Shihabuddin Lamya S
Genzyme Corporation, Boston, MA, USA.
Methods Mol Biol. 2008;438:55-66. doi: 10.1007/978-1-59745-133-8_6.
Self-renewing multipotent stem/progenitor cells have been isolated from various areas of the adult central nervous system, both neurogenic and non-neurogenic, in vitro by using a variety of mitogens and culture conditions. Mitogenic growth factors stimulate the proliferation of multipotent stem/progenitor cells from embryonic and adult rodent, primates, and human. Neural stem cells were isolated from the adult rat spinal cord as follows: Adult rat spinal cords were dissected, and tissue was mechanically and enzymatically dissociated. The cell suspension was filtered and further purified from debris and enriched for progenitors by using a Percoll density gradient. The dissociated cells were grown on polyornithine- and laminin-coated plates in N2 medium containing fibroblast growth factor-2. The cells grew as attached layers. Cultures were passaged by trypsinization, and rapidly proliferating cells that could be passaged, frozen, and thawed were isolated. Clonal cultures derived from single founder cells identified by marker genes generate neurons, astrocytes, and oligodendrocytes, confirming the multipotent nature of the parent cell. Subcloning analysis showed that after serial passaging, recloning, and expansion, these cells retained multipotentiality, indicating that they are self-renewing.
通过使用多种促分裂原和培养条件，在体外已经从成年中枢神经系统的各个区域（包括神经源性和非神经源性区域）分离出了自我更新的多能干细胞/祖细胞。促有丝分裂生长因子刺激来自胚胎和成年啮齿动物、灵长类动物及人类的多能干细胞/祖细胞的增殖。从成年大鼠脊髓中分离神经干细胞的方法如下：解剖成年大鼠脊髓，将组织进行机械和酶解处理。细胞悬液经过过滤，通过使用Percoll密度梯度从碎片中进一步纯化，并富集祖细胞。解离后的细胞接种在包被有聚鸟氨酸和层粘连蛋白的培养板上，置于含有成纤维细胞生长因子-2 的N2培养基中培养。细胞贴壁生长。通过胰蛋白酶消化进行传代培养，分离出能够传代、冷冻和解冻的快速增殖细胞。由标记基因鉴定的单个起始细胞形成的克隆培养物可产生神经元、星形胶质细胞和少突胶质细胞，证实了亲代细胞的多能性。亚克隆分析表明，经过连续传代、再克隆和扩增后，这些细胞仍保留多能性，表明它们具有自我更新能力。
