ENRICHMENT AND CHARACTERIZATION OF THYMUS-REPOPULATING CELLS IN STROMA-DEPENDENT CULTURES OF RAT BONE-MARROW

The bone marrow precursor cells seeding the thymus have been difficult to investigate using fresh bone marrow and in vivo thymus reconstitut ion assays. We have therefore designed a short-term bone marrow cultur e system allowing the study of thymus-repopulating cells in the marrow microenvironment. Low-density rat bone marrow cells were grown on pre -established mouse bone marrow stromal cell layers. Cocultured cells w ere maintained either under steroid-free conditions (Whitlock/Witte-ty pe culture) or in the presence of 10(-7) M hydrocortisone (Dexter-type culture). After 3 days in vitro, the unanchored cell fractions were t ested for their ability to colonize and repopulate fetal mouse thymic lobes in vitro. Both fresh low-density cells and Whitlock/Witte-type c ultures, but not Dexter-type cultures, gave rise intrathymically to si gnificant numbers of rat donor-type Thy-1.1high CD2+ CD5low CD43+ cell s accounting for 50% to 90% of the organ-cultured cells at day 14. Rep opulation of fetal mouse thymic lobes by rat Thy-1.1high cells could b e used as a readout assay for initiation of thymopoiesis from bone mar row precursor cells, since 90% of the cells were CD3-/low and TCRalpha beta-/low and 15% of the cells co-expressed CD4 and CD8. Dose-response analysis showed that thymus repopulating cells were at least maintain ed, if not amplified during the 3-day culture period, leading to at le ast a 10-fold enrichment as compared to unfractionated bone marrow. Un like fresh low-density cells before culture, short-term Whitlock/Witte -type cultures were depleted in myeloid-restricted precursor cells. In culture, the thymus-repopulating activity was predominantly associate d with a 10% lymphoid cell subset which did not express the B-lineage- associated antigens revealed by HIS24 (the rat B220 equivalent) and HI S50 mAbs. We propose that unanchored thymus-repopulating cells enriche d in Whitlock/Witte-type cultures may represent lymphoid-restricted, T -cell precursors of the bone marrow capable of emigrating and colonizi ng the thymus.