Rapid generation of sub-type, region-specific neurons and neural networks from human pluripotent stem cell-derived neurospheres.
Rapid generation of sub-type, region-specific neurons and neural networks from human pluripotent stem cell-derived neurospheres.
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DOI：
10.1016/j.scr.2015.10.014
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发表时间：
2015-11
影响因子：
1.2
通讯作者：
Hong Y
中科院分区：
文献类型：
--
作者：
Begum AN;Guoynes C;Cho J;Hao J;Lutfy K;Hong Y
基于干细胞的神经元分化为疾病建模和再生医学提供了独特的机会。神经球是最常用的用于神经分化的神经祖细胞，但它们在培养过程中经常结块，这一直是神经分化的一个挑战。在这项研究中，我们报告了一种新的方法和确定的培养条件，可以在没有任何遗传操作的情况下，从人类胚胎和诱导多能干细胞衍生的神经球中产生亚型或区域特异性神经元。在添加10% CO2的敲除血清替代培养基（SKSRM）中生成圆形和边缘明亮的神经球，与5% CO2培养相比，NESTIN， PAX6和FOXG1基因的表达增加了一倍。此外，引入了一个额外的步骤（AdSTEP）来分裂神经球，并促进神经上皮型单层的形成，我们称之为“神经球皮”。大神经管型玫瑰花结（NTTR）结构形成于神经球层，与神经外胚层来源的神经祖细胞相比，NTTR表达了更高水平的PAX6、SOX2和NESTIN基因。神经球皮在27天内出现了皮层、锥体、gaba能、谷氨酸能、胆碱能等不同层次的神经元，这比传统的神经分化方案（42-60天）要短。在额外的补充和时间安排下，多巴胺能神经元和浦肯野神经元也在培养中产生。此外，我们的体内实验结果表明，与非碎片化的神经球相比，碎片化的神经球在严重联合免疫缺陷（SCID）小鼠大脑中促进了更好的神经发生。因此，这种基于神经球的神经分化方案是研究神经分化、神经元移植和高通量筛选分析的有价值的工具。
Stem cell-based neuronal differentiation has provided a unique opportunity for disease modeling and regenerative medicine. Neurospheres are the most commonly used neuroprogenitors for neuronal differentiation, but they often clump in culture, which has always represented a challenge for neurodifferentiation. In this study, we report a novel method and defined culture conditions for generating sub-type or region-specific neurons from human embryonic and induced pluripotent stem cells derived neurosphere without any genetic manipulation. Round and bright-edged neurospheres were generated in supplemented knockout serum replacement medium (SKSRM) with 10% CO2, which doubled the expression of the NESTIN, PAX6 and FOXG1 genes compared to those cultured with 5% CO2. Furthermore, an additional step (AdSTEP) was introduced to fragment the neurospheres and facilitate the formation of a neuroepithelial-type monolayer that we termed the “neurosphederm”. The large neural tube-type rosette (NTTR) structure formed from the neurosphederm, and the NTTR expressed higher levels of the PAX6, SOX2 and NESTIN genes compared to the neuroectoderm-derived neuroprogenitors. Different layers of cortical, pyramidal, GABAergic, glutamatergic, cholinergic neurons appeared within 27 days using the neurosphederm, which is a shorter period than in traditional neurodifferentiation-protocols (42–60 days). With additional supplements and timeline dopaminergic and Purkinje neurons were also generated in culture too. Furthermore, our in vivo results indicated that the fragmented neurospheres facilitated significantly better neurogenesis in severe combined immunodeficiency (SCID) mouse brains compared to the non-fragmented neurospheres. Therefore, this neurosphere-based neurodifferentiation protocol is a valuable tool for studies of neurodifferentiation, neuronal transplantation and high throughput screening assays.