VARIABLE SELF-RENEWAL OF RECONSTITUTING STEM-CELLS IN LONG-TERM BONE-MARROW CULTURES

Long-term bone marrow cultures (LTBMC) serve as a valuable in vitro mo del of the bone marrow microenvironment. The stromal layer supports th e growth of immature and mature cell populations through production of colony-stimulating factors and cell:cell interactions. LTBMC are devo id of mature lymphoid cells but contain stem cells capable of restorin g lymphoid and myeloid function in suitable recipients. Severe combine d immune deficiency (scid) mice provide a useful environment to study lymphocyte development, as their autosomal recessive mutation on chrom osome 16 leaves them with a severe deficiency of B and T lymphocytes. To determine the presence of different classes of stem cells in LTBMC, adherent cells from the cultures were grafted into sublethally irradi ated scid mice and lineage reconstitution was evaluated 6 weeks to 3 m onths postengraftment. Self-renewal of donor stem cells was tested by serial transfer of scid bone marrow to secondary C.B-17 and scid recip ients. Mature lymphoid and myeloid cells were isolated from reconstitu ted mice and a restriction fragment length polymorphism (RFLP) at the C mu immunoglobulin locus was used to distinguish donor and host cells . We found that LTBMC contained both long-term and short-term reconsti tuting stem cells. The long-term stem cells had significant self-renew al potential and fully reconstituted all lineages in both primary and secondary recipients. The short-term stem cells produced mostly lympho id progeny at the time of analysis and their limited self-renewal capa city led to partial reconstitution of only the primary recipients. The short-term reconstituting cells may be lymphoid-restricted stem cells .