Minger S L, Fisher L J, Ray J, Gage F H
Salk Institute, Laboratory of Genetics, San Diego, California 92186-5800, USA.
Exp Neurol. 1996 Sep;141(1):12-24. doi: 10.1006/exnr.1996.0134.
The intracerebral transplantation of freshly dissected fetal tissue containing cholinergic neurons of the developing basal forebrain has been reported to reverse lesion-induced or age-related cognitive deficits in animal models of cholinergic neuronal degeneration. Grafts of cultured fetal neurons, however, have generally shown poor cellular survival and limited therapeutic benefit. We tested the hypothesis that recent advances in the identification of growth factors that promote the survival and propagation of fetal precursor cells in vitro would improve the long-term survival of cultured neurons following intracerebral implantation. Dissociated cells from gestational Day 14 rodent basal forebrain were grown in chemically defined media supplemented with 20 ng/ml basic fibroblast growth factor. Two weeks postplating, numerous cells were present in the cultures and showed immunoreactive labeling for a variety of markers, including glutamic acid decarboxylase, neuron-specific enolase, neurofilament proteins, glial fibrillary acidic protein and, occasionally, choline acetyltransferase. To determine if cultured basal forebrain cells would survive intracerebral implantation, the cells were implanted homotypically into the nucleus basalis magnocellularis. To enhance the potential for graft survival in vivo, cells were also implanted into the nucleus basalis magnocellularis following an ibotenic acid lesion and into the denervated frontal cortex. Animals sacrificed between 2 weeks and 7 months following transplantation showed good and comparable graft survival in all sites. Immunocytochemical analysis revealed that representative populations of cells observed in vitro survived for prolonged periods in vivo, even in sites distal from their normal cellular targets. Thus, neuronal populations expanded in vitro can successfully survive and maintain cellular phenotypes post-transplantation. These results suggest a potential for isolating and growing specific neuronal populations in vitro for intracerebral transplantation.
据报道，将含有发育中的基底前脑胆碱能神经元的新鲜解剖胎儿组织进行脑内移植，可逆转胆碱能神经元变性动物模型中损伤诱导的或与年龄相关的认知缺陷。然而，培养的胎儿神经元移植通常显示细胞存活率低且治疗效果有限。我们检验了这样一个假设：在体外促进胎儿前体细胞存活和增殖的生长因子鉴定方面的最新进展，将提高脑内植入后培养神经元的长期存活率。从妊娠第14天的啮齿动物基底前脑分离的细胞，在添加了20 ng/ml碱性成纤维细胞生长因子的化学限定培养基中培养。接种两周后，培养物中有大量细胞，并对多种标志物显示免疫反应性标记，包括谷氨酸脱羧酶、神经元特异性烯醇化酶、神经丝蛋白、胶质纤维酸性蛋白，偶尔还有胆碱乙酰转移酶。为了确定培养的基底前脑细胞在脑内植入后是否能存活，将这些细胞同型植入大细胞基底核。为了提高体内移植存活的可能性，在注射鹅膏蕈氨酸损伤后，也将细胞植入大细胞基底核以及去神经支配的额叶皮质。移植后2周和7个月之间处死的动物显示，所有部位的移植存活率良好且相当。免疫细胞化学分析显示，体外观察到的代表性细胞群体在体内能长时间存活，即使在远离其正常细胞靶点的部位也是如此。因此，体外扩增的神经元群体在移植后能够成功存活并维持细胞表型。这些结果表明，体外分离和培养特定神经元群体用于脑内移植具有潜力。
