Efficient Generation of Mature Cerebellar Purkinje Cells From Mouse Embryonic Stem Cells
Efficient Generation of Mature Cerebellar Purkinje Cells From Mouse Embryonic Stem Cells
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DOI：
10.1002/jnr.22208
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发表时间：
2010-02-01
影响因子：
4.2
通讯作者：
Okano, Hideyuki
中科院分区：
文献类型：
--
作者：
Tao, Osamu;Shimazaki, Takuya;Okano, Hideyuki
用BMP4、Fgf8b和Wnt3a处理的无血清培养胚状体样聚集体（SFEB）漂浮培养系统中，小鼠胚胎干细胞（ESCs）可以产生小脑神经元，包括浦肯野细胞（PCs）及其前体细胞。在此，我们成功建立了一个共培养系统，在SFEB中诱导esc来源的浦肯野细胞（EDPC）前体中的PCs成熟，使用出生后(P) 6-8天的小鼠小脑分离培养物作为饲养层。当贴壁培养物不含饲养细胞或饲养层来自新生儿小脑时，PC成熟不完全或异常。与出生后的小脑喂食器共培养4周后，EDPCs表现出成熟的PCs形态，并与其他小脑神经元突触发生。此外，这些edpc的电生理特性与体外天然成熟pc的电生理特性一致，例如电流注射和兴奋性或抑制性突触后电流引发的Na+或Ca2+峰值，这些特性通过全细胞膜片钳记录进行评估。因此，SFEB中的EDPC前体可以在体外成熟为与天然pc性能相当的pc。(C) 2009 Wiley-Liss, Inc。
Mouse embryonic stem cells (ESCs) can generate cerebellar neurons, including Purkinje cells (PCs) and their precursor cells, in a floating culture system called serum-free culture of embryoid body-like aggregates (SFEB) treated with BMP4, Fgf8b, and Wnt3a. Here we successfully established a coculture system that induced the maturation of PCs in ESC-derived Purkinje cell (EDPC) precursors in SFEB, using as a feeder layer a cerebellum dissociation culture prepared from mice at postnatal day (P) 6-8. PC maturation was incomplete or abnormal when the adherent culture did not include feeder cells or when the feeder layer was from neonatal cerebellum. In contrast, EDPCs exhibited the morphology of mature PCs and synaptogenesis with other cerebellar neurons when grown for 4 weeks in coculture system with the postnatal cerebellar feeder. Furthermore, the electrophysiological properties of these EDPCs were compatible with those of native mature PCs in vitro, such as Na+ or Ca2+ spikes elicited by current injections and excitatory or inhibitory postsynaptic currents, which were assessed by whole-cell patch-clamp recordings. Thus, EDPC precursors in SFEB can mature into PCs whose properties are comparable with those of native PCs in vitro. (C) 2009 Wiley-Liss, Inc.