Phenotypic and functional analyses of CD34NEG hematopoietic precursors from mobilized peripheral blood.

Methods are described for the characterization of CD34 antigen modulation and its relationship to cell proliferation in early human hematopoietic cells. Toward that end, quiescent primitive CD34+ and CD34NEG cells are purified from mobilized peripheral blood (MoPB). Unlike CD34NEG cells from other sources, those from MoPB grow readily in stroma-free culture, facilitating their analysis. Using a lineage-depleted, low-density mononuclear cell fraction, CD34NEGCD38NEGLINNEG and CD34+CD38NEGLINNEG cells are purified by cell sorting. Cells are cultured in serum-free medium supplemented with early acting cytokines. Up- and downmodulation of CD34 antigen can be observed within 40 h of incubation. Samples are removed for analysis of expression of CD34, CD38 and lineage-commitment antigens as well as for cell proliferation as determined by expression of Ki67 antigen and uptake of pyronin Y. This approach permits an assessment of changes in CD34 and CD38 antigen expression by primitive LINNEG cells as they are activated for growth or remain in a quiescent state.