Pluripotent Stem Cells 101

How Do We Get Pluripotent Stem Cells?

Pluripotent stem cells can be created in several ways, depending on the type.

Genetic reprogramming (induced pluripotent cells):
Several labs, including that of George Q. Daley, MD, PhD, Director of Stem Cell Transplantation Program, have shown that it requires only a handful of genes to reprogram an ordinary cell from the body, such as a skin cell, into what’s known as an induced pluripotent cell (iPS cell). Currently, these genes (Oct4, Sox2, Myc, and Klf4) are most commonly brought into the cell using viruses, but there are newer methods that do not use viruses.

Although skin cells are probably the number-one source of iPS cells currently, lines are also being created from blood cells and mesenchymal stem cells (a type of multipotent adult stem cell that gives rise to a variety of connective tissues). Laboratories in the Stem Cell Program at Children’s Hospital Boston are exploring whether iPS lines made from different kinds of patient cells are easier to work with, or can more readily form the particular kind of cell a patient might need for treatment.

Children’s researchers are also continuing to experiment with more efficient programming techniques, so they can get a higher yield of true pluripotent stem cells.

IVF donations of unused/discarded embryos (ES cells):
Another major source of pluripotent stem cells for research purposes is unused embryo donated by couples undergoing in vitro fertilization (IVF). Some of these may be poor-quality embryos that would otherwise be discarded. The resulting cells are considered to be “true” embryonic stem cells (ES cells).

The donated embryos are placed in a media preparation in special dishes and allowed to develop for a few days. At about the fifth day the embryo reaches the blastocyst stage and forms a ball of 100-200 cells. At this stage, ES cells are derived from the blastocyst’s inner cell mass. In some cases, the ES cells can be isolated even before the blastocyst stage.

To date, Children’s has created more than a dozen new ES cell lines using this approach, which we are now making available to other scientists. These ES cells are not genetically matched to a particular patient, but instead are used to advance our knowledge of how stem cells behave and differentiate.

Some people question the ethics of using discarded IVF embryos for research. For more discussion, see Policy and Ethics.

Somatic cell nuclear transfer:
The process called nuclear transfer involves combining a donated human egg with a cell from the body (typically a skin cell) to create a type of embryonic stem cell, sometimes called an ntES cell. Nuclear transfer requires an egg donor.

First, an incredibly thin microscopic needle is used to remove the egg’s nucleus, which contains all the egg’s genetic material, and replace it with the nucleus from the body cell. The process of transferring the nucleus into the egg reprograms it, reactivating the full set of genes for making all the tissues of the body. How this happens isn’t well understood yet, and researchers in the Stem Cell Program at Boston Children’s Hospital are trying to understand it better.

Next, the resulting reprogrammed cell is encouraged to develop and divide in the lab, and by about day five, it forms a blastocyst, a ball of 100-200 cells. The inner cells of the blastocyst are then isolated to create ntES cells.

Of all the techniques for making pluripotent cells, nuclear transfer is the most technically demanding and therefore the least efficient. To date, it has only been successful in lower animals, not in humans. But because the stem cells created would be an exact genetic match to the patient, nuclear transfer may eliminate the tissue matching and tissue rejection problems that are currently a serious obstacle to successful tissue transplantation. For this reason, nuclear transfer is an important area of research at Children’s.

Because ntES cells created from human patients would match them genetically, nuclear transfer is sometimes called therapeutic cloning—not to be confused with the concept of reproductive cloning.

Parthenogenesis (unfertilized eggs):
Using a series of chemical treatments, it’s possible to trick an egg into developing into an embryo without being fertilized by sperm. This process, called parthenogenesis, sometimes happens in nature, allowing many plants and some animals to reproduce without the contribution of a male.

By inducing parthenogenesis artificially, researchers have been able to create parthenogenetic embryonic stem cells, or pES cells, in mice. The embryos created, known as parthenotes, are grown for about five days until they reach the blastocyst stage. Development is then stopped and pES cells were derived from the blastocyst’s inner core of cells.

Parthenogenesis hasn’t been accomplished in human eggs yet, at least not by choice (a Korean team is thought to have created human pES cells accidentally in 2007). But researchers at Children’s are trying to do so, since pES cells, if carefully typed genetically, could potentially be used to create master banks of pluripotent stem cells. Doctors could then choose a cell line that’s genetically compatible with the patient’s immune system. (For details, see How do pluripotent stem cells get turned into treatments?).

Of more immediate concern is the possibility that parthenogenesis could be used to make pES cells for the egg donor herself or a sibling. However, before using these cells in patients, researchers need to know more about the safety of this approach.

Giving

Spotlight

  • When is an iPS cell really an iPS cell?

    The production of induced pluripotent stem cells is often imprecise, yielding many incompletely reprogrammed cells. Now, Thorsten Schlaeger, PhD, and George Daley, MD, PhD, of Children’s Stem Cell Program have developed a battery of tests to ensure that he has the real thing: pure pluripotent stem cells. The new work creates a standard of analysis in the field. Read more.

  • Reprogramming: Breakthrough of the Year

    The Stem Cell Program at Children’s Hospital Boston was one of the first three labs to successfully make human iPS cells. Read more about genetic reprogramming in Science magazine, which named this advance 2008′s Breakthrough of the Year, and watch Science’s in-depth video, featuring interviews with Children’s George Q. Daley and others.

  • IVF embryos

    In addition to healthy embryos
    donated by couples, discarded poor-quality embryos from fertility clinics have a role in stem cell research. Typically, when a couple undergoes in vitro fertilization, or IVF, about half of the seven-odd embryos per cycle are discarded because they are judged not to be viable. In 2008, Paul Lerou, MD, a neo-natologist at Children’s Hospital Boston and an investigator in the Daley Lab, showed that it is possible to salvage these embryos and derive embryonic stem cell lines
    from them for research purposes. Several of the lines created by the Daley team are now under active study. Read more.

  • Is nuclear transfer the same as cloning?

    Creating embryonic stem cells through nuclear transfer, if achieved in humans, could provide patients with cells that match them genetically. For that reason, this technique is sometimes been called therapeutic cloning. Nuclear transfer is definitively not an attempt to create a new human being: Lab development is ended no later than the blastocyst
    stage, and the embryo
    created through nuclear transfer is never implanted into a womb, so could never develop much further. This kind of reproductive cloning has, however, been done in animals like the famous Dolly the Sheep.

  • An egg’s potential: parthenogenesis

    The Stem Cell Program at Children’s Hospital Boston was the first to show that parthenogenesis could be coupled with careful genetic typing to make pluripotent stem cells that match the egg donor and potentially other patients too. This technique, using eggs alone, could possibly provide some patients with cell-based treatments that wouldn’t be rejected by their immune system. Read more in our news release and watch a series of video clips where George Q. Daley, MD, PhD, explains the parthenogenesis technique, its place in the embryonic stem cell field, and his vision for creating embryonic stem cell banks.