人脑类器官正在成为研究人脑健康和疾病的转化相关模型。然而，人类特异性蛋白质加工在人脑类器官中是否保守还有待证明。在此，我们证明，非引导脑类器官的细胞命运和组成是由拟胚体形成过程中的培养条件决定的，并且可以优化此阶段的培养条件，以导致神经胶质细胞相关蛋白和神经网络活动的存在。体外三个月。在这些优化的条件下，由来自男性和女性同胞的诱导多能干细胞 (iPSC) 生成的无引导脑类器官在生长速度、大小和总蛋白含量方面相似，并且在细胞组成和代谢方面表现出最小的批次间差异。神经元、小胶质细胞和大胶质细胞（星形胶质细胞和少突胶质细胞）标记物的比较表明，这些大脑类器官中的特征与尸检的人类皮质和小脑特征比小鼠皮质样本中的特征更相似，这首次证明了人类特异性蛋白质加工在非引导的大脑类器官中大部分是保守的。因此，我们的类器官协议提供了四种主要细胞类型，它们似乎以与人脑非常相似的方式处理蛋白质，并且它们所需的时间是其他协议所需的一半。这种独特的人脑副本和基本特征为未来研究奠定了基础，旨在研究人脑特异性蛋白质模式（例如异构体、剪接变体）以及在类原位环境中调节神经胶质和神经元过程。版权© 2024 Wenzel 和 Mousseau。

Human brain organoids are emerging as translationally relevant models for the study of human brain health and disease. However, it remains to be shown whether human-specific protein processing is conserved in human brain organoids. Herein, we demonstrate that cell fate and composition of unguided brain organoids are dictated by culture conditions during embryoid body formation, and that culture conditions at this stage can be optimized to result in the presence of glia-associated proteins and neural network activity as early as three-months in vitro. Under these optimized conditions, unguided brain organoids generated from induced pluripotent stem cells (iPSCs) derived from male-female siblings are similar in growth rate, size, and total protein content, and exhibit minimal batch-to-batch variability in cell composition and metabolism. A comparison of neuronal, microglial, and macroglial (astrocyte and oligodendrocyte) markers reveals that profiles in these brain organoids are more similar to autopsied human cortical and cerebellar profiles than to those in mouse cortical samples, providing the first demonstration that human-specific protein processing is largely conserved in unguided brain organoids. Thus, our organoid protocol provides four major cell types that appear to process proteins in a manner very similar to the human brain, and they do so in half the time required by other protocols. This unique copy of the human brain and basic characteristics lay the foundation for future studies aiming to investigate human brain-specific protein patterning (e.g., isoforms, splice variants) as well as modulate glial and neuronal processes in an in situ-like environment.Copyright © 2024 Wenzel and Mousseau.