| The lineage restriction of prospectively isolated hematopoietic progenitors traditionally has been assessed by bulk in vitro culture and transplantation of large numbers of cells in vivo, but these methods cannot distinguish between homogenous multipotent or heterogeneous restricted populations. While bulk cell assays have yielded useful information about the development of the mature cells of the blood and immune systems from the hematopoietic stem cell (HSC), the development of single cell techniques is needed for the finer resolution of various subpopulations. Using clonal assays from 1 or 5 cells in vitro, single cell quantitative gene expression analyses, and engraftment of mice with low numbers of cells in vivo, we now redefine the common myeloid progenitor (CMP), as cells that are lin-c-Kit+Sca-1 loCD27+Flk-2-CD150lo (SL-CMP; Sca-1lo CMP), and the granulocyte macrophage progenitor (GMP), as cells that are lin-c-Kit+Sca-1loCD27 +Flk-2+CD150-/lo (SL-GMP; Sca-1 lo GMP). We find that mast cell potential is present in the redefined CMP (SL-CMP) fraction but not in the more differentiated SL-GMP population and it is closely related to megakaryocyte/erythrocyte specification. Our data provide new criteria for the prospective isolation of CMP and GMP and support the conclusion that mast cells are specified in hematopoiesis earlier than and independently from granulocytes. Our novel single cell approach for analyzing lineage determination allows for a more refined understanding of hematopoietic development, and by extension hematopoietic malignancies as well as for the dissection of other cell and tissue differentiation pathways. |
