Quantitative Microplate Assay for Studying Mesenchymal Stromal Cell-Induced Neuropoiesis
Quantitative Microplate Assay for Studying Mesenchymal Stromal Cell-Induced Neuropoiesis
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DOI：
10.5966/sctm.2012-0119
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发表时间：
2013-03-01
影响因子：
6
通讯作者：
Case, Casey C.
中科院分区：
文献类型：
--
作者：
Aizman, Irina;McGrogan, Michael;Case, Casey C.
在神经退行性环境中移植间充质基质细胞（MSC）或其衍生物被认为是有益的，因为它们提供营养支持、迁移指导和神经源性刺激。越来越需要间充质-神经细胞相互作用的体外模型，以鉴定MSC活性的介质和定量评估MSC制剂的神经生成潜能。在这里，我们的特点是一个微孔板格式的共培养系统中，原代胚胎大鼠皮质细胞直接与人骨髓间充质干细胞在细胞衍生的细胞外基质（ECM）在没有外源性生长因子的情况下共培养。在该系统中，大鼠神经干细胞/早期祖细胞标记物巢蛋白以及神经元和星形胶质细胞标记物的表达水平直接依赖于MSC剂量，而少突胶质细胞标记物表现出双相MSC剂量反应，如使用种特异性定量逆转录聚合酶链反应在总细胞裂解物中测量并使用免疫染色确认的。神经细胞的增殖和分化均参与了MSC介导的神经细胞生成。ECM的硫酸乙酰肝素蛋白聚糖是巢蛋白阳性细胞群生长所必需的。中和研究表明，MSC衍生的成纤维细胞生长因子2是一个主要的和可扩散的诱导大鼠巢蛋白，而MSC衍生的骨形态发生蛋白（BMP），特别是BMP 4，是星形细胞的介质，主要作用于共培养设置。该系统能够分析多因素MSC-神经细胞相互作用，并可用于阐明MSC及其衍生物制剂的神经生成潜能。干细胞翻译医学2013;2：223-232
Transplanting mesenchymal stromal cells (MSCs) or their derivatives in a neurodegenerative environment is believed to be beneficial because of the trophic support, migratory guidance, and neurogenic stimuli they provide. There is a growing need for in vitro models of mesenchymal-neural cell interactions to enable identification of mediators of the MSC activity and quantitative assessment of neuropoietic potency of MSC preparations. Here, we characterize a microplate-format coculture system in which primary embryonic rat cortex cells are directly cocultured with human MSCs on cell-derived extracellular matrix (ECM) in the absence of exogenous growth factors. In this system, expression levels of the rat neural stem/early progenitor marker nestin, as well as neuronal and astrocytic markers, directly depended on MSC dose, whereas an oligodendrogenic marker exhibited a biphasic MSC-dose response, as measured using species-specific quantitative reverse transcription-polymerase chain reaction in total cell lysates and confirmed using immunostaining. Both neural cell proliferation and differentiation contributed to the MSC-mediated neuropoiesis. ECM's heparan sulfate proteoglycans were essential for the growth of the nestin-positive cell population. Neutralization studies showed that MSC-derived fibroblast growth factor 2 was a major and diffusible inducer of rat nestin, whereas MSC-derived bone morphogenetic proteins (BMPs), particularly, BMP4, were astrogenesis mediators, predominantly acting in a coculture setting. This system enables analysis of multifactorial MSC-neural cell interactions and can be used for elucidating the neuropoietic potency of MSCs and their derivative preparations. STEM CELLS TRANSLATIONAL MEDICINE 2013;2:223-232