Phillips André W, Falahati Sina, DeSilva Roshi, Shats Irina, Marx Joel, Arauz Edwin, Kerr Douglas A, Rothstein Jeffrey D, Johnston Michael V, Fatemi Ali
Hugo W. Moser Research Institute at Kennedy Krieger, Johns Hopkins University.
J Vis Exp. 2012 Jun 20(64):3462. doi: 10.3791/3462.
This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.
这是一份从E13期小鼠胎儿脊髓中提取神经胶质限制前体细胞（GRP）的实验方案。这些细胞是少突胶质细胞谱系中的早期前体细胞。最近，这些细胞已被作为白质疾病恢复性治疗的潜在细胞来源进行研究。脑室周围白质软化症（PVL）是儿童非遗传性白质疾病的主要病因，影响高达50%的极早产儿。数据表明，发育中的大脑对缺氧缺血、氧化应激和兴奋性毒性的易感性增加，这些因素会选择性地损害新生白质。神经胶质限制前体细胞（GRP）、少突胶质前体细胞（OPC）和未成熟少突胶质细胞（preOL）似乎是PVL发病机制中的关键因素，也是持续研究的对象。此外，先前的研究已经确定了中枢神经系统组织中的一个亚群，该亚群对谷氨酸兴奋性毒性的易感性增加，并且存在这种易感性的发育模式。我们实验室目前正在研究少突胶质前体细胞在PVL中的作用，并使用处于GRP发育阶段的细胞。我们在几种实验范式中利用这些提取的GRP细胞，以测试它们对与PVL一致的特定应激的反应。GRP细胞可以在体外被诱导分化为OPC和preOL，用于小鼠PVL模型的移植实验以及PVL样损伤（包括缺氧缺血）的体外模型实验。通过使用培养细胞和体外研究，实验之间的变异性会降低，这有助于数据的解读。培养细胞还可以富集GRP群体，同时将非GRP表型污染细胞的影响降至最低。
