CD133 Expression by Neural Progenitors Derived from Human Embryonic Stem Cells and Its Use for Their Prospective Isolation
CD133 Expression by Neural Progenitors Derived from Human Embryonic Stem Cells and Its Use for Their Prospective Isolation
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DOI：
10.1089/scd.2008.0124
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发表时间：
2009-03-01
影响因子：
4
通讯作者：
Hawes, Susan M.
中科院分区：
文献类型：
--
作者：
Peh, Gary S. -L.;Lang, Richard J.;Hawes, Susan M.
产生纯化的神经祖细胞的能力对于开发基于胚胎干细胞的疗法以缓解人类神经系统疾病至关重要。虽然已经描述了许多用于将人胚胎干 (hES) 细胞定向分化为神经细胞的细胞培养方案，但大多数产生混合群体，一些含有不同胚胎胚层谱系的细胞，甚至是未分化的胚胎干细胞。在这项研究中，我们描述了一种通过流式细胞术分离、分离单细胞以及随后从 hES 细胞中培养神经祖细胞的方法。正如之前报道的，用骨形态发生蛋白（BMP）拮抗剂头蛋白处理的 hES 细胞以相对较高的频率产生神经球。然而，这些经过 noggin 处理的胚胎干细胞培养物是异质的，即使在分化 14 天后，表达胚胎干标记物的细胞仍然可以检测到。为了分离纯人类神经祖细胞，我们根据假定的神经干细胞标记 CD133 以及 GCTM-2 和 SSEA-1 抗原的表达，对经 noggin 处理的 ES 细胞进行分级，这些抗原分别标记来自 hES 细胞培养物的多能干细胞和分化细胞。与CD133(-)细胞相比，CD133(+)细胞形成更大的球体。 CD133(+)SSEA1(+)细胞和CD133(+)SSEA-1(-)细胞表达相似水平的神经基因并以相似的频率形成神经球。相比之下，CD133(+)GCTM-2(+)细胞表达高水平的OCT4，但不表达神经谱系基因，并且无法形成神经球。 CD133(+)GCTM-2(-)细胞形成神经球的频率相对最高。因此，GCTM-2 的阴性选择可用于纯化从 hES 细胞分化的特定细胞类型。
The ability to generate purified neural progenitors is critical to the development of embryonic stem cell-based therapies to alleviate human neurological disorders. While many cell culture protocols for directed differentiation of human embryonic stem (hES) cells into neural cells have been described, most yield mixed populations, some containing cells of different embryonic germ layer lineages, or even undifferentiated embryonic stem cells. In this study, we describe a method for single-cell dissociation, isolation by flow cytometry, and subsequent culture of neural progenitors from hES cells. As reported earlier, hES cells treated with the bone morphogenetic protein (BMP) antagonist noggin gave rise to neurospheres at a relatively high frequency. However, these noggin-treated embryonic stem cell cultures were heterogeneous, with cells expressing embryonic stem markers still detectable even following 14 days of differentiation. In order to isolate pure human neural progenitors, we fractionated noggin-treated ES cells on the basis of their expression of the putative neural stem cell marker, CD133, and the GCTM-2, and SSEA-1 antigens, which mark pluripotent stem cells and differentiated cells respectively from hES cell culture. CD133(+) cells formed larger spheres compared to CD133(-) cells. CD133(+)SSEA1(+) cells and CD133(+)SSEA-1(-) cells expressed similar levels of neural genes and formed neurospheres at similar frequencies. By contrast, CD133(+)GCTM-2(+) cells expressed high levels of OCT4 but not neural lineage genes and failed to form neurospheres. CD133(+)GCTM-2(-) cells formed neurospheres at the relative highest frequency. Thus, negative selection with GCTM-2 may be useful for the purification of specific cell types differentiated from hES cells.