Shofuda Tomoko, Fukusumi Hayato, Kanematsu Daisuke, Yamamoto Atsuyo, Yamasaki Mami, Arita Norio, Kanemura Yonehiro
Division of Stem Cell Research, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka, Japan.
Neuroreport. 2013 Jan 23;24(2):84-90. doi: 10.1097/WNR.0b013e32835cb677.
In vitro, human neural stem cells can be selectively expanded from fetal or adult neural tissues as neurospheres consisting of immature neural progenitor cells. Access to human neural tissues is limited, making it difficult to propagate and use primary neural stem or progenitor cells (NSPCs) from human neural tissues (hN-NSPCs). It was recently demonstrated that hN-NSPCs can be differentiated from either human embryonic stem cells (hESC-NSPCs) or human-induced pluripotent stem cells (hiPSC-NSPCs), and that hESC-NSPCs and hiPSC-NSPCs are adaptable, powerful substitutes for hN-NSPCs in both regenerative medicine and pharmacological or neurotoxicological assays. We here describe a new protocol to generate neurospheres consisting of hiPSC-NSPCs using microsphere arrays, the surface of which is modified with polyethylene glycol to render it nonadhesive to cells. Primary hiPSCs treated with noggin formed neurospheres on the microsphere arrays and could be stably propagated as free-floating spheroids. The hiPSC-NSPCs proliferating in these neurospheres were almost identical in phenotype to hN-NSPCs, in both cell-surface marker expression and their ability to differentiate into neuronal cells, although gene expression profiles showed that the hiPSC-NSPCs had higher neural and lower glial gene expression, along with mid-hindbrain-like regional specificity. This convenient propagation protocol can be used to evaluate the neurosphere-forming efficiency of hiPSC clones. This method will support the generation of neurospheres from hESCs and hiPSCs and contribute to the use of hESC-NSPCs and hiPSC-NSPCs in research.
在体外，人类神经干细胞可以从胎儿或成人神经组织中选择性地扩增为神经球，这些神经球由未成熟的神经祖细胞组成。获取人类神经组织受到限制，这使得从人类神经组织（hN - NSPCs）中扩增和使用原代神经干细胞或祖细胞变得困难。最近有研究表明，hN - NSPCs可以从人类胚胎干细胞（hESC - NSPCs）或人类诱导多能干细胞（hiPSC - NSPCs）分化而来，并且hESC - NSPCs和hiPSC - NSPCs在再生医学以及药理学或神经毒理学检测中都是hN - NSPCs适用且强大的替代品。我们在此描述一种使用微球阵列生成由hiPSC - NSPCs组成的神经球的新方法，该微球阵列的表面用聚乙二醇进行了修饰，使其对细胞不具有粘附性。用诺金处理的原代hiPSCs在微球阵列上形成神经球，并可以作为自由漂浮的球体稳定扩增。在这些神经球中增殖的hiPSC - NSPCs在细胞表面标志物表达以及分化为神经元细胞的能力方面，其表型与hN - NSPCs几乎相同，尽管基因表达谱显示hiPSC - NSPCs具有更高的神经基因表达和更低的胶质基因表达，同时具有中后脑样区域特异性。这种便捷的扩增方法可用于评估hiPSC克隆形成神经球的效率。该方法将有助于从hESCs和hiPSCs生成神经球，并促进hESC - NSPCs和hiPSC - NSPCs在研究中的应用。
