人类皮层类器官（hCO）表型的可重复性对模拟神经发育性疾病仍然存在一定的担忧。尽管受控的hCO方案能在一个地方的多个细胞系中可重复地产生皮层细胞类型，但尚未评估在协同的方案下不同地点的可变性。我们提出了一项hCO跨地点可重复性研究，研究了多种表型。三个独立的研究团队使用协同的小型旋转生物反应器方案从一种诱导多能干细胞（iPSC）系列中产生了hCO。我们使用单细胞RNA测序、三维荧光成像、相差成像、定量PCR和流式细胞术来表征不同地点的三个月分化过程中的细胞。在所有的地点，hCO主要表型是可重复的皮层前体和神经细胞类型，这些类型与体内脑的组织结构相一致，并且一直以皮层壁状芽的形式存在。我们在hCO的大小和形态方面检测到了地点间的差异。差异基因表达显示了不同地点间代谢和细胞应激的差异。虽然iPSC培养条件一致且未分化，但分化之前活化的干细胞标记物表达与hCO的细胞类型比例相关。我们确定了使用协同分化方案在不同地点可重复的hCO表型。同时，我们也发现了先前所描述的hCO模型的局限性，包括目标不符合的分化、坏死核、细胞应激等。改善对干细胞状态如何影响hCO早期细胞类型的理解可能提高hCO分化的可靠性。hCO细胞类型比例和组织结构的跨地点可重复性为未来协同进行神经发育性疾病模拟研究的前景性元分析研究奠定了基础。

Reproducibility of human cortical organoid (hCO) phenotypes remains a concern for modeling neurodevelopmental disorders. While guided hCO protocols reproducibly generate cortical cell types in multiple cell lines at one site, variability across sites using a harmonized protocol has not yet been evaluated. We present an hCO cross-site reproducibility study examining multiple phenotypes.Three independent research groups generated hCOs from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol. scRNA-seq, 3D fluorescent imaging, phase contrast imaging, qPCR, and flow cytometry were used to characterize the 3 month differentiations across sites.In all sites, hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions with moderate to high fidelity to the in vivo brain that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and morphology. Differential gene expression showed differences in metabolism and cellular stress across sites. Although iPSC culture conditions were consistent and iPSCs remained undifferentiated, primed stem cell marker expression prior to differentiation correlated with cell type proportions in hCOs.We identified hCO phenotypes that are reproducible across sites using a harmonized differentiation protocol. Previously described limitations of hCO models were also reproduced including off-target differentiations, necrotic cores, and cellular stress. Improving our understanding of how stem cell states influence early hCO cell types may increase reliability of hCO differentiations. Cross-site reproducibility of hCO cell type proportions and organization lays the foundation for future collaborative prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.