Kleppner S R, Robinson K A, Trojanowski J Q, Lee V M
Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical School, Philadelphia 19104, USA.
J Comp Neurol. 1995 Jul 10;357(4):618-32. doi: 10.1002/cne.903570410.
Retinoic acid (RA) induces a human teratocarcinoma cell line (NTera-2 or NT2) to give rise exclusively to post-mitotic neuron-like (NT2N) cells, but NT2N cells never acquire a fully mature neuronal phenotype in vitro. To determine whether NT2N cells can mature into adult neuron-like cells in vivo, purified NT2N cells were grafted into different regions of the central nervous system (CNS) of adult and neonatal athymic mice, and the grafts were examined immunohistochemically by light, confocal, and electron microscopy using antibodies to a panel of developmentally regulated neuronal polypeptides. NT2N grafts were distinguished from endogenous mouse neurons with antibodies that recognize human or murine specific epitopes in selected neuronal polypeptides. Viable NT2N cells were identified in > 89% of graft recipients (N = 90), and some grafts survived 14 months. Within 3 weeks of implantation, grafted NT2N cells re-extended their processes, and the location of the grafts (e.g., septum versus neocortex) appeared to determine the extent to which processes were elaborated. Within the early post-transplantation period, grafted NT2N cells expressed the same neuronal polypeptides as their in vitro counterparts. However, between 6 weeks and 4-6 months post-implantation, the grafted NT2N cells progressively acquired the molecular phenotype of fully mature in vivo neurons as evidenced by dramatically increased expression of the most highly phosphorylated isoforms of the heavy neurofilament subunit, and the de novo expression of adult CNS tau. Notably, the time course for the extension of processes and the expression of neuronal polypeptides by NT2N grafts was similar in neonatal and adult mice. Although grafted NT2N cells formed synapse-like structures and elaborated dendrites and axons, these axons remained unmyelinated. Finally, none of the transplanted NT2N cells reverted to a neoplastic state. These studies demonstrate that pure populations of grafted human NT2N cells acquire a fully mature neuronal phenotype in vivo, and that these cells integrate and survive for > 1 year post-implantation in the mouse CNS. These human neuron-like cells are an attractive model system for studies of neuronal development, polarity and transplantation.
视黄酸（RA）可诱导人畸胎瘤细胞系（NTera - 2或NT2）仅产生有丝分裂后的神经元样（NT2N）细胞，但NT2N细胞在体外从未获得完全成熟的神经元表型。为了确定NT2N细胞在体内是否能成熟为成年神经元样细胞，将纯化的NT2N细胞移植到成年和新生无胸腺小鼠中枢神经系统（CNS）的不同区域，并使用针对一组发育调控神经元多肽的抗体，通过光镜、共聚焦显微镜和电子显微镜对移植组织进行免疫组织化学检查。利用识别选定神经元多肽中人类或小鼠特异性表位的抗体，将NT2N移植组织与内源性小鼠神经元区分开来。在超过89%的移植受体（N = 90）中鉴定出存活的NT2N细胞，一些移植组织存活了14个月。植入后3周内，移植的NT2N细胞重新伸出其突起，移植组织的位置（例如，隔区与新皮质）似乎决定了突起的伸展程度。在移植后的早期，移植的NT2N细胞表达与其体外对应细胞相同的神经元多肽。然而，在植入后6周和4 - 6个月之间，移植的NT2N细胞逐渐获得了体内完全成熟神经元的分子表型，这表现为重神经丝亚基的最高磷酸化异构体的表达显著增加，以及成年中枢神经系统tau蛋白的从头表达。值得注意的是，NT2N移植组织突起伸展的时间进程和神经元多肽的表达在新生小鼠和成年小鼠中相似。虽然移植的NT2N细胞形成了突触样结构，并形成了树突和轴突，但这些轴突仍未髓鞘化。最后，没有一个移植的NT2N细胞恢复到肿瘤状态。这些研究表明，移植的人NT2N细胞纯群体在体内获得了完全成熟的神经元表型，并且这些细胞在小鼠中枢神经系统植入后整合并存活超过1年。这些人神经元样细胞是研究神经元发育、极性和移植的有吸引力的模型系统。
