Xie Xiao-Yan, Xie Chao, Shi Wei, Li Jin, Li Yan-Hua, Wang Dong-Mei, Bai Ci-Xian, Chen Lin, Pei Xue-Tao
Department of Stem Cell Biology, Beijing Institute of Transfusion Medicine, Beijing 100850, China.
Sheng Li Xue Bao. 2004 Jun 25;56(3):306-12.
Ex vivo maintainance of human stem cells is crucial for many clinical applications. Current culture conditions provide some level support but cytokines induce most quiescent stem cells to proliferate and differentiate. Better control of primitive cells is needed to extend the time and range of manipulation of such cells. A recently identified plant lectin Flt3 receptor-interacting lectin (FRIL) present may a special ability to preserve primitive CB progenitors for extended periods in culture without exogenous cytokines. But the mechanisms of FRIL preserving quiescent primitive cells are still unknown. Recently a novel protein HTm4 and its alternatively spliced variant HTm4S, which serve as hematopoietic cell cycle regulators, have been identified. In this report we studied the effect of FRIL on the in vitro maintenance of quiescent human cord blood stem cells and the expression of the novel hematopoietic cell cycle regulator HTm4 and HTm4S in progenitor cells cultured in FRIL. We analyzed the proliferation and the HPP-CFC proportion of CD34(+) cells treated with FRIL. The human HTm4 and HTm4S mRNA expression was detected by semi-quantitative RT-PCR, and the cell cycle status of CB CD34(+) cells was analyzed by FACS. The results showed that incubation of CD34(+) cells in FRIL resulted in a low proliferation of progenitor cells and fewer cycling cells, but FRIL selectively maintained a higher number of primitive cells with proliferative potential in suspension culture. CB CD34(+) cells cultured in FRIL showed significant diversity in the expression of HTm4 and HTm4S during 0~14 d. On d 0, HTm4 was detected at high level, downregulated on d 1, but upregulated during d 3 to d 14, and reaching the highest level on d 7. But the expression levels of HTm4S changed little in the cells cultured in FRIL except the obviously increased expression on d 7. Exogenous expression showed that HTm4 was localized around the karyon while HTm4S scatted in the cytoplasm, respectively, which may be responsible for their difference in function. Thus, FRIL can preserve quiescent primitive CD34(+), and FRIL's ability to preserve quiescent primitive cells in a reversible manner may significantly expand the time and range of ex vivo manipulations of human stem cells for clinical applications. In other words, HTm4 and HTm4S may play a crucial role in the cell cycle modulation of CD34(+) progenitor cells maintained with FRIL in vitro.
人干细胞的体外维持对于许多临床应用至关重要。当前的培养条件提供了一定程度的支持，但细胞因子会诱导大多数静止的干细胞增殖和分化。需要更好地控制原始细胞，以延长此类细胞的操作时间和范围。最近发现的一种植物凝集素Flt3受体相互作用凝集素（FRIL）可能具有一种特殊能力，即在无外源性细胞因子的情况下，能在培养中长时间保存原始脐血祖细胞。但FRIL保存静止原始细胞的机制仍不清楚。最近已鉴定出一种新型蛋白质HTm4及其可变剪接变体HTm4S，它们作为造血细胞周期调节因子。在本报告中，我们研究了FRIL对静止人脐血干细胞体外维持的影响，以及在FRIL培养的祖细胞中新型造血细胞周期调节因子HTm4和HTm4S的表达。我们分析了用FRIL处理的CD34(+)细胞的增殖情况和HPP-CFC比例。通过半定量RT-PCR检测人HTm4和HTm4S mRNA的表达，并通过流式细胞术分析脐血CD34(+)细胞的细胞周期状态。结果表明，将CD34(+)细胞置于FRIL中培养会导致祖细胞增殖率低且处于细胞周期中的细胞较少，但FRIL在悬浮培养中选择性地维持了更多具有增殖潜力的原始细胞。在FRIL中培养的脐血CD34(+)细胞在0至14天期间HTm4和HTm4S的表达表现出显著差异。在第0天，HTm4检测到高水平表达，第1天下调，但在第3天至第14天上调，并在第7天达到最高水平。但在FRIL培养的细胞中，HTm4S的表达水平变化不大，除了在第7天明显增加。外源性表达显示，HTm4分别定位于细胞核周围，而HTm4S散布于细胞质中，这可能是它们功能差异的原因。因此，FRIL可以保存静止的原始CD34(+)细胞，并且FRIL以可逆方式保存静止原始细胞的能力可能会显著扩大人干细胞用于临床应用的体外操作时间和范围。换句话说，HTm4和HTm4S可能在体外FRIL维持的CD34(+)祖细胞的细胞周期调节中起关键作用。
