ADDITION OF A BONE-MARROW FACILITATING CELL-POPULATION INCREASES STEMCELL-DERIVED COBBLESTONE AREA FORMATION IN IMPAIRED LONG-TERM BONE-MARROW CULTURE STROMA

Treatment of mouse bone marrow (BM) with rabbit anti-mouse brain serum (RAMBS) plus complement (C') depletes several cell types, including T cells and facilitating cells (FCs), that is, cells that facilitate en graftment of sorted allogeneic stem cells (SCs) in vivo. In the presen t study, treatment of BM with RAMBS+C' resulted in the depletion of ap proximately half of the late cobblestone area (CA)-forming stem cells as assayed on irradiated long-term bone marrow culture (LTBMC) stroma. In addition, LTBMC of RAMBS+C'-treated BM produced functionally impai red stroma with reduced ability to support CA formation by nontreated exogenous SCs. This stromal impairment was not due to depletion of TCR alpha beta T cells in the BM, because BM cultures from TCR alpha-chai n knockout mice supported normal numbers of exogenous CAs. Because CD8 (+)/TCR- cells are enriched for FCs, we tested the effect of adding th ese cells back to the treated BM prior to culture. The sorted FCs alon e did not produce CAs, but did improve the ability of the impaired str oma to support late CA formation by sorted SCs. These studies provide a new model for dissecting the roles of different cellular components of BM in producing functional stroma that sup ports CA formation by SC s, and show that the number of CAs formed depends on the ''quality'' o f the stroma as well as the number of SCs seeded. These findings furth er suggest that CD8(+)/TCR- BM cells may be important for the establis hment of functional stroma.