FUNCTIONAL, PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF CYTOKINE LOW-RESPONDING CIRCULATING CD34(+) HEMATOPOIETIC PROGENITORS

Circulating CD34(+) cell populations characterized by a low rate (up t o live) or high rate (more than five) of cell divisions were isolated from 8d cultures in the presence of stem cell factor (SCF), interleuki n-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), Flt3 ligand and Peg-rHu megakaryocyte growth and development factor (P eg-rHuMGDF) using the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and now cytometric cell sorting, Phenoty pic characterization of cells which had experienced up to five divisio ns (CFDA-SEbright) showed a similar surface antigen expression to star ting, freshly isolated CD34(+) cells, Conversely, cells which experien ced more than five divisions (CFDA-SEdim) showed a differentiating beh aviour, downregulating CD34 antigen and acquiring differentiation mark ers. CFDA-SEbright cells were significantly enriched in CD105 (endogli n) positive precursors as compared to both freshly isolated CD34(+) an d CFDA-SEdim cells, Functional analysis indicated that CFDA-SEbright h ad a 3-fold and 10-fold greater cumulative cloning efficiency as compa red to freshly isolated CD34(+) cells and CFDA-SEdim cells, respective ly. CFDA-SEbright cells retained the vast majority of LTC-IC and showe d a LTC-IC frequency 2.8-fold higher than that found in freshly isolat ed CD34(+) cells. RT-PCR and Western blot analyses showed significantl y higher bcl-2 RNA and protein levels in CFDA-SEbright cells as compar ed to freshly isolated CD34(+) and CFDA-SEdim cells. This study indica tes that cytokine low-responding circulating CD34(+) cells (CFDA-SEbri ght cells) represent a functionally, phenotypically and molecularly di stinct multipotent progenitor population with biological properties as sociated with primitive precursors.