In vitro cell density-dependent clonal growth of EGF-responsive murine neural progenitor cells under serum-free conditions
In vitro cell density-dependent clonal growth of EGF-responsive murine neural progenitor cells under serum-free conditions
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DOI：
10.1006/exnr.1997.6672
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发表时间：
1997-11-01
影响因子：
5.3
通讯作者：
Quesenberry, PJ
中科院分区：
文献类型：
--
作者：
Hulspas, R;Tiarks, C;Quesenberry, PJ
研究表明，对表皮生长因子(EGF)有反应的神经前体细胞群具有增殖潜能，可分化为神经元、星形胶质细胞和少突胶质细胞。我们对EGF反应性神经前体细胞进行了鉴定，这些细胞产生了双线神经元/神经胶质细胞集落(克隆形成单位神经元-胶质细胞；CFU-NeG1)和单线神经元集落(CFU-Ne)。使用来自Balb/c和rosa26(转β-半乳糖苷酶基因)小鼠的脑细胞的混合物来证实克隆。除少数例外，X-Gal染色后，集落显示全部蓝色细胞或全部透明细胞。10-11天后，通过测定集落大小和接种效率，分析克隆生长与细胞密度的关系。生长依赖于密度(10,000个/毫升以下没有生长)，因此无法完成单细胞克隆。当培养浓度为12,500~200,000个/ml时，来自15-18天小鼠胚胎的EGF反应神经细胞的平均培养效率为4%，而出生后1天的神经细胞也有类似的结果。按集落大小分类，大于50个细胞的集落相对数量在50,000个/ml时最多，培养11天后，94%、96%和78%的集落分别含有表达Nestin、神经丝和GFAP的细胞。双标记实验显示>；62%的集落同时含有GFAP和神经丝表达细胞。这些研究证实，在胎儿和出生后的小鼠脑中至少存在两种克隆神经前体细胞：CFU-Ne和CFU-NeGl。(C)1997年学术出版社。
Neural progenitor cell populations responsive to epidermal growth factor (EGF) have been shown to have proliferative potential and give rise to neurons, astrocytes, and oligodendrocytes. We have characterized EGF-responsive neural progenitor cells that give rise to bilineage neuronal/glial colonies (colony-forming unit neuron-glia; CFU-NeGl) and unilineage neuronal colonies (CFU-Ne). Clonality was confirmed utilizing mixtures of brain cells from Balb/c and ROSA26 (transgenic for beta-galactosidase) mice. With a few exceptions, colonies showed either all blue cells or all clear cells after staining with X-Gal. Clonal growth was analyzed after 10-11 days in relation to cell density by determining colony size and plating efficiency. Growth was density dependent (no growth below 10,000 cell/ ml) and thus single cell cloning was not accomplished. An average plating efficiency of 4% was found for EGF-responsive neural cells derived from day 15-18 murine embryos when cultured at 12,500 to 200,000 cells/ml. Similar results were obtained with 1-day-old postnatal neural cells. When colonies were categorized by size, the relative number of colonies over 50 cells appeared to be maximum at 50,000 plated cells/ml. After 11 days in culture, 94, 96, and 78% of the colonies contained cells that expressed nestin, neurofilament, and GFAP, respectively. Double-label experiments revealed that > 62% of the colonies contained both GFAP and neurofilament expressing cells. These studies establish the existence of at least two populations of clonal neural progenitors: CFU-Ne and CFU-NeGl in fetal and postnatal murine brain. (C) 1997 Academic Press.