建立人胚胎干细胞神经定向分化过程中的谱系追踪系统

One of the most critical scientific questions during early embryonic development is how various cell lineages are sequentially produced. More importantly, understanding cell fate determination mechanisms during early development is also the foundation of establishing robust techniques for targeted lineage differentiation from puripotent stem cells and therefore benefits regenerative medicine. By taking advantage of the Cre/Loxp recombination system in transgenic mice, a body of progresses has been made in understanding cell fate determination mechanisms during early mouse embryonic development. However, such a powerful system is still lacking in the human system because of ethical problems and inefficient gene targeting in human cells. Here, we propose to build such a system based on our well established human embryonic stem cell maintenance and neural differentiation system as well as newly-emerging TALEN/Cas9 homologous recombination system. We will first build a human embryonic stem cell line with the CAG-Loxp-Stop-Loxp-eGFP cassette inserted in the AAVS1 locus. Based on this line, a couple of lines with Cre recombinase driven by promoters of specific genes of interest will also be built through homologous recombination. We will more focus on neural genes, such as Pax6, Nkx2.1, Lhx6 and Lhx8, which is aiming to untangle the conservation and uniqueness of human neural induction and regional patterning mechanisms.

理解发育过程中细胞谱系间的衍生规律是发育学的核心，也是靶向多能干细胞分化为特定的细胞类型并应用于再生医学的理论基础。基于Cre/Loxp转基因小鼠的谱系追踪，由于其较好的结合了基因表达的时空特征和便捷的报告体系，被广泛应用于发育过程中细胞命运决定机制的研究并取得了令人瞩目的成就。然而，对于人类细胞，针对不同谱系细胞的发育规律，我们却缺乏有力的研究手段。本申请拟结合我们现有的人多能干细胞体外培养和神经定向分化技术与新兴的基于TALEN/Cas9的同源重组技术，体外构建针对人类早期发育过程的谱系追踪系统。这包括建立AAVS1位点的CAG-Loxp-Stop-Loxp-eGFP同源重组人胚胎干细胞系，和基于该细胞系的靶基因启动子驱动的Cre重组细胞系。在靶基因的选择上，聚焦神经发育，研究Pax6、Nkx2.1、Lhx6/8阳性神经上皮或前体细胞的谱系转换，探讨人早期神经发育过程中的基础理论问题。

构建出可诱导的人胚胎干细胞谱系示踪系统。利用同源重组方法，在人多能干细胞（hPSCs）特异的染色体开放位点AAVS1整合受重组酶Cre或Flp控制的转录终止调控元件片段LoxP-STOP-LoxP（LSL）或FRT-STOP-FRT（FSF），同时选取GFP作为报告基因，构成可控制的报告基因系统；在谱系特异性表达的调控开关方面，我们选取了人特异的早期神经外胚层（neuroectoderm，NE）转录因子PAX6和早期神经管底板（floor plate，FP）转录因子FOXA2作为驱动重组酶Cre或Flp表达的基因。通过2A连接重组酶替换PAX6和FOXA2终止密码子，以及将重组酶替换PAX6和FOXA2起始密码子，构建成PAX6和FOXA2的谱系特异性表达的GFP示踪的谱系示踪系统。此外，采用重组酶羧基末端连接突变雌激素受体片段（estrogen receptor，ER）的策略，构建出可诱导的重组酶入核的细胞系，提高谱系示踪的灵活性与可控性。通过体外神经定向分化系统和体内畸胎瘤实验，证明上述的多种方法可以成功用于谱系示踪，为构建谱系示踪人细胞系提供更灵活、精确的选择。基于这一系统，我们证明了PAX6阳性的NE是人神经系统NP的起源，揭示了人脑发育的特有属性。

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