High-resolution video monitoring of hematopoietic stem cells cultured in single-cell arrays identifies new features of self-renewal
High-resolution video monitoring of hematopoietic stem cells cultured in single-cell arrays identifies new features of self-renewal
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DOI：
10.1073/pnas.0602548103
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发表时间：
2006-05-23
影响因子：
11.1
通讯作者：
Jervis, Eric
中科院分区：
文献类型：
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作者：
Dykstra, Brad;Ramunas, John;Jervis, Eric
为了寻找自我更新造血干细胞（HSC）的新指标，从成年小鼠骨髓中分离高度纯化的群体，显微操作到专门设计的显微阵列中，并在300 ng/ml钢因子、20 ng/ml IL-11和1 ng/ml flt 3-配体中培养4天。在此期间，每个细胞及其后代通过使用数字延时摄影以3分钟的间隔成像。然后收获单个克隆，并通过使用4个月的多谱系再增殖终点（对淋巴和骨髓谱系的贡献> 1%）测定小鼠中的HSC。在第一个实验中，14个初始细胞中的6个（43%）和61个克隆中的17个（28%）具有HSC活性，表明HSC自我更新分裂已经在体外发生。与HSC活性相关的特征包括较长的细胞周期时间和在培养的最后12小时期间克隆内的大多数细胞上没有尾足。结合这些标准，最大限度地区分克隆与HSC活性，从那些没有，并确定了一个子集的61个克隆的27。这27个克隆包括具有HSC活性的所有17个克隆;检测效率为63%（比原始组的频率高2.26倍）。在两个独立的实验中证实了这些特征用于区分含HSC克隆的效用，其中所有含HSC克隆以相似的2至3倍更高的效率被鉴定。这些研究说明了这种监测系统的潜力，以检测增殖的HSC的新功能，是预测自我更新的部门。
To search for new indicators of self-renewing hematopoietic stem cells (HSCs), highly purified populations were isolated from adult mouse marrow, micromanipulated into a specially designed microscopic array, and cultured for 4 days in 300 ng/ml Steel factor, 20 ng/ml IL-11, and 1 ng/ml flt3-ligand. During this period, each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (> 1% contribution to lymphoid and myeloid lineages). In a first experiment, 6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity, demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions.