Skin Grown from Human Embryonic Stem Cells

Findings Identify Barriers to Therapy and Potential Paths Around Them

In an important step toward the therapeutic use of embryonic stem cells, HMS researchers have found a way to improve the production of skin cells from a human embryonic stem (ES) cell line. In the study, the scientists identify some unexpected barriers to the formation of useful skin tissue from ES cells, including a lower-than-expected capacity to multiply and difficulty isolating pure cell populations. Both of these problems were overcome by introducing growth-promoting genes into the ES-derived skin cells, an approach that may also be necessary to ensure sufficient growth of other cell types.

Photo by Graham Ramsay

Skin cell expert Howard Green (front) and colleagues (rear, from left) Shiro Iuchi, James Rheinwald, and Karen Easley are applying their know-how to the problem of producing therapeutically useful keratinocytes from human embryonic stem cell lines.

The findings, which were published in the online edition of Proceedings of the National Academy of Sciences during the week of Jan. 30, show that despite the excitement over the medical uses of ES cells, much work remains to turn the cells into actual therapies.

“The total amount of our experience with cultured cells as therapeutic measures is extremely limited, so while the possibility of using ES cells and their derivatives is very important, there remains a fair amount of work to be done before we get there,” said Howard Green, the George Higginson professor of cell biology and lead investigator on the study. “In this work we have tried to make a logical analysis of some of the problems and how to deal with them.”

ES Cell Future
The promise and challenge of ES cell–derived therapies rest on their ability to transform into any cell type desired for transplantation or other medical application. During development, the embryonic environment efficiently steers stem cells to their fate. But until this work, researchers had not had success in replicating those conditions in a lab dish to produce the pure populations of differentiated cells that would be acceptable for therapeutic use.

ES cells aside, only two examples exist of the successful use of cultured cells of any type to treat injury or disease, and one of them originated in Green’s lab. More than 20 years ago, Green and his colleagues pioneered a technique for growing sheets of keratinocytes in vitro for use as skin grafts. His work gained worldwide attention in 1983 when the grafts saved two young brothers who had both suffered burns over 90 percent of their bodies. Starting with a postage stamp–sized piece of undamaged skin from each boy, Green and his colleagues grew half a square meter of graftable cells in just a few weeks. The youngsters survived, and the technique has been used to treat thousands of burn victims since then and to heal wounds and skin conditions.

Green knew from his previous work that ES cells cultured under conditions that support the growth of postnatal keratinocytes from skin would give rise to differentiated cells that in many ways resemble those keratinocytes. But to be viable therapeutic candidates, the ES-derived cells must be purified, since residual undifferentiated ES cells run the risk of forming tumors when transplanted.

The problem came when the researchers tried to purify the cells. The ES-derived skin cells grew so poorly that they could not be separated from contaminating ES cells. After a short time in culture, they stopped growing altogether. This was not anticipated based on the behavior of embryonic stem cells or postnatal keratinocytes, both of which grow for long periods in vitro.

To boost cell replication, Green turned to longtime collaborator James Rheinwald, an HMS associate professor of dermatology at Brigham and Women’s Hospital and the Harvard Skin Disease Research Center, to introduce viral growth-stimulating genes into the ES-derived cells. The results were striking. Within one week, the cultures were full of rapidly growing cells that could be purified and that carried several markers of mature keratinocytes. One line formed a continuous sheet of cells in culture, mimicking the structure of skin grafts grown from normal cells.

Deviant Behavior
Despite improvements in their growth, the ES-derived keratinocytes still do not behave exactly like normal postnatal keratinocytes. They show a reduced ability to differentiate, perhaps due in part to the powerful effects of the HPV E6 and E7 genes used to help the cells bypass internal growth-arrest mechanisms. For this reason, and because the presence of viral genes is problematic in cells to be used in people, other methods of improving cell proliferation and extending life span are now under study, Green said.

According to David Scadden, co-director of the Harvard Stem Cell Institute and director of the MGH Center for Regenerative Medicine, the study shows the attention to detail that will be necessary to bring ES-derived cells to clinical use. “The optimism surrounding embryonic stem cells is deserved, but it shouldn’t be naive,” he said. “One of the points of caution this study raises is that we can’t assume that medical applications of stem cells are all going to be slam dunks. Issues will arise, but good cell biology focusing on these tissues and other cell systems will provide insights so that these issues can be overcome.”

Adult stem cells have been in use for 40 years, and researchers are still working out their vagaries, Scadden points out. “The blood stem cell that is clinically used to cure people with deadly diseases remains a very difficult cell to work with outside the body. And yet we have been able to make accommodation for that biology and use it effectively in a clinical setting.”

The question of whether genetic manipulation of other ES-derived cells will be necessary to improve their growth and purification is wide open, according to Green. His work examined just one ES cell line, differentiated under a particular set of conditions. “Now,” he said, “it’s up to the people who are working with different ES-derived cell types to see if this kind of genetic modification is going to be necessary for their cells as well.”

—Pat McCaffrey

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