Functional,phenotypic and molecular characterization of cytokine low-responding circulating CD34+ haemopoietic progenitors

Circulating CD34⁺ cell populations characterized by a low rate (up to five) or high rate (more than five) of cell divisions were isolated from 8 d cultures in the presence of stem cell factor (SCF), interleukin-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 (Peg-rHuMGDF) using the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and flow cytometric cell sorting. Phenotypic characterization of cells which had experienced up to five divisions (CFDA-SE^bright) showed a similar surface antigen expression to starting, freshly isolated CD34⁺ cells. Conversely, cells which experienced more than five divisions (CFDA-SE^dim) showed a differentiating behaviour, down-regulating CD34 antigen and acquiring differentiation markers. CFDA-SE^bright cells were significantly enriched in CD105 (endoglin) positive precursors as compared to both freshly isolated CD34⁺ and CFDA-SE^dim cells. Functional analysis indicated that CFDA-SE^bright had a 3-fold and 10-fold greater cumulative cloning efficiency as compared to freshly isolated CD34⁺ cells and CFDA-SE^dim cells, respectively. CFDA-SE^bright cells retained the vast majority of LTC-IC and showed a LTC-IC frequency 2.8-fold higher than that found in freshly isolated CD34⁺ cells. RT-PCR and Western blot analyses showed significantly higher bcl-2 RNA and protein levels in CFDA-SE^bright cells as compared to freshly isolated CD34⁺ and CFDA-SE^dim cells. This study indicates that cytokine low-responding circulating CD34⁺ cells (CFDA-SE^bright cells) represent a functionally, phenotypically and molecularly distinct multipotent progenitor population with biological properties associated with primitive precursors.