Liu Xin-Chun, Zhu Yue
Department of Orthopedics, First Affiliated Hospital, China Medical University, Shenyang, China.
Sheng Li Xue Bao. 2006 Aug 25;58(4):384-90.
Neural stem cells are a potential therapeutic source for cellular transplantation therapy in neurological diseases. The present paper was aimed to investigate whether neural stem cells could be obtained from the spinal cords of low temperature preserved abortuses. Fourteen weeks old abortuses were stored in a refrigerator at 4 degrees C without any additional treatments for 2, 6 and 12 h before use. The spinal cords were anatomized out and divided into cervical cords, thoracic cords and lumbar/sacral cords. Then the spinal cord segments were used for cell culture separately. Neural stem cells were isolated from the segments and cultured in bFGF, EGF and N2 supplement containing free-serum DMEM/F12 (1:1) medium. In order to examine the differentiation potential, the stem cells were induced to differentiate with 5% fetal bovine serum on poly-l-lysine substrate. Clonal culture was carried out to demonstrate that the isolated cells met the standard of stem cells. Indirect fluorescent immunocytochemistry was used to examine the expressions of neural stem cell marker (nestin), neuron marker (MAP2), astrocyte marker (GFAP) and cholinergic marker (ChAT). The stem cells in different cultures were compared. One-way analysis of variance and Kruskal-Wallis test were used for the statistical comparison. As a result, neural stem cells were obtained from all the spinal cord segments with different postmortem intervals. Both the cells on the surface and inside the neurospheres showed nestin immunoreactivity. Therefore, nearly all the cells that composed the neurospheres were nestin-positive undifferentiated cells. When the spheres were induced to differentiate, they could yield GFAP-positive astrocytes and MAP2-positive neurons including ChAT-positive cholinergic neurons. Primary neurospheres could be dissociated mechanically, expand in subcultures and maintain the differentiation potential. In clonal cultures, single cells from a single primary sphere could give rise to new neurospheres, which had the same differentiation potential as the primary spheres. The lumbar/sacral cord cultures gave rise to the most abundant primary neurospheres. When the preservation time of the fetus was prolonged to 12 h, the number of primary neurospheres decreased sharply. The clonal formation and phenotype capacity were similar in all cultures. In conclusion, spinal neural stem cells can be isolated from low temperature preserved abortuses and represent an alternative source for experimental and potential therapeutic purposes.
神经干细胞是神经疾病细胞移植治疗的一种潜在治疗来源。本文旨在研究能否从低温保存流产胎儿的脊髓中获取神经干细胞。14周龄的流产胎儿在4℃冰箱中保存，使用前未经任何额外处理，分别保存2、6和12小时。解剖出脊髓并分为颈髓、胸髓和腰骶髓。然后将脊髓节段分别用于细胞培养。从节段中分离神经干细胞，并在含有碱性成纤维细胞生长因子（bFGF）、表皮生长因子（EGF）和N2添加剂的无血清DMEM/F12（1:1）培养基中培养。为了检测分化潜能，将干细胞在聚-L-赖氨酸底物上用5%胎牛血清诱导分化。进行克隆培养以证明分离的细胞符合干细胞标准。采用间接荧光免疫细胞化学法检测神经干细胞标志物（巢蛋白）、神经元标志物（微管相关蛋白2，MAP2）、星形胶质细胞标志物（胶质纤维酸性蛋白，GFAP）和胆碱能标志物（胆碱乙酰转移酶，ChAT）的表达。比较不同培养物中的干细胞。采用单因素方差分析和Kruskal-Wallis检验进行统计学比较。结果显示，在不同的死后间隔时间下，所有脊髓节段均能获取神经干细胞。神经球表面和内部的细胞均显示巢蛋白免疫反应性。因此，几乎所有构成神经球的细胞都是巢蛋白阳性的未分化细胞。当神经球被诱导分化时，它们能产生GFAP阳性的星形胶质细胞和MAP2阳性的神经元，包括ChAT阳性的胆碱能神经元。原代神经球可以机械解离，在传代培养中扩增并保持分化潜能。在克隆培养中，单个原代神经球的单个细胞可以产生新的神经球，其分化潜能与原代神经球相同。腰骶髓培养物产生的原代神经球最多。当胎儿保存时间延长至12小时时，原代神经球数量急剧减少。所有培养物中的克隆形成和表型能力相似。总之，脊髓神经干细胞可以从低温保存的流产胎儿中分离出来，是实验和潜在治疗目的的一种替代来源。
