suk: Seeing Double, December 2005; Scientists Clone Human Embryos to Create Stem Cells, February 2004.]

While other efforts to produce human stem cells by using the SCNT method have been more legitimate, they failed to hit the mark due to technical complications. For example, New York Stem Cell Foundation specialist Dieter Egli produced a line of human stem cells in 2011. However, since the egg’s nucleus remained in the cell, the resulting stem cells had an abnormal number of chromosomes.
Breakfast of Champions

Shoukhrat Mitalipov, senior scientist at the Oregon National Primate Research Center and lead author of the Cell paper, has been investigating stem cells since the 1990s. In 2007, his team used the SCNT method to clone embryonic stem cell lines in monkeys. The scientists then used knowledge gleaned from those studies to successfully clone human embryonic stem cells, the feat described in the Cell paper.

Mitalipov took egg cells that had been donated by volunteers to serve as the basis for future stem cells. Since egg quality is essential for this process to work, the eggs came from healthy, young donors. He then cleared out the DNA from the cells and replaced it with DNA from the skin cells of other individuals (the first DNA came from fetuses and the rest came from an eight-month-old with Leigh syndrome, a rare metabolic disorder). “The idea is that the egg cytoplasm has…the ability to reset the cell’s identity,” Mitalipov explained in an interview with Fox News. “It basically erases all this memory, and now we can derive them and make them into stem cells.”

From previous experience, Mitalipov knew he had to be extra careful with the DNA exchange process, timing it to take place when the egg was most likely to accept the new genetic material. An inactivated Sendai virus helped fuse the egg with the body cells, and an electric jolt helped jump-start embryonic development.

An ingredient that played an important role was, oddly enough, a substance we ourselves often rely on to get going in the morning—caffeine. Since caffeine inhibits an enzyme responsible for breaking down the “maturation promoting factor,” adding it to the mix helped prevent premature activation during spindle removal and somatic cell fusion. Harvard University scientist George Daley jokingly calls this “the Starbuck’s experiment.” “This little change in the cocktail,” he adds, “was what really allowed the experiment to really ultimately succeed.”

While each step was important, the most challenging part of the process was actually the combination of steps. As Mitalipov explains, “there is no one trick to making this work. It is like winning the lottery, all the numbers have to line up the right way to win.” Mitalipov’s monkey-cell research was especially useful for this purpose: before switching to human cells, which are more expensive and more difficult to obtain than monkey cells, Mitalipov fine-tuned the process for monkey cells by testing over a thousand combinations of steps.
Speedy Success

Having set up the cell colonies in December 2012, the scientists were excited to see that four of them started growing. The growth continued past the eight-cell stage, which had been the farthest scientists could go in previous attempts. Once the newly cloned embryos were five or six days old, Mitalipov and his team could manipulate them to create particular types of somatic cells.

Knowing how hard it has been for others to make this process work, Mitalipov was surprised to get results relatively quickly: “I thought we would need about 500 to 1,000 eggs to optimize the process and anticipated it would be a long study that would take several years. But in the first experiment we got a blastocyst and within a couple of months we already had an [embryonic] stem cell line.”

Mitalipov describes the successful outcome: “A thorough examination of the stem cells derived through this technique demonstrated their ability to convert just like normal embryonic stem cells, into several different types, including nerve cells, liver cells, and heart cells.” And while the technical details remain to be worked out, the team sees the results as “a significant step forward in developing the cells that could be used in regenerative medicine.”
Not So Fast

“Our finding offers new ways of generating stem cells for patients with dysfunctional or damaged tissues and organs,” Mitalipov explained in an interview. “Such stem cells can regenerate and replace those damaged cells and tissues and alleviate diseases that affect millions of people.”

The study was immediately hailed as an important breakthrough. However, a week later things took an unexpected turn. An anonymous commenter on PubPeer (a website that allows scientists to comment on the work of their colleagues) pointed out four mistakes involving images that had been mislabeled or duplicated.

Arnold Kriegstein, a researcher from the University of California, San Francisco, says that this situation “is really like déjà vu all over again.” But while it does add to the existing controversy surrounding the whole subject of stem cell research, the mistakes in Mitalipov’s paper are clearly accidental and don’t affect the validity of his results. More than anything, they appear to be the result of a rush to publish: the study was accepted by Cell within days of its submission.

In response to the criticism, Cell editors, as well as OHSU representatives, have called the inaccuracies “minor errors.” According to OHSU spokesman Jim Newman, “Neither OHSU nor Cell editors believe these errors impact the scientific findings of the paper in any way. We also do not believe there was any wrongdoing.”
Batch of Cells or Future Human?

But even if these details are ironed out, broader disagreements about Mitalipov’s approach are likely to persist. While raising hopes, the study has also stirred up ethical concerns that have been part of the stem cell debate from the beginning. As Daley puts it, “This is a huge scientific advance…But it’s going to, I think, raise the specter of controversy again.”

And, sure enough, it did. For one thing, using human embryos—even ones generated without fertilization—for medical research remains a bone of contention. When seen as a potentially revolutionary medical treatment, the procedure becomes more palatable. As University of Pennsylvania scientist John Gearhart says, “Where you can improve [a patient’s] quality of life tremendously through this kind of technology, I personally believe that it is ethical to use material like this.”

Moreover, Mitalipov argues that his method of obtaining stem cells is qualitatively different from one that uses fertilized embryos. However, he also recognizes that not everyone might agree with him: “We think it’s more ethically acceptable, but you never know. Some people [might] say now they’re trying to use cloned embryos.”

Indeed, those who believe that cloned embryos amount to potential human beings are hesitant to accept their use in the lab. The idea of “manufacturing” human life at any stage doesn’t sit well with everyone. Instead, opponents urge scientists to focus on methods that don’t involve embryos at all and argue that the discovery of iPS cells as an alternative way to produce stem cells should make methods involving embryos obsolete. Miodrag Stojkovic, a Serbian scientist and a fertility clinic director, is very frank in his assessment: “Honestly, the most surprising thing [about this paper] is that somebody is still doing human [SCNT] in the era of iPS cells.”

However, creating stem cells via cloning has significant advantages when compared with iPS. Instead of iPS’s reprogramming of the body cell of an adult, the nuclear transfer method provides a clean slate for the stem cells it creates. As Mitalipov explains, “If you take a cell from a 90-year-old patient, the battery is kind of drained. You could make new cells by reprogramming it, but the energy is gone.” However, “the idea behind using egg cells is that you’re fully recharging the battery and making cells that could probably live another 90 years. We’re taking you back to having some young cells.”

In order to get a more definitive answer about the way the two techniques compare, more studies comparing iPS and SCNT techniques have to be done. Along with team member Masahito Tachibana, Mitalipov plans to conduct a comparison study between the two methods using cells from the same donor.
The Clones Are Not Coming

While creating and destroying embryos at will might seem questionable to critics, allowing them to fully develop is even more unsettling. Cloning a batch of particular cells is a huge leap forward for medical science. However, cloning entire humans by using the same method could become a disaster.

In an effort to reassure people that this is not what he is doing, Mitalipov emphasizes the fact that his goals are solely “therapeutic,” rather than “reproductive,” cloning. The embryos he created will not be implanted: “We never tried that in humans and it’s not our intention,” he explains. Likewise, Daley insists that “no legitimate scientists would want to use this technology for reproductive purposes. They would see it not only as unethical, but unsafe and probably illegal.” Moreover, since, in Mitalipov’s work with monkeys, implanting cloned embryos didn’t produce viable offspring, Mitalipov argues that it’s very unlikely that this technique could be used for this purpose in humans.

However, just because Mitalipov isn’t planning to clone humans doesn’t mean nobody else is planning to try. It’s theoretically possible that someone will figure out how to use this method to clone humans in the future. (After all, SCNT itself didn’t initially seem to work for human cells.) “This study shows that human cloning can be done….The more important debate is whether it should be done,” said Richard Doeflinger at a meeting of the U.S. Conference of Catholic Bishops. Those attending the meeting were concerned that the findings “will be taken up by those who want to produce cloned children as ‘copies’ of other people.”

Stem cell research has been dogged by controversy from the start, and is likely to remain controversial for quite some time. If Mitalipov’s research leads to new therapies that are as significant as supporters of the research anticipate, that will certainly have a big effect on the debate.
Discussion Questions

Can you think of any explanations for why human cloning is more difficult than cloning other mammals?

Some cloned animals have had various problems, including shorter lifespans, as compared with animals born through natural processes—why do you think this is?

A factsheet on cloning from the National Human Genome Research Institute notes that telomeres (the tips of chromosomes) may have something to do with the shorter lifespans—why do you think this is?
Journal Abstracts and Articles

(Researchers’ own descriptions of their work, summary or full-text, on scientific journal websites).

“Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer” http://www.cell.com/abstract/S0092-8674(13)00571-0.

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