Lundberg Cecilia, Englund Ulrica, Trono Didier, Björklund Anders, Wictorin Klas
Wallenberg Neuroscience Center, Lund University, S-221 84, Sweden.
Exp Neurol. 2002 Jun;175(2):370-87. doi: 10.1006/exnr.2002.7888.
The rat neural cell line RN33B has a remarkable ability to undergo region-specific neuronal differentiation after transplantation into the CNS. To further study its neurogenic properties in vivo, we used a recombinant lentiviral vector to genetically label the cells with the Green Fluorescent Protein (GFP) gene before implantation into the striatum/cortex, hippocampus, or mesencephalon of newborn rats. Three weeks after implantation, about 1-2% of the GFP-expressing cells had developed morphologies typical of neurons, astrocytes, or oligodendrocytes, the rest remained as either immature or undifferentiated nestin-positive cells. At 15-17 weeks postgrafting, the immature cells had disappeared in most graft recipients and only cells with neuronal or glial morphologies remained in similar numbers as at 3 weeks. The GFP distributed throughout the expressing cells, revealing fine morphological details, including dendrites with spines and extensive axonal projections. In all forebrain regions, the grafted cells differentiated into neurons with morphologies characteristic for each site, including large numbers of pyramidal-like cells in the cortex and the hippocampus, giving rise to dense projections to normal cortical target regions and to the contralateral hippocampus, respectively. In lower numbers, it was also possible to identify GFP-positive granulelike cells in the hippocampus, as well as densely spiny neurons in the striatum. In the mesencephalon by contrast, cells with astrocytic features predominated. The ability of the grafted RN33B cells to undergo region-specific differentiation into highly specialized types of forebrain projection neurons and establish connections with appropriate targets suggests that cues present in the microenvironment of the neonatal rat brain can effectively guide the development of immature progenitors, also in the absence of ongoing neurogenesis.
大鼠神经细胞系RN33B在移植到中枢神经系统后具有显著的区域特异性神经元分化能力。为了进一步研究其在体内的神经发生特性，我们在将细胞植入新生大鼠的纹状体/皮质、海马体或中脑之前，使用重组慢病毒载体用绿色荧光蛋白（GFP）基因对细胞进行基因标记。植入后三周，约1 - 2%表达GFP的细胞已发育出典型的神经元、星形胶质细胞或少突胶质细胞形态，其余细胞仍为未成熟或未分化的巢蛋白阳性细胞。移植后15 - 17周，大多数移植受体中的未成熟细胞消失，仅留下具有神经元或神经胶质形态的细胞，数量与3周时相似。GFP分布于整个表达细胞中，揭示了精细的形态细节，包括有棘的树突和广泛的轴突投射。在所有前脑区域，移植细胞分化为具有各部位特征形态的神经元，包括皮质和海马体中大量的锥体细胞样细胞，分别向正常皮质靶区域和对侧海马体发出密集投射。在海马体中也能鉴定出少量GFP阳性的颗粒样细胞，以及纹状体中多棘神经元。相比之下，在中脑，具有星形胶质细胞特征的细胞占主导。移植的RN33B细胞能够进行区域特异性分化，形成高度特化的前脑投射神经元类型，并与适当的靶标建立连接，这表明新生大鼠脑微环境中的线索能够有效地引导未成熟祖细胞的发育，即使在没有持续神经发生的情况下也是如此。
