Hunt C P J, Pouton C W, Haynes J M
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Australia.
Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Australia.
Neurochem Int. 2017 Jun;106:3-13. doi: 10.1016/j.neuint.2017.01.003. Epub 2017 Jan 5.
In the developing ventral telencephalon, cells of the lateral ganglionic eminence (LGE) give rise to all medium spiny neurons (MSNs). This development occurs in response to a highly orchestrated series of morphogenetic stimuli that pattern the resultant neurons as they develop. Striatal MSNs are characterised by expression of dopamine receptors, dopamine-and cyclic AMP-regulated phosphoprotein (DARPP32) and the neurotransmitter GABA. In this study, we demonstrate that fine tuning Wnt and hedgehog (SHH) signaling early in human embryonic stem cell differentiation can induce a subpallial progenitor molecular profile. Stimulation of TGFβ signaling pathway by activin-A further supports patterning of progenitors to striatal precursors which adopt an LGE-specific gene signature. Moreover, we report that these MSNs also express markers associated with mature neuron function (cannabinoid, adenosine and dopamine receptors). To facilitate live-cell identification we generated a human embryonic stem cell line using CRISPR-mediated gene editing at the DARPP32 locus (DARPP32). The addition of dopamine to MSNs either increased, decreased or had no effect on intracellular calcium, indicating the presence of multiple dopamine receptor subtypes. In summary, we demonstrate greater control over early fate decisions using activin-A, Wnt and SHH to direct differentiation into MSNs. We also generate a DARPP32 reporter line that enables deeper pharmacological profiling and interrogation of complex receptor interactions in human MSNs.
在发育中的腹侧端脑中，外侧神经节隆起（LGE）的细胞产生所有的中等棘状神经元（MSN）。这种发育是对一系列高度协调的形态发生刺激的反应，这些刺激在神经元发育过程中对其进行模式化。纹状体MSN的特征是表达多巴胺受体、多巴胺和环磷酸腺苷调节的磷蛋白（DARPP32）以及神经递质GABA。在本研究中，我们证明在人类胚胎干细胞分化早期微调Wnt和刺猬信号通路（SHH）可以诱导亚 pallial祖细胞分子特征。激活素A对TGFβ信号通路的刺激进一步支持祖细胞向采用LGE特异性基因特征的纹状体前体的模式化。此外，我们报告这些MSN还表达与成熟神经元功能相关的标志物（大麻素、腺苷和多巴胺受体）。为了便于活细胞鉴定，我们在DARPP32基因座（DARPP32）使用CRISPR介导的基因编辑生成了一个人类胚胎干细胞系。向MSN中添加多巴胺对细胞内钙的影响要么增加、要么减少、要么没有影响，这表明存在多种多巴胺受体亚型。总之，我们证明使用激活素A、Wnt和SHH对早期命运决定有更大的控制，以指导分化为MSN。我们还生成了一个DARPP32报告系，能够对人类MSN中的复杂受体相互作用进行更深入的药理学分析和研究。
