Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity despite the tremendous upsurge in derivation methods, suggesting inadequate patterning of early neural stem cells (NSCs). Here we show that a short and early Dual SMAD and WNT inhibition course is necessary and sufficient to establish robust and lasting cortical organoid NSC identity, efficiently suppressing non-cortical NSC fates, while other widely used methods are inconsistent in their cortical NSC-specification capacity. Accordingly, this method selectively enriches for outer radial glia NSCs, which cyto-architecturally demarcate well-defined outer sub-ventricular-like regions propagating from superiorly radially organized, apical cortical rosette NSCs. Finally, this method culminates in the emergence of molecularly distinct deep and upper cortical layer neurons, and reliably uncovers cortex-specific microcephaly defects. Thus, a short SMAD and WNT inhibition is critical for establishing a rich cortical cell repertoire that enables mirroring of fundamental molecular and cyto-architectural features of cortical development and meaningful disease modelling.

尽管推导方法激增，但大脑类器官表现出广泛的区域异质性，同时伴随着有限的皮质细胞多样性，这表明早期神经干细胞 (NSC) 的模式化不足。在这里，我们表明，短期和早期的双 SMAD 和 WNT 抑制过程是必要和充分的，以建立稳健和持久的皮质类器官 NSC 身份，有效抑制非皮质 NSC 命运，而其他广泛使用的方法在其皮质 NSC 规范能力方面不一致. 因此，该方法选择性地丰富了外放射状神经胶质神经干细胞，这些神经干细胞在细胞结构上划定了明确的外部亚心室样区域，这些区域从径向组织良好的心尖皮质玫瑰花结神经干细胞传播而来。最后，这种方法最终导致分子上不同的深层和上皮层层神经元的出现，并可靠地揭示了皮层特异性小头畸形缺陷。因此，短暂的 SMAD 和 WNT 抑制对于建立丰富的皮质细胞库至关重要，该库能够反映皮质发育的基本分子和细胞结构特征以及有意义的疾病建模。