Cellular Neurobiology Research Branch, Intramural Research Program (IRP), National Institute on Drug Abuse, National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Baltimore, MD 21244, USA.
Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine, Hannover 30173, Germany.
Dis Model Mech. 2014 Dec;7(12):1397-405. doi: 10.1242/dmm.017251. Epub 2014 Oct 2.
Neocortical development involves ordered specification of forebrain cortical progenitors to various neuronal subtypes, ultimately forming the layered cortical structure. Modeling of this process using human pluripotent stem cells (hPSCs) would enable mechanistic studies of human neocortical development, while providing new avenues for exploration of developmental neocortical abnormalities. Here, we show that preserving hPSCs aggregates - allowing embryoid body formation - while adding basic fibroblast growth factor (bFGF) during neuroepithelial development generates neural rosettes showing dorsal forebrain identity, including Mash1(+) dorsal telencephalic GABAergic progenitors. Structures that mirrored the organization of the cerebral cortex formed after rosettes were seeded and cultured for 3 weeks in the presence of FGF18, BDNF and NT3. Neurons migrated along radial glia scaffolding, with deep-layer CTIP2(+) cortical neurons appearing after 1 week and upper-layer SATB2(+) cortical neurons forming during the second and third weeks. At the end of differentiation, these structures contained both glutamatergic and GABAergic neurons, with glutamatergic neurons being most abundant. Thus, this differentiation protocol generated an hPSC-based model that exhibits temporal patterning and a neuronal subtype ratio similar to that of the developing human neocortex. This model was used to examine the effects of cocaine during neocorticogenesis. Cocaine caused premature neuronal differentiation and enhanced neurogenesis of various cortical neuronal subtypes. These cocaine-induced changes were inhibited by the cytochrome P450 inhibitor cimetidine. This in vitro model enables mechanistic studies of neocorticogenesis, and can be used to examine the mechanisms through which cocaine alters the development of the human neocortex.
新皮层的发育涉及到前脑皮质祖细胞向各种神经元亚型的有序特化，最终形成分层的皮质结构。使用人类多能干细胞（hPSC）对这一过程进行建模，将使我们能够对人类新皮层发育进行机制研究，同时为探索发育性新皮层异常提供新途径。在这里，我们表明，在神经上皮发育过程中保留 hPSC 集落（允许胚体形成），同时添加碱性成纤维细胞生长因子（bFGF），可产生具有背侧前脑特征的神经玫瑰花结，包括 Mash1（+）背侧端脑 GABA 能祖细胞。在玫瑰花结形成后，将这些结构播种并在存在 FGF18、BDNF 和 NT3 的情况下培养 3 周，可模拟大脑皮层的组织。神经元沿着放射状胶质支架迁移，深皮层 CTIP2（+）皮质神经元在 1 周后出现，而上皮层 SATB2（+）皮质神经元在第 2 周和第 3 周形成。在分化结束时，这些结构包含谷氨酸能和 GABA 能神经元，其中谷氨酸能神经元最为丰富。因此，该分化方案生成了一种基于 hPSC 的模型，该模型表现出类似于发育中人类新皮层的时间模式和神经元亚型比例。该模型用于研究可卡因在新皮层发生过程中的作用。可卡因导致神经元过早分化，并增强了各种皮质神经元亚型的神经发生。这些可卡因诱导的变化被细胞色素 P450 抑制剂西咪替丁抑制。这种体外模型使我们能够对新皮层发生进行机制研究，并可用于研究可卡因改变人类新皮层发育的机制。
