Koller M R, Oxender M, Brott D A, Palsson B O
Aastrom Biosciences, Inc., Ann Arbor, MI 48106, USA.
J Hematother. 1996 Oct;5(5):449-59. doi: 10.1089/scd.1.1996.5.449.
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.
c-kit和flt-3酪氨酸激酶受体在原始造血细胞上表达，且两种受体的配体均已克隆。在本研究中，使用人骨髓单个核细胞（BMMNC）和CD34富集细胞的频繁换液培养，比较了在白细胞介素-3（IL-3）、粒细胞-巨噬细胞集落刺激因子（GM-CSF）和促红细胞生成素（EPO，即3/GM/EPO）存在的情况下，c-kit配体（KL）和flt-3配体（FL）的作用。在单个核细胞培养中，与仅含3/GM/EPO的对照培养相比，KL使细胞产量增加1.7倍（p < 10⁻⁴，n = 13），集落形成单位 - 粒细胞/巨噬细胞（CFU - GM）产量增加2.4倍（p < 10⁻³）。类似地，FL使细胞产量增加1.3倍（p < 10⁻³），CFU - GM产量增加4.4倍（p < 10⁻⁶）。因此，FL对CFU - GM产量的作用比KL更强，但两者均未改变所产生集落的谱系组成（粒细胞、单核细胞、巨噬细胞）。将KL或FL直接添加到集落测定中，仅使CFU - GM生长增加1.2倍，这表明对CFU - GM产量增加的作用发生在祖细胞前期阶段。在CD34富集细胞培养中，KL和FL对CFU - GM产量的作用相似（比对照高9倍）。然而，单个核细胞培养（含有等量的CD34⁺lin⁻细胞）总是比CD34富集细胞的平行培养产生更多的细胞（2倍至4倍）和CFU - GM（3倍至6倍）。在单个核细胞培养中FL（相对于KL）的更大作用可能是由于与内源性产生的生长因子协同作用，而这些生长因子在CD34富集细胞培养中不存在。含FL的培养物（±KL）产生的细胞形成更大的集落，并且这些细胞在重新接种到二级和三级培养物中时具有更大的增殖潜力。此外，与3/GM/EPO培养相比，FL使长期培养起始细胞（LTC - IC）产量增加2.1倍（p < 0.01），CD34⁺lin⁻细胞产量增加6倍（p < 0.05）。相比之下，KL不影响LTC - IC产量，仅轻微增加CD34⁺lin⁻细胞产量（1.4倍）。红细胞数量因KL而增加（2.8倍），因FL而减少（0.6倍），而粒细胞和单核细胞数量因KL（1.4倍）和FL（2.0倍）均增加。当一起使用时，KL和FL在细胞、CFU - GM和LTC - IC产量以及谱系组成方面完全具有加和性。结果表明，对于单个核细胞扩增，FL是比KL更有效的协同生长因子，并且KL和FL以独立、直接、加和的方式起作用。
