Kentroti S, Vernadakis A
Department of Pharmacology, University of Colorado School of Medicine, Denver 80262, USA.
Glia. 1996 Oct;18(2):79-91. doi: 10.1002/(SICI)1098-1136(199610)18:2<79::AID-GLIA1>3.0.CO;2-7.
We examined properties of glia derived from very early neurogenesis in the chick embryo, as well as their behavior in response to extended cell passages and at various periods in culture. Primary cultures derived from the telencephalic region of 3-day-old chick embryo (stage 19) exhibited intense staining for vimentin (Vim; indicative of immature glial phenotypes) throughout the 17-day culture period, transitioning to Vim-positive/glial fibrillary acidic protein (GFAP)-positive astroblasts after a single cell passage at 13 days in vitro (DIV). With subsequent passage (P; through P6), cell continued to coexpress Vim/GFAP with the occasional appearance of fibronectin (Fib). By P11, Vim staining was faint, whereas GFAP staining gained in intensity, indicating the presence of mature astrocytes. These cultures also featured significant activity of glutamine synthetase (GS), which increased slightly with both cell passage and days in culture, correlating well with immunocytochemical findings. The activity of 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNP) remained low, indicating a low percentage of mature oligodendrocytes. Thus, embryonic day (E)3H glia cultures mature into astrocytes given sufficient time in culture. To obtain a stable population of glia at various stages of astrocytic differentiation, we immortalized and subcloned homogenous colonies of E3H glia precursors at specific stages of development. A cell suspension was electroporetically transfected with the pSV40neo gene for large T antigen (E3H.SV5). After 5 passages, the parent colonies consisted of a heterogeneous population of cells, most of which were vim and GFAP positive. Following subcloning, one line (colony 7) displayed the staining pattern of mature astrocytes. However, with advancing age in culture, staining for both vim and GFAP became increasingly faint. Compared with control (normal) cultures, transfected cells exhibited a significantly lower activity of GS that did not fluctuate with days in culture but decreased with advancing cell passage. Furthermore, CNP activity in E3H.SV5 colony 7 was approximately double that observed in normal cultures at the same cell passage. This observation suggests that the phenotype of transfected glia was less stable than that observed in normal glial cultures.
我们研究了源自鸡胚极早期神经发生的神经胶质细胞的特性，以及它们在细胞传代延长和培养不同时期的反应。来自3日龄鸡胚（第19阶段）端脑区域的原代培养物在整个17天的培养期内对波形蛋白（Vim；指示未成熟神经胶质细胞表型）呈现强烈染色，在体外培养13天（DIV）进行一次细胞传代后转变为Vim阳性/胶质纤维酸性蛋白（GFAP）阳性的星形胶质细胞。在随后的传代（P；至P6）过程中，细胞继续共表达Vim/GFAP，偶尔出现纤连蛋白（Fib）。到P11时，Vim染色变淡，而GFAP染色强度增加，表明存在成熟的星形胶质细胞。这些培养物还具有显著的谷氨酰胺合成酶（GS）活性，其随细胞传代和培养天数略有增加，与免疫细胞化学结果密切相关。2',3'-环核苷酸3'-磷酸水解酶（CNP）的活性保持较低，表明成熟少突胶质细胞的比例较低。因此，胚胎第3天（E）的神经胶质细胞培养物在培养足够时间后会成熟为星形胶质细胞。为了在星形胶质细胞分化的不同阶段获得稳定的神经胶质细胞群体，我们在发育的特定阶段对E3H神经胶质细胞前体的同质集落进行了永生化和亚克隆。用携带大T抗原的pSV40neo基因（E3H.SV5）对细胞悬液进行电穿孔转染。传代5次后，亲代集落由异质细胞群体组成，其中大多数细胞vim和GFAP呈阳性。亚克隆后，一个细胞系（集落7）呈现出成熟星形胶质细胞的染色模式。然而，随着培养时间的延长，vim和GFAP的染色都变得越来越淡。与对照（正常）培养物相比，转染细胞的GS活性显著降低，其活性不随培养天数波动，但随细胞传代增加而降低。此外，在相同细胞传代时，E3H.SV5集落7中的CNP活性约为正常培养物中观察到的活性的两倍。这一观察结果表明，转染的神经胶质细胞的表型比正常神经胶质细胞培养物中观察到的表型更不稳定。
