Generation of region-specific neural cells from Embryonic Stem Cells

最近，在非洲爪哇和果蝇神经诱导的分子理解方面取得了重大进展。相比之下，对哺乳动物神经诱导和不同神经元生成的分子机制知之甚少。利用哺乳动物胚胎干细胞，我们正在研究这些问题。首先，我们建立了一个诱导小鼠和灵长类ES细胞神经分化的体外实验系统。利用共培养体系，我们在一些基质细胞中鉴定了ES细胞的神经诱导活性，并将这种活性命名为基质细胞衍生诱导活性(SDIA)。尤其是PA6细胞(来源于颅骨骨髓的基质细胞)能有效地(90%)诱导ES细胞的神经分化。接下来，我们分析了SDIA诱导的神经细胞的区域特性。SDlA处理的ES细胞表达前脑、中脑和后脑标志物，但不表达脊髓标志物。它们通过背腹轴表达各种背腹神经标志物。最后，我们检测了SDIA处理的ES细胞产生各种神经细胞类型的能力。维甲酸修饰的SDIA处理的ES细胞向尾部方向。Shh抑制背神经标记物，诱导腹侧中枢神经系统组织，如运动神经元和底板细胞。BMP4抑制腹侧神经标志物，诱导背侧和神经脊标志物。因此，SDIA处理的ES细胞具有对维甲酸、Shh和BMP4等构型因子的反应能力，并可以被修饰为区域特异性神经细胞。

Recently significant progress has been made in the molecular understanding of neural induction in Xenopus and Drosophila. By contrast, relatively little is known about molecular mechanisms of mammalian neural induction and generation of diverse neurons. Using mammalian Embryonic Stem (ES) cells, we are studying these issues.To begin with, we established an in vitro experimental system that induces neural differentiation of mouse and primate ES cells. Using co-culture system, we have identified neural inducing activity of ES cells in some stromal cells and named this activity Stromal Cell-Derived Inducing Activity (SDIA). Especially PA6 cells (stromal cells derived from skull bone marrow)efficiently (>90%) induce neural differentiation of ES cells.We next analyzed regional identities of SDIA-induced neural cells. SDlA-treated ES cells express forebrain, midbrain and hindbrain markers but not spinal cord markers. They express a variety of dorsal-ventral neural markers in terms of dorsal-ventral axis.Finally, we examined the capacity of SDIA-treated ES cells to generate a wide variety of neural cell types. Retinoic Acid modified SDIA-treated ES cells into the caudal direction. Shh suppressed dorsal neural markers and induce ventral Central Nervous System tissues, such as motor neurons and floor plate cells. BMP4 suppressed ventral neural markers and induced dorsal and neural crest markers. Thus SDIA-treated ES cells have the competence to respond to patterning factors, such as Retinoic Acid, Shh and BMP4, and can be modified to region-specific neural cells.