Molecular Endocrinology, MKIII, University Clinic Dresden, Fiedlerstr. 42, 01307 Dresden, Germany.
Mech Ageing Dev. 2010 Feb;131(2):124-32. doi: 10.1016/j.mad.2010.01.001. Epub 2010 Jan 13.
The potential use of neural stem cells in basic research, drug testing and for development of therapeutic strategies requires large scale in vitro amplification, increasing the probability of genetic instability and transformation. Little is known, however, about potential correlations between long-term culture of neural stem and progenitor cells (NSPCs), changed differentiation and self-renewal capacities, and the occurrence of chromosomal instability. This study investigates the effect of extended culture time on self-renewal, differentiation capacity, cell cycle phase distribution, telomere length, telomerase activity and chromosomal stability on fetal brain-derived cells that form floating sphere colonies (neurospheres). We observed that increased sphere-forming capacity indicative of increased proliferation was accompanied by a decreased ability to differentiate into neural lineages. The high mobility group A (Hmga2) gene positively regulates self-renewal via repression of p16(Ink4a) and p19(ARF) gene expression. This study discerned an upregulation of Hmga2 gene and protein expression and decreased p16(Ink4a) and p19(ARF) gene expression, suggesting that Hmga2 might promote the proliferation of neurosphere cells in long-term culture. Further, our analyses revealed a significant decrease in telomere length after 4 weeks of culturing that is paralleled by a moderate upregulation of telomerase activity. Importantly, regular gain of chromosome 1 with random structural chromosomal aberrations was observed within 16 weeks of neurosphere cell culture. Genetic instability and diminished differentiation capacity seem to be a consequence of long-term culture of neurosphere cells. These data indicate the necessity to analyze self-renewal, differentiation capacity, telomere length, tumor suppressor genes and chromosomal stability in neurosphere cultures prior to their usage in basic research, drug testing or the development of therapeutic strategies.
神经干细胞在基础研究、药物测试和治疗策略开发中的潜在应用需要大规模的体外扩增，这增加了遗传不稳定性和转化的可能性。然而，关于神经干细胞和祖细胞（NSPC）的长期培养、分化和自我更新能力的变化，以及染色体不稳定性的发生之间的潜在相关性，人们知之甚少。本研究调查了延长培养时间对自我更新、分化能力、细胞周期相分布、端粒长度、端粒酶活性和染色体稳定性的影响，这些影响是胎儿脑源性细胞形成悬浮球集落（神经球）时发生的。我们观察到，增殖能力增加（指示增殖增加）伴随着向神经谱系分化的能力降低。高迁移率族蛋白 A（Hmga2）基因通过抑制 p16（Ink4a）和 p19（ARF）基因的表达，正向调节自我更新。本研究发现 Hmga2 基因和蛋白表达上调，p16（Ink4a）和 p19（ARF）基因表达下调，提示 Hmga2 可能促进神经球细胞在长期培养中的增殖。此外，我们的分析显示，培养 4 周后端粒长度显著缩短，同时端粒酶活性中度上调。重要的是，在神经球细胞培养 16 周内，经常观察到 1 号染色体的获得，伴有随机的结构染色体异常。遗传不稳定性和分化能力下降似乎是神经球细胞长期培养的结果。这些数据表明，在将神经球细胞用于基础研究、药物测试或治疗策略开发之前，有必要分析神经球培养物中的自我更新、分化能力、端粒长度、肿瘤抑制基因和染色体稳定性。
