Skin Cells Engineered to Mimic Thymus In Producing Mature T Cells

Findings Suggest Promising Approach to Immunotherapy

Stem cell–based therapies are the hottest of the hot new approaches for attempting to treat a host of diseases. But in some fields of medicine, stem cells are old hat. The routine success of bone marrow transplantation, for instance, relies on a few marrow-derived stem cells to take up residence in a recipient and grow and mature into many distinct types of blood and immune cells. The reconstitution is not perfect, though. After a transplant, adults often take a long time, or fail completely, to replenish the T cells of the immune system. The reason is simple—like children, stem cells need guidance and education to grow up into productive, mature T cells. The schooling of T cells normally takes place in the thymus, an organ that by adulthood has shrunk to little more than a vestige of its young self.

Photo by Graham Ramsay Rachael Clark and Thomas Kupper have found a novel niche in a three-dimensional skin cell culture system that turns undifferentiated human bone marrow stem cells into mature, functional immune T cells.

To ease the T cell shortage, researchers from Brigham and Women’s Hospital have come up with an alternative educational opportunity for stem cells. By adapting skin cells as a stand-in for those in the thymus, the scientists can produce fully functional, mature T cells from bone marrow in a laboratory dish. When skin cells were coaxed to grow on a rigid three-dimensional support, they created a thymuslike environment that apparently suited the stem cells to a T.

“This work shows for the first time that skin cells, when cultured on a 3-D matrix, can recapitulate the important function of the thymus during T cell development,” said Thomas Kupper, the Thomas B. Fitzpatrick professor of dermatology. Kupper and lead author Rachael Clark describe their work in an Oct. 13 posting in the Journal of Clinical Investigation online.

“There are many, many more experiments to do, but the exciting aspect is that a simple skin biopsy and a bone marrow biopsy, both of which are done as outpatient procedures, can give you the raw materials for a new T cell repertoire,” Kupper said. Having a system that uses solely patient tissue opens up the possibility that physicians could one day create a fresh crop of T cells for anyone who needs them after bone marrow transplantation, or to fight cancer or HIV.

Redesigning Skin Cells
Previous attempts to raise T cells in vitro from bone marrow hematopoietic stem cells relied on mouse thymus or fetal human thymus tissue. Several years ago, Harvard’s Mark Poznansky, Richard Evans, and David Scadden showed that mouse thymus tissue could support the generation of human T cells if the cells were grown in a 3-D environment, rather than as a flat layer. But for therapeutic use, mouse tissue would never pass muster, so Kupper and Clark, an HMS instructor in dermatology, came up with what seemed like a wild idea.

They knew that flat skin cultures could support the production of many kinds of cells from bone marrow, but never T cells. They also knew well that there were many similarities between the skin and thymus in the kinds of cells they contain and in the genes they express. “In ways, the thymus looks like skin rolled up into a ball,” Clark explained. “So we thought, can we take skin and make it behave like a thymus?”

Clark began by trying to grow two types of skin cells, keratinocytes and fibroblasts, because similar cells were known to play a role in T cell development in the thymus. Thinking that the important difference between skin and thymus could be their architecture, she aimed to array the cells on the same scaffold used by Poznansky and his colleagues.

It took two years just to work out the conditions to get the different kinds of cells to grow together. “It was like getting Italian, French, and German people to eat lunch together—and all like the food,” said Clark. But when she did discover just the right menu, she found that a culture system that included both kinds of cells growing in three dimensions did, in fact, reproduce much of the thymus microenvironment. When Clark added hematopoietic progenitor cells from human bone marrow, within three weeks, the culture was producing new, mature T cells. Experiments proved that the T cells arose de novo from bone marrow stem cells, had the potential to recognize a wide variety of antigens, and became active when faced with bacterial antigens or foreign cells.

T Cell Training
Just as important as recognizing foreign antigens is the ability of T cells to tolerate self, and one of the critical functions of the thymus is to get rid of self-reactive T cells that if let loose, trigger autoimmune disease. Clark and Kupper showed that their T cells did not tend to attack cells derived from the same bone marrow sample, an indication of at least some measure of successful tolerance training. They also found for the first time that skin cells shared with the thymus the talent of training T cells to ignore auto--antigens from many other organs and tissues. The safety of T cells in clinical use will be tied to how well they are educated to bypass normal healthy tissue, and this part of their development will be the focus of continued scrutiny by the researchers.

Before T cells go live in humans, the process will need to be scaled up many hundreds of times. For skin cells, this presents no problem. “From one routine biopsy, you can grow enough skin cells to cover a tennis court,” Kupper said. “The potential for future development of this process is enormous. We’ve shown proof of concept, but we haven’t played at all with the variables. And this is, as far as I know, the only system that has the potential to make T cells suitable for use in humans.”

“This is elegant work, and it’s remarkable that you can generate T cells this way,” said Poznansky, now an HMS assistant professor of medicine at MGH. “This work shows that it’s possible to manipulate a niche in a 3-D artificial material and then get stem cells to do something unexpected.” The idea that putting stem cells in a carefully engineered microenvironment, including control of the geometry and cellular makeup of the niche, can unlock their hidden potential is one that aspiring developers of other stem cell–based therapies can take a lesson from, Poznansky said.

— Pat McCaffrey