Watanabe T, Voyvodic J T, Chan-Ling T, Sagara H, Hirosawa K, Mio Y, Matsushima S, Uchimura H, Nakahara K, Raff M C
Department of Clinical Pathology, Kyorin University School of Medicine, Tokyo, Japan.
J Comp Neurol. 1997 Jan 20;377(3):341-50. doi: 10.1002/(sici)1096-9861(19970120)377:3<341::aid-cne3>3.0.co;2-2.
We previously developed a reaggregate cell culture system (pellet cultures) in which retinal neuroepithelial cells proliferate and give rise to rod photoreceptor cells (rods) in vitro (Watanabe and Raff, 1990, Neuron 4:461-467). In the present study, we analyzed cell differentiation and morphogenesis in pellet cultures by using both cell-type-specific markers with immunofluorescence and electron microscopy. We demonstrated that, in addition to rods, the other major retinal cell types, including amacrine cells, bipolar cells, Müller cells, and ganglion cells were all present in the pellets, where most were able to develop from dividing precursor cells in vitro. The different cell types in the pellets became organized into two distinct structures: dark rosettes and pale rosettes. The cellular composition of these structures indicated that the dark rosettes correspond to the outer nuclear layer and the pale rosettes to the inner nuclear layer of the normal retina. Ultrastructural studies have indicated that the thin layer of neuronal processes surrounding the dark rosettes correspond to the outer plexiform layer, and the central region of the pale rosettes correspond to the inner plexiform layer of the normal retina. Other features of normal retinal development also occurred in the pellets, including programmed cell death and the formation of inner and outer rod cell segments and synapses. Thus, pellet cultures provide a convenient way to study different aspects of retinal development where one can control the size and the cellular composition of the initial reaggregate.
我们之前开发了一种重聚集细胞培养系统（微团培养），在该系统中，视网膜神经上皮细胞能够增殖并在体外产生视杆光感受器细胞（视杆细胞）（Watanabe和Raff，1990年，《神经元》4:461 - 467）。在本研究中，我们通过使用免疫荧光和电子显微镜的细胞类型特异性标记物，分析了微团培养中的细胞分化和形态发生。我们证明，除了视杆细胞外，其他主要的视网膜细胞类型，包括无长突细胞、双极细胞、穆勒细胞和神经节细胞，都存在于微团中，其中大多数能够在体外从分裂的前体细胞发育而来。微团中的不同细胞类型形成了两种不同的结构：暗玫瑰花结和淡玫瑰花结。这些结构的细胞组成表明，暗玫瑰花结对应于正常视网膜的外核层，淡玫瑰花结对应于内核层。超微结构研究表明，围绕暗玫瑰花结的薄层神经突对应于外网状层，淡玫瑰花结的中心区域对应于正常视网膜的内网状层。正常视网膜发育的其他特征也在微团中出现，包括程序性细胞死亡以及内外视杆细胞段和突触的形成。因此，微团培养提供了一种方便的方法来研究视网膜发育的不同方面，在这种方法中，可以控制初始重聚集体的大小和细胞组成。
