Self-renewal and differentiation of a novel bipotent myeloid progenitor clone in the stroma-dependent culture.
To understand regulation of myeloid development, it is necessary to obtain the myeloid progenitor cell lines with self-renewal and differentiation capacities. Because prolonged hematopoiesis occurs with the production of myeloid cells at all stages of differentiation in the Dexter-type long-term bone marrow cultures, we tried to obtain stroma-dependent myeloid progenitor cells starting from the long-term bone marrow culture. 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 monolayer of established spleen stromal cell line, MSS62. After 10-12 passages of DFC cells on MSS62, several clones were obtained by colony formation on MSS62 cell layer. Among these clones, DFC-a cells could be maintained for a long period by coculturing with the established stromal cell line, MSS62.DFC-a cells proliferated by forming cobblestones and contained blast cells, granulocytes, and macrophages. Cell sorting and coculture experiments indicated that the blast type cells exhibiting c-Kit(+) Gr-1(-) Mac-1(-), stroma-dependently self-renewed, and spontaneously differentiated toward granulocytes (c-Kit(+) Gr-1(+) Mac-1(+)) and macrophages (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 GM-CSF or IL-3 but could not be maintained constantly by these cytokines. The present study demonstrated that DFC-a is a novel bipotent myeloid progenitor cell clone as a simple model system of stroma-dependent myeloid development. It may reflect distinct properties for the earliest myeloid progenitor cells in vivo. It is of interest to know what signals are provided by MSS62 stromal cells to maintain the myeloid progenitor cells.
Okubo
T
T
Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
Yanai
N
N
Obinata
M
M
D000818:Animals; D000943:Antigens, Differentiation / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002999:Clone Cells / Q000166:cytology / Q000187:drug effects; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006410:Hematopoiesis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D007377:Interleukin-3 / Q000494:pharmacology; D008747:Methylcellulose; D051379:Mice; D008810:Mice, Inbred C57BL; D013154:Spleen / Q000166:cytology*; D017154:Stromal Cells / Q000502:physiology
D000943:Antigens, Differentiation; D007377:Interleukin-3; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D008747:Methylcellulose