Kawata M, Sekiya S, Kera K, Kimura H, Takamizawa H
Department of Obstetrics and Gynecology, School of Medicine, Chiba University, Japan.
Cancer Res. 1991 May 15;51(10):2655-69.
From the human teratocarcinoma-derived cell line PA-1, we established a clonal line, PA-1/NR, that stably produced a distinct cellular arrangement of neural rosettes when cultured as in vitro multicellular spheroids for 3 weeks. On immunofluorescence staining and fluorescence-activated cell sorter analyses, PA-1/NR cells in monolayer expressed the neuroectoderm-associated antigens HNK-1, NC-1, and A2B5 and the neuroblastoma-associated antigens KP-NAC8 and KP-NAC10 but lacked human embryonal carcinoma antigens, SSEA-3 or K21 antigen. Here, we investigated the developmental process of rosette formation with respect to morphological features, distribution of mitotic cells, and expression of multiple lineage-related markers and extracellular matrix (ECM) components. Ultrastructural examination of these rosettes disclosed a well-defined cavity radially surrounded by wedge-shaped or pseudostratified cells, apical microvilli and junctional complexes, and basal laminae and collagen fibrils at their basal surface. In these rosettes, many proliferating cells were detected by the immunohistochemical staining of cells incorporating bromodeoxyuridine. PA-1/NR spheroids consistently displayed neuron-specific enolase, S-100 protein, and vimentin but not glial fibrillary acidic protein, neurofilament proteins, or myelin basic protein. The rosette formation accompanied a strikingly polarized and overlapped deposition of ECM components including tenascin-carrying HNK-1 epitopes, laminin, type IV collagen, heparan, and chondroitin sulfate proteoglycans. Immunoblotting analyses showed that laminin B1 and B2 chains were constitutively expressed, whereas a fully assembled form of laminin and type IV collagen appeared only after spheroid development, suggesting that these ECM components play a morphogenetically important role in rosette formation. Close similarities between these rosettes and the neural tube of humans and experimental animals in the morphogenetic process and ECM formation lead us to propose that the PA-1/NR spheroids provide an in vitro model for the study of the earliest stage of human neurogenesis.
从人畸胎癌衍生细胞系PA-1中，我们建立了一个克隆系PA-1/NR，当将其作为体外多细胞球体培养3周时，该克隆系能稳定产生独特的神经玫瑰花结细胞排列。在免疫荧光染色和荧光激活细胞分选分析中，单层培养的PA-1/NR细胞表达神经外胚层相关抗原HNK-1、NC-1和A2B5，以及神经母细胞瘤相关抗原KP-NAC8和KP-NAC10，但缺乏人胚胎癌抗原SSEA-3或K21抗原。在此，我们从形态特征、有丝分裂细胞分布以及多种谱系相关标志物和细胞外基质（ECM）成分的表达方面，研究了玫瑰花结形成的发育过程。对这些玫瑰花结的超微结构检查发现，有一个明确界定的腔，其周围呈放射状排列着楔形或假复层细胞、顶端微绒毛和连接复合体，以及基底表面的基膜和胶原纤维。在这些玫瑰花结中，通过掺入溴脱氧尿苷的细胞免疫组织化学染色检测到许多增殖细胞。PA-1/NR球体始终显示神经元特异性烯醇化酶、S-100蛋白和波形蛋白，但不显示胶质纤维酸性蛋白、神经丝蛋白或髓鞘碱性蛋白。玫瑰花结的形成伴随着ECM成分的显著极化和重叠沉积，这些成分包括携带HNK-1表位的腱生蛋白、层粘连蛋白、IV型胶原、硫酸乙酰肝素和硫酸软骨素蛋白聚糖。免疫印迹分析表明，层粘连蛋白B1和B2链组成性表达，而层粘连蛋白和IV型胶原的完全组装形式仅在球体发育后出现，这表明这些ECM成分在玫瑰花结形成中发挥着形态发生上的重要作用。这些玫瑰花结与人类和实验动物神经管在形态发生过程和ECM形成方面的密切相似性，使我们提出PA-1/NR球体为研究人类神经发生的最早阶段提供了一个体外模型。
