Sutherland H J, Lansdorp P M, Henkelman D H, Eaves A C, Eaves C J
Terry Fox Laboratory, Cancer Control Agency, British Columbia, Vancouver, Canada.
Proc Natl Acad Sci U S A. 1990 May;87(9):3584-8. doi: 10.1073/pnas.87.9.3584.
A major goal of current hematopoiesis research is to develop in vitro methods suitable for the measurement and characterization of stem cells with long-term in vivo repopulating potential. Previous studies from several centers have suggested the presence in normal human or murine marrow of a population of very primitive cells that are biologically, physically, and pharmacologically different from cells detectable by short-term colony assays and that can give rise to the latter in long-term cultures (LTCs) containing a competent stromal cell layer. In this report, we show that such cultures can be used to provide a quantitative assay for human "LTC-initiating cells" based on an assessment of the number of clonogenic cells present after 5-8 weeks. Production of derivative clonogenic cells is shown to be absolutely dependent on the presence of a stromal cell feeder. When this requirement is met, the clonogenic cell output (determined by assessment of 5-week-old cultures) is linearly related to the input cell number over a wide range of cell concentrations. Using limiting dilution analysis techniques, we have established the frequency of LTC-initiating cells in normal human marrow to be approximately 1 per 2 X 10(4) cells and in a highly purified CD34-positive subpopulation to be approximately 1 per 50-100 cells. The proliferative capacity exhibited by individual LTC-initiating cells cultured under apparently identical culture conditions was found to be highly variable. Values for the number of clonogenic cells per LTC-initiating cell in 5-week-old cultures ranged from 1 to 30 (the average being 4) with similar levels being detected in positive 8-week-old cultures. Some LTC-initiating cells are multipotent as evidenced by their generation of erythroid as well as granulopoietic progeny. The availability of a system for quantitative analysis of the proliferative and differentiative behavior of this newly defined compartment of primitive human hematopoietic cells should facilitate future studies of specific genetic or microenvironmental parameters involved in the regulation of these cells.
当前造血研究的一个主要目标是开发适合测量和表征具有长期体内重建造血潜能干细胞的体外方法。几个中心先前的研究表明，在正常人或小鼠骨髓中存在一群非常原始的细胞，它们在生物学、物理和药理学特性上与短期集落测定法可检测到的细胞不同，并且在含有合适基质细胞层的长期培养（LTC）中能够产生后者。在本报告中，我们表明这样的培养物可用于基于对5 - 8周后存在的克隆形成细胞数量的评估，为人“LTC启动细胞”提供定量测定。衍生克隆形成细胞的产生绝对依赖于基质细胞饲养层的存在。当满足这一要求时，克隆形成细胞产量（通过评估5周龄培养物确定）在很宽的细胞浓度范围内与输入细胞数量呈线性相关。使用极限稀释分析技术，我们确定正常人骨髓中LTC启动细胞的频率约为每2×10⁴个细胞中有1个，在高度纯化的CD34阳性亚群中约为每50 - 100个细胞中有1个。发现在明显相同培养条件下培养的单个LTC启动细胞所表现出的增殖能力高度可变。在5周龄培养物中，每个LTC启动细胞产生的克隆形成细胞数量值范围为1至30（平均为4），在8周龄阳性培养物中检测到类似水平。一些LTC启动细胞是多能的，这可由它们产生红系以及粒系后代得到证明。一个用于定量分析这种新定义的原始人类造血细胞区室增殖和分化行为的系统的可用性，应有助于未来对参与这些细胞调控的特定遗传或微环境参数的研究。
