胚胎干细胞定向分化为神经上皮干细胞和皮层投射神经元的体系建立

发布时间：2019-01-23 12:53

【摘要】：在胚胎发育过程中,脊索诱导其背侧中线的外胚层(神经外胚层)形成神经板并发育成神经管。紧接着神经管闭合,管腔特化成脑室,背部区域特化成端脑。当端脑起始发育的时候,组成神经管的神经上皮干细胞,不断的增殖扩增,逐渐转变为放射状胶质前体细胞,最后这些胶质前体细胞进行迁移分化最终形成了大脑皮层。神经管发育异常将导致一系列疾病。因此,利用多能性干细胞,发展一种灵长类神经管发育模型,能够帮助我们研究神经管疾病。本研究建立了一种稳健的体系,该体系能够在单细胞水平上进行克隆扩增并形成微小的类神经管结构：另外,单细胞不仅能够在体外产生功能神经元,还能在体内存活并广泛再生神经元轴突：除此之外,本研究还发现神经上皮干细胞特性的维持与类神经管结构的形成依赖高能量代谢：通过Notch信号与细胞粘附分子来调控Wnt信号通路,单个神经上皮干细胞能够转变为放射状胶质前体细胞：最后,利用"NESC-TO-NTs"体系,本研究成功模拟了叶酸在神经管闭合过程中的作用,表明叶酸可以调控多种机制预防神经管疾病。可见,本研究建立了一个能够在体外研究神经管发育和疾病理想的体系。大脑皮层是大脑中最复杂的结构之一,其损伤会导致一系列的神经发育和神经退行性疾病。在结构上,大脑皮层可以分为6层,第1层为cajal细胞层,2、3、4层为上层投射神经元,5、6层为底层投射神经元。根据神经元的位置和突触连接方式,5、6层又称为离皮质投射神经元。离皮质投射神经元的分化方法缺乏,限制了研究其在大脑皮层中的发育和功能。本研究开发了“两步法”能够从人胚胎干细胞快速诱导离皮质神经元：(1)诱导胚胎干细胞转变为神经上皮干细胞；(2)神经上皮干细胞分化产生8096的高纯度离皮质投射神经元。本方法获取的神经上皮干细胞不但能够在单细胞水平进行长时间的自我更新并自我重组形成类神经管结构,还能够稳定的分化为离皮质投射神经元和中间神经元。另外,本研究还发现：移植的神经上皮干细胞能成功整合到小鼠脑中,并分化为投射神经元,从而建立有效的突触连接和特殊的投射模式。因此,有效的产生离皮质投射神经元,能够促进人们了解大脑皮层的发育机制,并为细胞治疗提供充足的储备资源。
[Abstract]:During embryonic development, the notochord induces the ectoderm (neuroectoderm) of the dorsal midline to form a nerve plate and develop into a neural tube. Then the nerve tube is closed, the lumen becomes the ventricle, and the back area becomes the terminal brain. When the terminal brain begins to develop, the neural epithelial stem cells that make up the neural tube proliferate and expand, and gradually transform into radial glial precursor cells. Finally, these glial precursor cells migrate and differentiate and finally form the cerebral cortex. Dysplasia of the neural tube can lead to a series of diseases. Therefore, the use of pluripotent stem cells to develop a primate neural tube development model can help us to study neural tube diseases. This study established a robust system of cloning and amplification at the single cell level and the formation of tiny neuron-like structures: in addition, single cells not only produced functional neurons in vitro, Can also survive and regenerate a wide range of neuronal axons in the body: It was also found that the maintenance of neural epithelial stem cell properties and the formation of neuron-like tubular structures depended on high energy metabolism: Wnt signaling pathways were regulated by Notch signals and cell adhesion molecules. Single neural epithelial stem cells can be transformed into radial glial progenitor cells. Finally, using the "NESC-TO-NTs" system, we successfully simulated the role of folic acid in the process of neural tube closure. It is suggested that folic acid can regulate multiple mechanisms for the prevention of neural tube diseases. Thus, an ideal system for studying neurotubules and diseases in vitro has been established. The cerebral cortex is one of the most complex structures in the brain. Its damage can lead to a series of neurodevelopmental and neurodegenerative diseases. In structure, the cerebral cortex can be divided into six layers, the first layer is the cajal cell layer, the second layer is the upper layer, the third layer is the upper layer, and the 5 layer is the bottom layer. According to the location and synaptic connection of neurons, the 5th layer is also called the apocortical projective neurons. The lack of differentiation of apocortical projective neurons limits the development and function of these neurons in the cerebral cortex. In this study, a "two-step" method was developed to rapidly induce dissociated neurons from human embryonic stem cells: (1) inducing embryonic stem cells into neural epithelial stem cells; (2) the differentiation of neural epithelial stem cells (NSCs) produced 8096 high purity detached cortical projective neurons. The neural epithelial stem cells obtained by this method can not only self-renew and self-recombine for a long time at the single cell level to form neuron-like structures, but also can steadily differentiate into apocortical projective neurons and intermediate neurons. In addition, we also found that the transplanted neural epithelial stem cells could be successfully integrated into the mouse brain and differentiated into projective neurons, thus establishing effective synaptic connections and special projection patterns. Therefore, the effective production of apocortical projective neurons can promote the understanding of the development mechanism of cerebral cortex and provide sufficient resources for cell therapy.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q42

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