Kaneko Y, Sakakibara S, Imai T, Suzuki A, Nakamura Y, Sawamoto K, Ogawa Y, Toyama Y, Miyata T, Okano H
Division of Neuroanatomy, Department of Neuroscience, Biomedical Research Center, Osaka University Graduate School of Medicine, Tokyo, Japan.
Dev Neurosci. 2000;22(1-2):139-53. doi: 10.1159/000017435.
In situ detection of neural progenitor cells including stem-like cells is essential for studying the basic mechanisms of the generation of cellular diversity in the CNS, upon which therapeutic treatments for CNS injuries, degenerative diseases, and brain tumors may be based. We have generated rat monoclonal antibodies (Mab 14H1 and 14B8) that recognize an RNA-binding protein Musashi1, but not a Musashi1-related protein, Musashi2. The amino acid sequences at the epitope sites of these anti-Musashi1 Mabs are remarkably conserved among the human, mouse, and Xenopus proteins. Spatiotemporal patterns of Musashi1 immunoreactivity in the developing and/or adult CNS tissues of frogs, birds, rodents, and humans indicated that our anti-Musashi1 Mabs reacted with undifferentiated, proliferative cells in the CNS of all the vertebrates tested. Double or triple immunostaining of embryonic mouse brain cells in monolayer cultures demonstrated strong Musashi1 expression in Nestin(+)/RC2(+) cells. The relative number of Musashi1(+)/Nestin(+)/RC2(+) cells increased fivefold when embryonic forebrain cells were cultured to form 'neurospheres' in which stem-like cells are known to be enriched through their self-renewing mode of growth. Nestin(+)/RC2(-) cells, which included Talpha1-GFP(+) neuronal progenitor cells and GLAST(+) astroglial precursor cells, were also Musashi1(+), as were GFAP(+) astrocytes. Young neurons showed a trace of Musashi1 expression. Cells committed to the oligodendroglial lineage were Musashi(-). Musashi1 was localized to the perikarya of CNS stem-like cells and non-oligodendroglial progenitor cells without shifting to cell processes or endfeet, and is therefore advantageous for identifying each cell and counting cells in situ.
原位检测包括干细胞样细胞在内的神经祖细胞对于研究中枢神经系统（CNS）细胞多样性产生的基本机制至关重要，而中枢神经系统损伤、退行性疾病和脑肿瘤的治疗可能基于此机制。我们制备了大鼠单克隆抗体（Mab 14H1和14B8），它们识别一种RNA结合蛋白Musashi1，但不识别与Musashi1相关的蛋白Musashi2。这些抗Musashi1单克隆抗体表位位点的氨基酸序列在人、小鼠和非洲爪蟾蛋白中显著保守。青蛙、鸟类、啮齿动物和人类发育中和/或成年中枢神经系统组织中Musashi1免疫反应性的时空模式表明，我们的抗Musashi1单克隆抗体与所有测试脊椎动物中枢神经系统中未分化的增殖细胞发生反应。单层培养的胚胎小鼠脑细胞的双重或三重免疫染色显示，Nestin(+)/RC2(+)细胞中Musashi1表达强烈。当胚胎前脑细胞培养形成“神经球”时，Musashi1(+)/Nestin(+)/RC2(+)细胞的相对数量增加了五倍，已知在“神经球”中干细胞样细胞通过其自我更新生长模式得以富集。包括Talpha1-GFP(+)神经祖细胞和GLAST(+)星形胶质前体细胞的Nestin(+)/RC2(-)细胞以及GFAP(+)星形胶质细胞也为Musashi1(+)。年轻神经元显示出微量的Musashi1表达。向少突胶质细胞谱系分化的细胞为Musashi(-)。Musashi1定位于中枢神经系统干细胞样细胞和非少突胶质祖细胞的胞体，而不转移至细胞突起或终足，因此有利于原位识别每个细胞并对细胞进行计数。
