Bone Marrow Derived Progenitor Cells and Their Contributions to the Thymic Stroma

The thymus serves as the primary lymphoid organ responsible for the development and selection of a self-tolerant T cell repertoire. In paradox to its critical functions for the adaptive immune response, the thymus undergoes a profound age associated decline beginning in early adult life resulting in significant decline in T-cell function.Thymic epithelial cells (TECs) are the most critical component of the thymic microenvironment and undergo rapid turn-over, so understanding the cellular mechanisms responsible for the maintenance of TEC number and organization will be critical in counteracting age associated involution, particularly in cancer patients, due to enhanced degeneration in response to therapy. In the search for stem/progenitor populations that contribute to TEC maintenance in the postnatal thymus, our lab has identified a new population of TEC progenitors that co-express the hematopoietic marker CD45 and the definitive thymic epithelial marker EpCAM, which is also highly enriched in stem cell markers such as, Sca1. CD45+ EpCAM+ cells are present in both the thymus and bone marrow (BM), suggesting that the CD45+EpCAM+ population may be recruited from a hematopoietic derived population in the BM, providing an additional source of progenitor cells that contribute to the maintenance of the thymic stroma. Immunofluorescence microscopy of FACS sorted CD45+ EpCAM+ cells from thymus and bone marrow confirmed surface protein expression of both markers, while Q-rtPCR confirmed co-expression of both genes at the RNA level. CD45+ EpCAM+ cells sorted from the BM or thymus of H2BGFP-expressing mice could contribute to fetal reaggregate thymus organ cultures and differentiate into keratin-expressing TECs and FSP-1 expressing fibroblasts after transplant under the kidney capsule of nude mice. Thus, BM derived CD45+EpCAM+ cells represent a potential new source of cells that may contribute to thymic homeostasis. When an intact fetal C57BL6 thymus was placed into the kidney capsule of an Actin H2BGFP mouse, H2BGFP-exressing cells homed to the fetal thymus and contributed to both Keratin+ and FoxN1+ TECs as well as FSP1+ thymic fibroblasts. Significantly, when Actin-H2BGFP fetal thymi were transplanted under the kidney capsule of a Rosa 26 mRFP transgenic mouse and allowed to grow for three weeks RFP-expressing CD45+EpCAM+ and CD45-EpCAM+ TECs were found to be present in the fetal thymus and were GFP-, indicating that the cells were not derived from cell fusion but represented cells that migrated in from the periphery of the Rosa26 mRFP mouse and differentiated into EpCAM expressing TECs. Very few cells were found to express both GFP and mRFP. Future studies will characterize the contribution of BM derived CD45+EpCAM+ stromal progenitors to distinct TEC microenvironments in both the steady state thymus and under conditions of demand, as well as their capacity to influence T cell selection and self-tolerance