Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations.
Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations.
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DOI：
10.1186/1471-2202-10-93
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发表时间：
2009-08-06
期刊：
影响因子：
2.4
通讯作者：
Dihné M
中科院分区：
文献类型：
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作者：
Illes S;Theiss S;Hartung HP;Siebler M;Dihné M
本研究旨在研究两种不同来源的胚胎干细胞来源的神经前体细胞在体外产生功能性神经网络的能力。第一批胚胎干细胞来源的神经前体细胞被培养成自由漂浮的神经聚集体，形成一个发育的生态位，包括不同类型的神经细胞，包括神经前体细胞、祖细胞和进一步成熟的细胞。这个生态位本身提供了多种不同的生长因子和细胞外基质蛋白，影响神经前体细胞和祖细胞的增殖和分化。第二个群体在单层培养中贴壁培养，以最严格地控制细胞外环境。这一群体包括高度同质性的神经前体细胞，这被认为是提供定义明确的神经元后代的一种有吸引力的方式。然而，到目前为止，这些不同的ES细胞来源的未成熟神经细胞群体产生功能神经元网络的能力还没有得到评估。虽然两种前体细胞群均可分化为足够数量的成熟Neun+神经元，也表达GABA或囊泡谷氨酸转运蛋白2(VGlut2)，但微电极阵列技术检测到，只有集合体来源的神经元群体在启动分化2-4周后表现出同步振荡的网络活动。在单层培养中，来自同质NPC的神经元即使在分化长达12周时也只显示出不相关的尖峰活动。我们证明，这些神经元表现出稀疏的分支轴突和胚胎vGlut2分布，表明终末神经元成熟受到抑制。相比之下，来自神经聚集体内异质群体的神经元看起来完全成熟，具有致密的轴突网络，并在突触前小泡中不时地表达vGlut2。此外，那些从贴壁神经聚集体迁移出来的NPC保持了生成同步振荡神经元网络的能力，即使它们与贴壁聚集体分离、解离和重新电镀也是如此。这些发现表明，异质神经细胞群体的小生境和聚集体内的复杂环境支持从ES细胞来源的神经细胞产生完全成熟的神经元和功能神经元网络。相比之下，单层培养中均一的ES细胞来源的神经前体细胞显示出功能成熟的受损。
The present work was performed to investigate the ability of two different embryonic stem (ES) cell-derived neural precursor populations to generate functional neuronal networks in vitro. The first ES cell-derived neural precursor population was cultivated as free-floating neural aggregates which are known to form a developmental niche comprising different types of neural cells, including neural precursor cells (NPCs), progenitor cells and even further matured cells. This niche provides by itself a variety of different growth factors and extracellular matrix proteins that influence the proliferation and differentiation of neural precursor and progenitor cells. The second population was cultivated adherently in monolayer cultures to control most stringently the extracellular environment. This population comprises highly homogeneous NPCs which are supposed to represent an attractive way to provide well-defined neuronal progeny. However, the ability of these different ES cell-derived immature neural cell populations to generate functional neuronal networks has not been assessed so far. While both precursor populations were shown to differentiate into sufficient quantities of mature NeuN+ neurons that also express GABA or vesicular-glutamate-transporter-2 (vGlut2), only aggregate-derived neuronal populations exhibited a synchronously oscillating network activity 2-4 weeks after initiating the differentiation as detected by the microelectrode array technology. Neurons derived from homogeneous NPCs within monolayer cultures did merely show uncorrelated spiking activity even when differentiated for up to 12 weeks. We demonstrated that these neurons exhibited sparsely ramified neurites and an embryonic vGlut2 distribution suggesting an inhibited terminal neuronal maturation. In comparison, neurons derived from heterogeneous populations within neural aggregates appeared as fully mature with a dense neurite network and punctuated vGlut2 expression within presynaptic vesicles. Also those NPCs that had migrated away from adherent neural aggregates maintained their ability to generate a synchronously oscillating neuronal network, even if they were separated from adherent aggregates, dissociated and re-plated. These findings suggest that the complex environment within niches and aggregates of heterogeneous neural cell populations support the generation of fully mature neurons and functional neuronal networks from ES cell-derived neural cells. In contrast, homogeneous ES cell-derived NPCs within monolayer cultures exhibited an impaired functional neuronal maturation.
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