Gothot A, Pyatt R, McMahel J, Rice S, Srour E F
Indiana Elks Cancer Research Center, Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-5121, USA.
Exp Hematol. 1998 Jul;26(7):562-70.
Exit of primitive hematopoietic progenitor cells (HPCs) from the G0 phase of the cell cycle in response to in vitro cytokine stimulation is a limiting step in successful ex vivo expansion. Simultaneous DNA/RNA staining with Hoechst 33342 and pyronin Y was used to separate human bone marrow CD34+ cells residing in G0 (G0CD34+) from those cycling in G1 and S/G2+M. Compared with CD34+ cells isolated in G1, G0CD34+ cells were characterized by a delayed response to cytokine stimulation and were enriched for long-term hematopoietic culture-initiating cells. We next compared the activation kinetics of individually sorted G0CD34+ cells stimulated with stem cell factor (SCF), flt3-ligand (FL), or interleukin-3 (IL-3) as single factors. In a novel clonal proliferation assay, the functional status of cells that had remained quiescent after an initial 7-day period and of those that had completed successive division cycles under each of these three factors was evaluated by assessment of subsequent proliferative capacity and maintenance of colony-forming cell precursor (pre-CFC) activity. All three cytokines were equally able to support the survival of primitive HPCs in the absence of cell division. Cells that did not respond to any cytokine stimulation for 7 days retained higher proliferative and pre-CFC activities than dividing cells. The hematopoietic function of cells that divided in response to SCF, FL, or IL-3 decreased after each division cycle. However, G0CD34+ cells displayed a heterogeneous response pattern to cytokine stimulation whereby SCF appeared to have a superior ability to promote the cycling of cells with high proliferative and pre-CFC activities. These results indicate that HPCs reside in opposing hierarchies of hematopoietic potential and responsiveness to cytokine stimulation. The data also begin to indicate relationships between cellular division in response to different stimuli and maintenance of hematopoietic function.
原始造血祖细胞（HPCs）在体外细胞因子刺激下从细胞周期的G0期退出是成功进行体外扩增的一个限制步骤。使用Hoechst 33342和派洛宁Y同时进行DNA/RNA染色，以将处于G0期的人骨髓CD34+细胞（G0CD34+）与处于G1期和S/G2+M期循环的细胞分离。与在G1期分离的CD34+细胞相比，G0CD34+细胞的特征是对细胞因子刺激的反应延迟，并且富含长期造血培养起始细胞。接下来，我们比较了用干细胞因子（SCF）、fms样酪氨酸激酶3配体（FL）或白细胞介素-3（IL-3）作为单一因子刺激的单独分选的G0CD34+细胞的激活动力学。在一项新的克隆增殖试验中，通过评估随后的增殖能力和集落形成细胞前体（pre-CFC）活性，对在最初7天保持静止的细胞以及在这三种因子中的每一种作用下完成连续分裂周期的细胞的功能状态进行了评估。在没有细胞分裂的情况下，所有三种细胞因子同样能够支持原始HPCs的存活。在7天内对任何细胞因子刺激都没有反应的细胞比分裂细胞保留了更高的增殖和pre-CFC活性。对SCF、FL或IL-3作出反应而分裂的细胞的造血功能在每个分裂周期后都会下降。然而，G0CD34+细胞对细胞因子刺激表现出异质性反应模式，由此SCF似乎具有更强的促进具有高增殖和pre-CFC活性的细胞循环的能力。这些结果表明，HPCs存在于造血潜能和对细胞因子刺激的反应性的相反层次结构中。数据还开始表明对不同刺激的细胞分裂与造血功能维持之间的关系。
