IntroductionProtocadherin-19 (PCDH19)-Clustering Epilepsy (PCE) is a developmental and epileptic encephalopathy caused by loss-of-function variants of the PCDH19 gene on the X-chromosome. PCE affects females and mosaic males while male carriers are largely spared. Mosaic expression of the cell adhesion molecule PCDH19 due to random X-chromosome inactivation is thought to impair cell–cell interactions between mutant and wild type PCDH19-expressing cells to produce the disease. Progress has been made in understanding PCE using rodent models or patient induced pluripotent stem cells (iPSCs). However, rodents do not faithfully model key aspects of human brain development, and patient iPSC models are limited by issues with random X-chromosome inactivation.MethodsTo overcome these challenges and model mosaic PCDH19 expression in vitro, we generated isogenic female human embryonic stem cells with either HA-FLAG-tagged PCDH19 (WT) or homozygous PCDH19 knockout (KO) using genome editing. We then mixed GFP-labeled WT and RFP-labeled KO cells and generated human cortical organoids (hCOs).ResultsWe found that PCDH19 is highly expressed in early (days 20–35) WT neural rosettes where it co-localizes with N-Cadherin in ventricular zone (VZ)-like regions. Mosaic PCE hCOs displayed abnormal cell sorting in the VZ with KO and WT cells completely segregated. This segregation remained robust when WT:KO cells were mixed at 2:1 or 1:2 ratios. PCE hCOs also exhibited altered expression of PCDH19 (in WT cells) and N-Cadherin, and abnormal deep layer neurogenesis. None of these abnormalities were observed in hCOs generated by mixing only WT or only KO (modeling male carrier) cells.DiscussionOur results using the mosaic PCE hCO model suggest that PCDH19 plays a critical role in human VZ radial glial organization and early cortical development. This model should offer a key platform for exploring mechanisms underlying PCE-related cortical hyperexcitability and testing of potential precision therapies.

中文翻译：

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Protocadherin-19 丛集性癫痫的人皮质类器官模型中细胞分选异常和早期神经发生改变

简介原钙粘蛋白-19 (PCDH19)-丛集性癫痫 (PCE) 是一种发育性癫痫性脑病，由 X 染色体上的 PCDH19 基因功能丧失变异体引起。 PCE 影响女性和马赛克男性，而男性携带者基本上不会受到影响。由于随机 X 染色体失活而导致的细胞粘附分子 PCDH19 的镶嵌表达被认为会损害突变型和野生型 PCDH19 表达细胞之间的细胞间相互作用，从而产生疾病。使用啮齿动物模型或患者诱导多能干细胞 (iPSC) 在理解 PCE 方面取得了进展。然而，啮齿类动物并不能忠实地模拟人脑发育的关键方面，并且患者 iPSC 模型受到随机 X 染色体失活问题的限制。方法为了克服这些挑战并在体外建立镶嵌 PCDH19 表达模型，我们用HA-FLAG 标记的 PCDH19 (WT) 或使用基因组编辑的纯合 PCDH19 敲除 (KO)。然后，我们混合 GFP 标记的 WT 和 RFP 标记的 KO 细胞，并生成人皮质类器官 (hCO)。结果我们发现 PCDH19 在早期（第 20-35 天）WT 神经玫瑰花结中高度表达，与 N-钙粘蛋白共定位于心室区（VZ）样区域。马赛克 PCE hCO 在 VZ 中表现出异常的细胞分选，KO 和 WT 细胞完全分离。当 WT:KO 细胞以 2:1 或 1:2 比例混合时，这种分离仍然很明显。 PCE hCO 还表现出 PCDH19（在 WT 细胞中）和 N-钙粘蛋白表达的改变，以及异常的深层神经发生。在仅混合 WT 或仅混合 KO（建模男性携带者）细胞生成的 hCO 中，没有观察到这些异常。讨论我们使用嵌合 PCE hCO 模型的结果表明，PCDH19 在人类 VZ 放射状胶质组织和早期皮质发育中发挥着关键作用。该模型应该为探索 PCE 相关皮质过度兴奋的机制和测试潜在的精准疗法提供一个关键平台。