Pleasure S J, Lee V M
Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia.
J Neurosci Res. 1993 Aug 15;35(6):585-602. doi: 10.1002/jnr.490350603.
We have identified a human cell line with a phenotype resembling committed CNS neuronal precursor cells. NTera 2/cl.D1 (NT2/D1) cells expressed nestin and vimentin, intermediate filament (IF) proteins expressed in neuroepithelial precursor cells, as well as MAP1b, a microtubule-associated protein (MAP) expressed in human neuroepithelium. NT2/D1 cells also expressed the cell adhesion molecules NCAM and N-cadherin which are thought to be important in cell-cell interactions within the neuroepithelium. These NT2/D1 cells also expressed small amounts of NF-L, alpha-internexin, NF-M, and MAP2c, indicating that they are committed to a neuronal fate. Previous studies have shown that, following RA treatment, a proportion of NT2/D1 cells terminally differentiate into neurons and that this occurs via an asymmetric stem cell mode of differentiation. In light of the identification of the neuroepithelial phenotype of NT2/D1 cells we decided to examine more closely the relationship of in vitro neurogenesis in NT2/D1 cells, during RA treatment to that of neurons in vivo. Three days after RA treatment, islands of NT2/D1 cells showed increased expression of neurofilament proteins and increased phosphorylation of NF-M. By 10-14 days, these cells began to resemble neurons morphologically, i.e., with rounded cell bodies and processes. These neuronal cells were clustered into clumps which rested on top of a layer of progenitor cells. In this upper layer, the neurons began to express MAP2b and tau and extinguished their expression of nestin. Recently, we developed a method for obtaining pure cultures of neurons from RA treated NT2/D1 cells. The phenotype of these postmitotic neurons is clearly dissociated from that of the untreated NT2/D1 cells. Given the data obtained in this study and the characterization of the neurons derived from NT2/D1 cells, we propose that NT2/D1 cells are a committed human neuronal precursor cell line which retains some stem cell characteristics and is capable only of terminal differentiation into neurons.
我们已鉴定出一种人类细胞系，其表型类似于定向中枢神经系统神经元前体细胞。NTera 2/cl.D1（NT2/D1）细胞表达巢蛋白和波形蛋白，这是神经上皮前体细胞中表达的中间丝（IF）蛋白，以及MAP1b，一种在人类神经上皮中表达的微管相关蛋白（MAP）。NT2/D1细胞还表达细胞粘附分子NCAM和N-钙粘蛋白，它们被认为在神经上皮内的细胞间相互作用中很重要。这些NT2/D1细胞也表达少量的NF-L、α-中间丝蛋白、NF-M和MAP2c，表明它们已定向为神经元命运。先前的研究表明，视黄酸（RA）处理后，一部分NT2/D1细胞会终末分化为神经元，且这一过程通过不对称干细胞分化模式发生。鉴于NT2/D1细胞神经上皮表型的鉴定结果，我们决定更深入地研究NT2/D1细胞在RA处理期间的体外神经发生与体内神经元神经发生之间的关系。RA处理三天后，NT2/D1细胞岛显示神经丝蛋白表达增加以及NF-M磷酸化增加。到10 - 14天时，这些细胞在形态上开始类似于神经元，即具有圆形细胞体和突起。这些神经元细胞聚集成团，位于一层祖细胞之上。在这上层中，神经元开始表达MAP2b和tau，并停止表达巢蛋白。最近，我们开发了一种从经RA处理的NT2/D1细胞中获得纯神经元培养物的方法。这些有丝分裂后神经元的表型与未处理的NT2/D1细胞明显不同。根据本研究获得的数据以及源自NT2/D1细胞的神经元的特征，我们提出NT2/D1细胞是一种定向人类神经元前体细胞系，它保留了一些干细胞特征，并且仅能终末分化为神经元。
