Atamian Alexander, Birtele Marcella, Hosseini Negar, Quadrato Giorgia
Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine University of Southern California, Los Angeles, CA, USA.
Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
Nat Protoc. 2024 Dec 2. doi: 10.1038/s41596-024-01093-w.
The advancement of research on human cerebellar development and diseases has been hindered by the lack of a cell-based system that mirrors the cellular diversity and functional characteristics of the human cerebellum. Here, we describe our protocol for a human pluripotent stem cell-derived human cerebellar organoid (hCerO) model, which successfully replicates the cellular diversity of the fetal cerebellum along with some of its distinct cytoarchitectural features. Our approach involves the patterning of human pluripotent stem cells, resulting in the generation of both cerebellar excitatory and inhibitory progenitor populations-specifically, the rhombic lip and ventricular zone progenitors, respectively. This patterning strategy leads to the reproducible differentiation of the major neurons of the cerebellum such as granule cells and Purkinje cells within just one month of culture. hCerOs serve as platforms for molecular, cellular and functional assays, including single-cell transcriptomics, immunohistochemistry and investigations into calcium dynamics and electrophysiological properties. Remarkably, the cultivation of hCerOs for up to 8 months enables the healthy survival and maturation of Purkinje cells, which exhibit molecular and electrophysiological features akin to their in vivo counterparts. Overall, our protocol generates and allows for the long-term culture of all major cell types within the cerebellum. Consequently, this significant advancement provides the developmental neurobiology field with a robust platform for exploring both cerebellar development and diseases within an all-human system. This protocol can be easily implemented by a technician with cell culture experience and takes 1-2 months to complete with an option for extended maturation over the course of several months.
由于缺乏能够反映人类小脑细胞多样性和功能特征的细胞系统，人类小脑发育与疾病的研究进展受到了阻碍。在此，我们描述了一种源自人类多能干细胞的人类小脑类器官（hCerO）模型的方案，该模型成功复制了胎儿小脑的细胞多样性及其一些独特的细胞结构特征。我们的方法涉及对人类多能干细胞进行模式化处理，从而分别产生小脑兴奋性和抑制性祖细胞群，具体而言，即分别产生菱唇祖细胞和脑室区祖细胞。这种模式化策略能够在仅一个月的培养时间内，使小脑的主要神经元如颗粒细胞和浦肯野细胞实现可重复的分化。hCerO可作为分子、细胞和功能分析的平台，包括单细胞转录组学、免疫组织化学以及对钙动力学和电生理特性的研究。值得注意的是，将hCerO培养长达8个月可使浦肯野细胞健康存活并成熟，这些细胞展现出与体内对应细胞相似的分子和电生理特征。总体而言，我们的方案能够生成并实现小脑中所有主要细胞类型的长期培养。因此，这一重大进展为发育神经生物学领域提供了一个强大的平台，用于在全人类系统中探索小脑发育和疾病。该方案具有细胞培养经验的技术人员即可轻松实施，完成时间为1 - 2个月，还可选择在数月内进行延长成熟培养。
