Experimental Hematology Unit, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.
Exp Hematol. 2010 Apr;38(4):301-310.e2. doi: 10.1016/j.exphem.2010.01.005. Epub 2010 Feb 4.
Recent evidence suggests that hematopoietic stem cells (HSCs) in the bone marrow (BM) are located in areas where the environment is hypoxic. Although previous studies have demonstrated positive effects by hypoxia, its role in HSC maintenance has not been fully elucidated, neither has the molecular mechanisms been delineated. Here, we have investigated the consequence of in vitro incubation of HSCs in hypoxia prior to transplantation and analyzed the role of hypoxia-inducible factor (HIF)-1alpha.
HSC and progenitor populations isolated from mouse BM were cultured in 20% or 1% O(2), and analyzed for effects on cell cycle, expression of cyclin-dependent kinase inhibitors genes, and reconstituting ability to lethally irradiated mice. The involvement of HIF-1alpha was studied using methods of protein stabilization and gene silencing.
When long-term FLT3(-)CD34(-) Lin(-)Sca-1(+)c-Kit(+) (LSK) cells were cultured in hypoxia, cell numbers were significantly reduced in comparison to normoxia. This was due to a decrease in proliferation and more cells accumulating in G(0). Moreover, the proportion of HSCs with long-term engraftment potential was increased. Whereas expression of the cyclin-dependent kinase inhibitor genes p21(cip1), p27(Kip1), and p57(Kip2) increased in LSK cells by hypoxia, only p21(cip1) was upregulated in FLT3(-)CD34(-)LSK cells. We could demonstrate that expression of p27(Kip1) and p57(Kip2) was dependent of HIF-1alpha. Surprisingly, overexpression of constitutively active HIF-1alpha or treatment with the HIF stabilizer agent FG-4497 led to a reduction in HSC reconstituting ability.
Our results imply that hypoxia, in part via HIF-1alpha, maintains HSCs by decreasing proliferation and favoring quiescence.
最近的证据表明，骨髓（BM）中的造血干细胞（HSCs）位于缺氧环境的区域。尽管先前的研究表明缺氧具有积极影响，但它在 HSC 维持中的作用尚未完全阐明，其分子机制也尚未阐明。在这里，我们研究了在移植前将 HSCs 在体外置于低氧环境中孵育的后果，并分析了缺氧诱导因子（HIF）-1α的作用。
从小鼠 BM 中分离出 HSC 和祖细胞群体，在 20%或 1%O2 中培养，并分析对细胞周期、细胞周期蛋白依赖性激酶抑制剂基因表达和重建致死性辐射小鼠的能力的影响。使用蛋白质稳定和基因沉默的方法研究 HIF-1α的参与。
当长期 FLT3(-)CD34(-)Lin(-)Sca-1(+)c-Kit(+)（LSK）细胞在低氧中培养时，与常氧相比，细胞数量明显减少。这是由于增殖减少和更多细胞进入 G0 期。此外，具有长期植入潜力的 HSC 比例增加。尽管低氧诱导 LSK 细胞中细胞周期蛋白依赖性激酶抑制剂基因 p21(cip1)、p27(Kip1)和 p57(Kip2)的表达增加，但仅在 FLT3(-)CD34(-)LSK 细胞中上调 p21(cip1)。我们可以证明 p27(Kip1)和 p57(Kip2)的表达依赖于 HIF-1α。令人惊讶的是，组成性激活的 HIF-1α的过表达或用 HIF 稳定剂 FG-4497 处理导致 HSC 重建能力降低。
我们的结果表明，缺氧部分通过 HIF-1α通过减少增殖和促进静止来维持 HSCs。
