Stroma-dependent maintenance of cytokine responsive hematopoietic progenitor cells derived from long-term bone marrow culture

Hematopoietic cells maintained for long periods on primary cultures of bone marrow stromal cells formed cobblestone colonies (Dexter's long-term bone marrow culture, LTBC), These stably maintained hematopoietic cells (for 4 m onths) were transferred to a coculture on an established spleen stromal cel l Line (MSS62), and maintained under stromal cell layer, where they retaine d their invasive ability in the restricted space between the stromal cell l ayer and culture substratum (DFC culture). DFC contained lineage-negative ( Lin(-)), c-Kit(+), Sca-1(+) cells and spontaneously produced Mac-1(+), Gr-1 (+) cells, DFC could not grow in the absence of MSS62 stromal cells, althou gh, GM-CSF, IL-3, or IL-7 stimulated its growth, Production of granulocyte and monocytic cells was maintained by CM-CSP or IL-3 while it was decreased by IL-7, RT-PCR analysis showed that the IL-7 responsive cell population e xpressed early lymphoid markers (Ikaros, Pax-5, Oct-2, Rag-1, TdT, IL-7R an d I mu), while lacking expression of receptors for G-CSF (G-CSFR) and for I CI-CSF (M-CSFR), or myeloperoxidase (MPO). These results suggested that DFC simultaneously contained lymphoid-committed progenitors and myeloid-commit ted progenitors, and that cytokines may expand their responding progenitor cells under the influence of signals provided by the stromal cells. Such a stromal cell-dependent culture system may be useful to analyze the switchin g mechanism from constitutive to inducible hematopoiesis in vitro.