Chung D J, Wong A, Hayashi K, Yellowley C E
Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Vet J. 2014 Jan;199(1):123-30. doi: 10.1016/j.tvjl.2013.10.020. Epub 2013 Oct 26.
Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are good candidates for cell therapy due to the accessibility of fat tissue and the abundance of AT-MSCs therein. Neurospheres are free-floating spherical condensations of cells with neural stem/progenitor cell (NSPC) characteristics that can be derived from AT-MSCs. The aims of this study were to examine the influence of oxygen (O2) tension on generation of neurospheres from canine AT-MSCs (AT-cMSCs) and to develop a hypoxic cell culture system to enhance the survival and therapeutic benefit of generated neurospheres. AT-cMSCs were cultured under varying oxygen tensions (1%, 5% and 21%) in a neurosphere culture system. Neurosphere number and area were evaluated and NSPC markers were quantified using real-time quantitative PCR (qPCR). Effects of oxygen on neurosphere expression of hypoxia inducible factor 1, α subunit (HIF1A) and its target genes, erythropoietin receptor (EPOR), chemokine (C-X-C motif) receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF), were quantified by qPCR. Neural differentiation potential was evaluated in 21% O2 by cell morphology and qPCR. Neurospheres were successfully generated from AT-cMSCs at all O2 tensions. Expression of nestin mRNA (NES) was significantly increased after neurosphere culture and was significantly higher in 1% O2 compared to 5% and 21% O2. Neurospheres cultured in 1% O2 had significantly increased levels of VEGF and EPOR. There was a significant increase in CXCR4 expression in neurospheres generated at all O2 tensions. Neurosphere culture under hypoxia had no negative effect on subsequent neural differentiation. This study suggests that generation of neurospheres under hypoxia could be beneficial when considering these cells for neurological cell therapies.
脂肪组织来源的间充质基质细胞（AT-MSCs）因其脂肪组织易于获取且其中AT-MSCs丰富，是细胞治疗的良好候选者。神经球是具有神经干/祖细胞（NSPC）特征的细胞自由漂浮球形聚集体，可从AT-MSCs中获得。本研究的目的是研究氧（O2）张力对犬AT-MSCs（AT-cMSCs）生成神经球的影响，并开发一种低氧细胞培养系统以提高所生成神经球的存活率和治疗效益。将AT-cMSCs在神经球培养系统中于不同氧张力（1%、5%和21%）下培养。评估神经球数量和面积，并使用实时定量PCR（qPCR）对NSPC标志物进行定量。通过qPCR定量氧对缺氧诱导因子1α亚基（HIF1A）及其靶基因促红细胞生成素受体（EPOR）、趋化因子（C-X-C基序）受体4（CXCR4）和血管内皮生长因子（VEGF）神经球表达的影响。通过细胞形态学和qPCR在21% O2条件下评估神经分化潜能。在所有O2张力下均成功从AT-cMSCs生成神经球。神经球培养后巢蛋白mRNA（NES）表达显著增加，且在1% O2条件下显著高于5%和21% O2条件。在1% O2条件下培养的神经球中VEGF和EPOR水平显著升高。在所有O2张力下生成的神经球中CXCR4表达均显著增加。低氧条件下的神经球培养对随后的神经分化没有负面影响。本研究表明，在考虑将这些细胞用于神经细胞治疗时，低氧条件下生成神经球可能有益。
