Self-renewal and differentiation of a novel bipotent myeloid progenitor clone in the stroma-dependent culture

Objective. To understand regulation of myeloid development, it is necessary to obtain the myeloid progenitor cell lines with self-renewal and differen tiation capacities. Because prolonged hematopoiesis occurs with the product ion of myeloid cells at all stages of differentiation in the Dexter-type lo ng-term bone marrow cultures, we tried to obtain stroma-dependent myeloid p rogenitor cells starting from the long-term bone marrow culture. Materials and Methods. Murine cobblestone areas generated in long-term bone marrow cultures were serially passaged every 10 days. After 4 months, the resultant hematopoietic cells, designated as DFC, were passaged on a monola yer of established spleen stromal cell line, MSS62, After 10-12 passages of DFC cells on MSS62, several clones were obtained by colony formation on MS S62 cell layer. Among these clones, DFC-a cells could be maintained for a l ong period by coculturing with the established stromal cell line, MSS62. Results. DFC-a cells proliferated by forming cobblestones and contained bla st cells, granulocytes, and macrophages. Cell sorting and coculture experim ents indicated that the blast type cells exhibiting c-Kit(+) Gr-1(-) Mac-1( -), stroma-dependently self-renewed, and spontaneously differentiated towar d granulocytes (c-Kit(+) Gr-1(+) Mac-1(+)) and macropahges (c-Kit(low/+) Gr -(1-) Mac-1(high)). Although most of DFC-a cells expressed c-Kit, SCF-c-Kit interaction was not always necessary for their growth. In the presence of stromal cells, growth and differentiation of DFC-a cells were stimulated by GM-CSF or IL-3. Without stromal cells, DFC-a was transiently expanded by G M-CSF or IL-3 but could not be maintained constantly by these cytokines. Conclusion. The present study demonstrated that DFC-a is a novel bipotent m yeloid progenitor cell clone as a simple model system of stroma-dependent m yeloid development. It may reflect distinct properties for the earliest mye loid progenitor cells in vivo. It is of interest to know what signals are p rovided by MSS62 stromal cells to maintain the myeloid progenitor cells. (C ) 2000 International Society for Experimental Hematology. Published by Else vier Science.