Daadi Marcel M, Maag Anne-Lise, Steinberg Gary K
Department of Neurosurgery and Stanford Stroke Center, Stanford University School of Medicine, Stanford, California, USA.
PLoS One. 2008 Feb 20;3(2):e1644. doi: 10.1371/journal.pone.0001644.
Human embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.
METHODS/PRINCIPAL FINDINGS: We isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.
CONCLUSIONS/SIGNIFICANCE: The SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research.
人类胚胎干细胞（hESCs）为神经疾病（如中风）的结构修复提供了几乎无限的神经细胞来源。神经细胞可以通过直接富集从hESCs中获得，也可以通过分离对特定生长因子有反应且可扩增的人类神经干细胞（hNSCs）群体来获得。研究表明，直接富集方法会产生异质性细胞群体，其中可能包含残留的未分化干细胞，这可能导致体内肿瘤形成。
方法/主要发现：我们使用添加了表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）和白血病抑制生长因子（LIF）的特定培养基，从hESCs中分离出了可扩增且同质的hNSCs群体（命名为SD56）。这些hNSCs以贴壁单层培养的方式生长。它们完全神经化，并均匀表达神经干细胞的分子特征，包括巢蛋白、波形蛋白和放射状胶质细胞标志物。这些hNSCs不表达多能性标志物Oct4或Nanog，也不表达中胚层或内胚层谱系的标志物。hNSCs的自我更新特性以细胞分裂的主要对称模式为特征。SD56 hNSCs在体外多次传代以及移植后均分化为神经元、星形胶质细胞和少突胶质细胞。这些标准共同证实了SD56细胞系的明确神经干细胞身份。重要的是，它们没有染色体异常，植入大鼠缺血性脑以及未处理的裸鼠脑和侧腹后也没有形成肿瘤。此外，在这些条件下分离的hNSCs向缺血损伤的成年脑实质迁移，并在移植后两个月改善了中风受损前肢的自主使用能力。
结论/意义：在报告的条件下获得的SD56人类神经干细胞是稳定的，在体内不形成肿瘤，并能在中风后实现功能恢复。这些特性表明，这种hNSC系可能提供一种可再生的、同质的神经细胞来源，这对于基础研究和转化研究将具有重要价值。
