The purpose of this grant is to regenerate and re-program the immune system by making specialized cells called thymic epithelial cells (TEC) from stem cells. TEC are supporting (stromal) cells in the thymus gland. TEC are required to 1) stimulate the growth of young T-cells and 2) select which T-cells are allowed to develop and become part of the immune system. Our hypothesis is that TEC can be generated from stem cells, transplanted into recipients, and be used to generate a new immune system that could replace an abnormal immune system. Examples of abnormal immune systems are those in which the T-cells attack cells of the body such as nerve-lining cells, resulting in multiple sclerosis.
TEC turn over at a rapid rate, i.e., a high percentage of TEC are dying at any moment, necessitating their replacement by new TEC that are generated from rapidly dividing immature TEC. We have shown that the high turnover is accelerated during aging, which explains why the thymus shrinks and loses the ability to make new T-cells. Furthermore, we have shown that the high turnover rate of TEC in aging is due to the action of two genes, NFB and HNF4, which become active in response to inflammation and fat, respectively. Thus, our research has been able to causally link two common disease processes, inflammation and obesity, to the aging of the thymus.
Our initial efforts were to generate new TEC from embryonic stem cells (ESC). These efforts used a complex system in which a gene called Tbx1 needed for TEC formation was introduced into ESC. This system was marked by a great deal of variability: different cultures would contain differing numbers of TEC-like cells ranging from almost none to a majority and the ability to robustly support the development of new T cells after transplantation was also inconsistent. The inability to consistently generate TEC was both a practical limit to the approach for pre-clinical studies and an anticipated problem in developing this approach further for clinical stem cell therapy. Because of the irreproducibility of the approach based on generating new TEC from ESC, we explored an alternative approach based on the isolation of adult TEC stem cells from the thymus.
For many if not all organs, populations of adult stem cells exist, which can contribute to the formation of the cells of that organ. The existence of TEC stem cells can be inferred from the high turnover rates of TEC, which can only be accounted for by the existence of an adult TEC stem cell population that can continually generate new TEC to take the place of the ones that are dying. This is analogous to the systems that maintain other types of epithelial cells like those of the skin and lining of the intestine. The potential advantage of using adult TEC stem cells is that they would not require the re-programming needed to turn ESC into TEC. Previous studies had demonstrated that there were immature TEC in the fetal thymus that could regenerate a new thymus upon transplantation. However, large numbers of these fetal TEC cells were required for transplantation, suggesting that these were not a pure TEC stem cell population, Furthermore, previous efforts to isolate TEC stem cells from the adult thymus have been unsuccessful.
We used a novel system to identify adult TEC stem cells, which is based on the Wnt signaling pathway, which is important for most stem cells and development. Wnt proteins are known to be important for the formation of TEC. In the genetically engineered mice that we used, TEC that were responding to Wnt signals at any time point could be switched by brief exposure to a hormone drug from expressing a protein that made them fluoresce red to expressing a different fluorescent protein that was green. When we switched the Wnt-responding cells in young or adult mice from red to green fluorescence, we found that a very small number of TEC were green while the vast majority of TEC remained red. This is consistent with the majority of TEC being Wnt-unresponsive and the rare Wnt-responsive cells being TEC stem cells. Analyses of mice for up to one year after switching showed that clusters of green TEC replaced the red TEC, consistent with the green TEC being derived from a single adult TEC stem cell that had been switched earlier in life and then developed into a group of mature TEC. Transplantation of as few as 60 of Wnt-responsive green cells into mice which lacked a thymus regenerates a robust new thymus and immune system in the recipient. To our knowledge, this is the highest level of purification of any TEC stem cell, and the first demonstration that TEC stem cells exist in the adult thymus. We have identified new markers that can be used to isolate TEC stem cells from any strain of mice, and potentially from humans. We have isolated sufficient numbers of cells to test whether the immune system of a mouse with an autoimmune disease that models MS, can be re-programmed by TEC stem cell transplantation.