C-KIT IS EXPRESSED BY PRIMITIVE HUMAN HEMATOPOIETIC-CELLS THAT GIVE RISE TO COLONY-FORMING CELLS IN STROMA-DEPENDENT CYTOKINE-SUPPLEMENTED CULTURE

Using monoclonal antibody (MAB) YB5.B8, we have examined the expressio n of the c-kit protein, the receptor for the hematopoietic cytokine st em cell factor (SCF), on primitive hematopoietic cells. Bone marrow mo nonuclear cells (BMMNC) enriched for immature cells by differential ag glutination using the lectin soybean agglutinin (SBA) were subjected t o multiparameter fluorescence activated cell sorting (FACS) based on l ight-scattering properties, the expression of the c-kit protein and th e CD34 antigen, and the retention of the vital fluorescent dye, Rhodam ine 123 (Rh123). Sorted populations were assayed for their content of directly clonogenic progenitor cells (colony-forming units-granulocyte /macrophage [CFU-GM], burst-forming units-erythroid [BFU-E], and multi potential colony-forming units [CFU-Mix]) and for the presence of more primitive progenitor cells (''pre-CFU''). The latter were assayed by (I) their ability to initiate and sustain hematopoiesis in a standard stromal cell-dependent culture system and(2) their capacity for de nov o generation of clonogenic progenitors in response to a combination of six recombinant hematopoietic cytokines in a stroma-independent suspe nsion culture assay. A mean of 76% of CD34(+) cells were found to coex press c-kit. The majority of directly clonogenic cells (98% of CFU-GM, 98% of CFU-Mix, and 85% of BFU-E) were found in the CD34(+)c-kit(+) f raction. Similarly, all pre-CFU were recovered in the CD34(+)c-kit(+)R h123(dull) fraction, irrespective of whether the cells were maintained on marrow stromal cells or in cytokine-supplemented liquid culture. A mean of 87% (range 70-100%) of the CD34(+)Rh123(dull) cells also expr essed c-kit. Since SCF has been reported to act as a growth factor for early lymphoid cells as well as myeloid cells, we looked for coexpres sion of c-kit and early lymphoid markers in the CD34(+) population by multiparameter flow cytometry. Coexpression of c-kit on a minority of cells with markers of B or T lineages was observed. The majority of ea rly lymphoid cells, however, appeared to lack c-kit expression. This w as confirmed by the finding that only 4% of c-kit(+)CD34(+) cells show ed terminal deoxynucleotidyl transferase (TdT) activity, compared with 25% of the c-kit(-)CD34(+) cells.