Udomsakdi C, Lansdorp P M, Hogge D E, Reid D S, Eaves A C, Eaves C J
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada.
Blood. 1992 Nov 15;80(10):2513-21.
The total number of clonogenic cells present in 5-week-old long-term cultures (LTC) initiated by seeding normal human marrow cells on competent adherent cell feeder layers allows for the quantitation of a more primitive hematopoietic input precursor cell type referred to as an LTC-initiating cell (LTC-IC). Previous studies have suggested that LTC-IC also circulate because production of clonogenic cells continues for many weeks when cells from the light-density (< 1.077 g/mL), T-cell-depleted fraction of normal blood are maintained on irradiated, marrow-derived feeder layers in LTC medium. We now show that the number of clonogenic cells present in such reconstructed LTC after 5 weeks is linearly related to the input number of peripheral blood (PB) cells over a wide range of cell concentrations, thereby permitting the quantitation of circulating LTC-IC by limiting dilution analysis. Using this approach, we have found the concentration of LTC-IC in the circulation of normal adults to be 2.9 +/- 0.5/mL. This is approximately 75-fold lower than the concentration of circulating clonogenic cells (ie, burst-forming units-erythroid plus colony-forming units [CFU] granulocyte-macrophage plus CFU-granulocyte, erythroid, monocyte, megakaryocyte) and represents a frequency of LTC-IC relative to all nucleated cells that is approximately 100-fold lower than that measured in normal marrow aspirate samples. Characterization studies showed most circulating LTC-IC to be small (low forward light scatter and side scatter), CD34+, Rh-123dull, HLA-DR-, and 4-hydroperoxycyclophosphamide-resistant cells, with differentiative and proliferative potentialities indistinguishable from LTC-IC in normal marrow. Isolation of the light-density, T-cell-depleted, CD34+, and either HLA-DR(low) or Rh-123(dull) fraction of normal blood yielded a highly enriched population of cells that were 0.5% to 1% LTC-IC (approximately 1,500-fold enriched beyond the light-density, T-cell-depletion step), a purity comparable to the most enriched populations of human marrow LTC-IC reported to date. However, purification of PB LTC-IC on the basis of these properties did not allow them to be physically separated from a substantial proportion (> 30%) of the clonogenic cells in the same samples, in contrast to previous findings for LTC-IC and clonogenic cells in marrow. These studies show the presence in the blood of normal adults of a relatively small but readily detectable population of functionally defined, primitive hematopoietic cells that share properties with marrow LTC-IC, a cell type thought to have in vivo reconstituting potential.(ABSTRACT TRUNCATED AT 400 WORDS)
通过将正常人骨髓细胞接种到有能力的贴壁细胞饲养层上启动的5周龄长期培养物（LTC）中存在的克隆形成细胞总数，使得对一种更原始的造血输入前体细胞类型（称为LTC起始细胞，LTC-IC）进行定量成为可能。先前的研究表明LTC-IC也在循环，因为当来自正常血液的低密度（<1.077 g/mL）、T细胞耗竭部分的细胞在LTC培养基中的辐照骨髓来源饲养层上维持时，克隆形成细胞的产生会持续数周。我们现在表明，在这样重建的LTC中5周后存在的克隆形成细胞数量在很宽的细胞浓度范围内与外周血（PB）细胞的输入数量呈线性相关，从而允许通过极限稀释分析对外周血LTC-IC进行定量。使用这种方法，我们发现正常成年人循环中LTC-IC的浓度为2.9±0.5/mL。这比循环克隆形成细胞（即红细胞爆式形成单位加上集落形成单位[CFU]粒细胞 - 巨噬细胞加上CFU - 粒细胞、红细胞、单核细胞、巨核细胞）的浓度低约75倍，并且相对于所有有核细胞，LTC-IC的频率比在正常骨髓抽吸样本中测得的低约100倍。特性研究表明，大多数循环LTC-IC体积小（前向光散射和侧向散射低）、CD34+、Rh-123暗淡、HLA-DR-且对4-氢过氧环磷酰胺耐药，其分化和增殖潜力与正常骨髓中的LTC-IC无法区分。分离正常血液的低密度、T细胞耗竭、CD34+以及HLA-DR（低）或Rh-123（暗淡）部分，得到了一个高度富集的细胞群体，其中LTC-IC占0.5%至1%（比低密度、T细胞耗竭步骤富集了约1500倍），纯度与迄今为止报道的最富集的人骨髓LTC-IC群体相当。然而，基于这些特性对PB LTC-IC进行纯化并不能使它们与同一样本中相当一部分（>30%）的克隆形成细胞物理分离，这与先前关于骨髓中LTC-IC和克隆形成细胞的研究结果相反。这些研究表明，正常成年人血液中存在相对少量但易于检测的功能明确的原始造血细胞群体，它们与骨髓LTC-IC具有共同特性，LTC-IC这种细胞类型被认为具有体内重建潜力。（摘要截短至400字）
