flt-3Ligand Is More Potent Thanc-kitLigand for the Synergistic Stimulation of Ex Vivo Hematopoietic Cell Expansion

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Abstract
The c-kit and flt-3 tyrosine kinase receptors are expressed on primitive hematopoietic cells, and ligands for both receptors have been cloned. In this study, the effects of c-kit ligand (KL) and flt-3 ligand (FL) were compared in the presence of IL-3, GM-CSF, and erythropoietin (3/GM/EPO), using frequent medium exchange cultures of human bone marrow mononuclear cells (BMMNC) and CD34-enriched cells. In MNC cultures, KL increased cell output by 1.7-fold (p < 10−4, n = 13) and CFU-GM output by 2.4-fold (p < 10−3) as compared with control cultures containing only 3/GM/EPO. Analogously, FL increased cell output by 1.3-fold (p < 10−3) and CFU-GM output by 4.4-fold (p < 10−6). Therefore, FL was more potent on CFU-GM output than KL, but neither altered the lineage composition (granulocyte, monocyte, macrophage) of the colonies produced. Direct addition of KL or FL to colony assays resulted in only a 1.2-fold increase in CFU-GM outgrowth, suggesting that the effects on increased CFU-GM output were at the preprogenitor stage. In CD34-enriched cell cultures, the effects of KL and FL on CFU-GM output were similar (9-fold above control). Nevertheless, MNC cultures (containing an equivalent number of CD34+lin−cells) always generated more cells (2-fold to 4-fold) and CFU-GM (3-fold to 6-fold) than did parallel cultures of CD34-enriched cells. The greater effect of FL (over KL) in MNC cultures was probably due to synergy with endogenously produced growth factors that were absent in CD34-enriched cell cultures. FL-containing cultures (±KL) generated cells that formed larger colonies, and these cells had more proliferative potential on replating into secondary and tertiary cultures. Furthermore, FL increased the output of LTC-IC by 2.1-fold (p < 0.01) and CD34+lin− cells by 6-fold (p < 0.05) as compared with 3/GM/EPO cultures. In contrast, KL did not affect the output of LTC-IC and only slightly increased CD34+lin− cell output (by 1.4-fold). Erythrocytes were increased by KL (2.8-fold) and decreased by FL (0.6-fold), whereas granulocytes and monocytes were increased by both KL (1.4-fold) and FL (2.0-fold). When used together, KL and FL were completely additive with respect to cell, CFU-GM, and LTC-IC output, as well as lineage composition. The results indicate that FL is a more potent synergistic growth factor than KL for MNC expansion and that KL and FL act in an independent, direct, additive manner.