Department of Molecular Cellular and Developmental Biology, Yale University , New Haven, Connecticut, USA.
Stem Cells Dev. 2010 Jul;19(7):999-1015. doi: 10.1089/scd.2009.0416.
One of the challenges in stem cell research is to avoid transformation during cultivation. We studied high passage subventricular zone derived neural stem cells (NSCs) cultures of adult rats in the absence of growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). We termed this culture exogenous growth factor independent neural stem cells (GiNSCs). GiNSCs expressed stemness markers, displayed a high constitutive NF-kappaB activity and an increased, aberrant, polyploid DNA content. GiNSCs showed a tumorigenic phenotype and formed colonies in a soft agar assay. Microarray analysis showed the up-regulation of the NF-kappaB target gene vascular endothelial growth factor (VEGF). In contrast, proneuronal genes were down-regulated. Under neuronal differentiation conditions GiNSCs adopted a glioma-like phenotype, with nuclear p53, preserving high amounts of Nestin positive cells and prolonged proliferation. Neutralization of VEGF strongly inhibited proliferation and induced differentiation. In a gain of function approach, the transfection of NSCs with constitutively active upstream kinase IKK-2 led to constitutively activated NF-kappaB, proliferation in absence of growth factors and augmented VEGF secretion. In a rescue experiment a reduction of NF-kappaB activity by overexpression of IkappaB-AA1 was able to shift the morphology toward an elongated cell form, increased cell death, and decreased proliferation. Thus GiNSCs may provide a potent tool in cancer research, as their exogenous cytokine independent proliferation and their constitutively high NF-kappaB expression presumes cancerous properties observed in gliomas. In addition, this study might add a novel mechanism for detecting oncogenic transformation in therapeutic stem cell cultures.
干细胞研究的挑战之一是避免在培养过程中的转化。我们研究了成年大鼠脑室下区衍生的神经干细胞（NSC）在缺乏生长因子表皮生长因子（EGF）和碱性成纤维细胞生长因子（bFGF）的情况下的高传代培养。我们将这种培养方法称为外源性生长因子独立的神经干细胞（GiNSCs）。GiNSCs 表达干细胞标志物，具有高组成性 NF-κB 活性和增加的、异常的、多倍体 DNA 含量。GiNSCs 表现出致瘤表型，并在软琼脂测定中形成集落。微阵列分析显示 NF-κB 靶基因血管内皮生长因子（VEGF）的上调。相反，神经前体细胞基因下调。在神经元分化条件下，GiNSCs 采用类似于神经胶质瘤的表型，核 p53 保留，Nestin 阳性细胞数量增加，增殖延长。VEGF 的中和作用强烈抑制增殖并诱导分化。在功能获得方法中，用组成型活性上游激酶 IKK-2 转染 NSCs 导致 NF-κB 的组成型激活、在没有生长因子的情况下增殖和增强的 VEGF 分泌。在挽救实验中，通过过表达 IkappaB-AA1 降低 NF-κB 活性能够将形态转向拉长的细胞形态，增加细胞死亡，并减少增殖。因此，GiNSCs 可能为癌症研究提供有力工具，因为它们的外源性细胞因子独立增殖和组成型高 NF-κB 表达假定了在神经胶质瘤中观察到的致癌特性。此外，本研究可能为检测治疗性干细胞培养中的致癌转化添加一种新的机制。
