Udomsakdi C, Eaves C J, Swolin B, Reid D S, Barnett M J, Eaves A C
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada.
Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6192-6. doi: 10.1073/pnas.89.13.6192.
In this report we describe a quantitative in vitro assay for the most primitive type of leukemic precursors yet defined in patients with chronic myeloid leukemia (CML). This assay is based on the recently described "long-term culture-initiating cell" (LTC-IC) assay for primitive normal human hematopoietic cells. Such cells, when cocultured with competent fibroblast feeder layers, give rise after a minimum of 5 weeks to multiple single and multilineage clonogenic progenitors detectable in secondary semisolid assay cultures. Similar cultures initiated by seeding a highly enriched source of leukemic cells from patients onto normal feeders showed the clonogenic cell output after 5 weeks to be linearly related to the input innoculum over a wide range down to limiting numbers of input cells, thus allowing absolute frequencies of leukemic LTC-ICs to be determined using standard limiting dilution analysis techniques. Leukemic LTC-IC concentrations in CML marrow were found to be decreased, on average to less than 10% of the normal LTC-IC concentration in normal marrow, but were greatly increased (up to greater than 10(5) times) in CML blood. Assessment of the number of clonogenic cells produced per leukemic LTC-IC by comparison to normal blood or marrow LTC-IC values showed this function to be unchanged in leukemic LTC-ICs [i.e., 3.1 +/- 0.4 clonogenic cells per CML LTC-IC (mean +/- SEM, n = 6) versus 3.7 +/- 1.2 (n = 3) and 4.3 +/- 0.4 (n = 5), respectively, for normal blood and marrow LTC-ICs]. In contrast, leukemic LTC-IC maintenance in LTC proved to be highly defective by comparison to normal LTC-IC of either blood or marrow origin. Thus, when cells from primary LTC were subcultured into secondary LTC-IC assays, leukemic LTC-IC rapidly declined (greater than 30-fold) within the first 10 days of culture, whereas normal LTC-IC numbers remained unchanged during this period. These findings illustrate how self-maintenance and differentiation events in primitive human hematopoietic cells can be differentially modulated by an oncogenic process and provide a framework for further studies of their manipulation, analysis, and therapeutic exploitation.
在本报告中，我们描述了一种针对慢性粒细胞白血病（CML）患者中迄今所定义的最原始类型白血病前体细胞的定量体外检测方法。该检测方法基于最近描述的用于原始正常人造血细胞的“长期培养起始细胞”（LTC - IC）检测方法。这类细胞与有能力的成纤维细胞饲养层共培养时，至少5周后会在二次半固体检测培养物中产生多个单谱系和多谱系克隆形成祖细胞。通过将患者高度富集的白血病细胞接种到正常饲养层上启动类似培养，结果显示5周后的克隆形成细胞产量在很宽的范围内，直至输入细胞的极限数量，都与输入接种物呈线性相关，从而可以使用标准的极限稀释分析技术确定白血病LTC - IC的绝对频率。发现CML骨髓中的白血病LTC - IC浓度平均降低至正常骨髓中正常LTC - IC浓度的不到10%，但在CML血液中则大幅增加（高达超过10⁵倍）。通过与正常血液或骨髓LTC - IC值比较，评估每个白血病LTC - IC产生的克隆形成细胞数量，结果显示白血病LTC - IC的这一功能未发生改变[即，每个CML LTC - IC产生3.1±0.4个克隆形成细胞（平均值±标准误，n = 6），而正常血液和骨髓LTC - IC分别为3.7±1.2（n = 3）和4.3±0.4（n = 5）]。相比之下，与血液或骨髓来源的正常LTC - IC相比，白血病LTC - IC在长期培养（LTC）中的维持能力被证明存在高度缺陷。因此，当将原代LTC中的细胞传代培养到二次LTC - IC检测中时，白血病LTC - IC在培养的前10天内迅速下降（超过30倍），而在此期间正常LTC - IC数量保持不变。这些发现说明了原始人类造血细胞中的自我维持和分化事件如何受到致癌过程的不同调节，并为进一步研究其操作、分析和治疗利用提供了框架。
