Department of Molecular Animal Physiology, Donders Institute for Brain, Cognition and Behavior, Centre for Neuroscience, Faculty of Science, Radboud University, 6525GA, Nijmegen, The Netherlands.
NeuroDrug Research Ltd, 6525ED, Nijmegen, The Netherlands.
Stem Cell Res Ther. 2023 Mar 23;14(1):50. doi: 10.1186/s13287-023-03261-3.
Three-dimensional (3D) human brain spheroids are instrumental to study central nervous system (CNS) development and (dys)function. Yet, in current brain spheroid models the limited variety of cell types hampers an integrated exploration of CNS (disease) mechanisms.
Here we report a 5-month culture protocol that reproducibly generates H9 embryonic stem cell-derived human cortical spheroids (hCSs) with a large cell-type variety.
We established the presence of not only neuroectoderm-derived neural progenitor populations, mature excitatory and inhibitory neurons, astrocytes and oligodendrocyte (precursor) cells, but also mesoderm-derived microglia and endothelial cell populations in the hCSs via RNA-sequencing, qPCR, immunocytochemistry and transmission electron microscopy. Transcriptomic analysis revealed resemblance between the 5-months-old hCSs and dorsal frontal rather than inferior regions of human fetal brains of 19-26 weeks of gestational age. Pro-inflammatory stimulation of the generated hCSs induced a neuroinflammatory response, offering a proof-of-principle of the applicability of the spheroids.
Our protocol provides a 3D human brain cell model containing a wide variety of innately developing neuroectoderm- as well as mesoderm-derived cell types, furnishing a versatile platform for comprehensive examination of intercellular CNS communication and neurological disease mechanisms.
三维（3D）人类脑球体对于研究中枢神经系统（CNS）的发育和（功能）障碍至关重要。然而，在现有的脑球体模型中，细胞类型的有限多样性阻碍了对中枢神经系统（疾病）机制的综合探索。
在这里，我们报告了一种 5 个月的培养方案，该方案可重复性地产生具有多种细胞类型的人类胚胎干细胞源性皮质球体（hCSs）。
通过 RNA 测序、qPCR、免疫细胞化学和透射电子显微镜，我们在 hCSs 中不仅建立了神经外胚层来源的神经祖细胞群体、成熟的兴奋性和抑制性神经元、星形胶质细胞和少突胶质细胞（前体细胞），还建立了中胚层来源的小胶质细胞和内皮细胞群体。转录组分析表明，5 个月大的 hCSs 与 19-26 周妊娠龄的人类胎儿大脑的额极而不是额极以下区域相似。生成的 hCSs 的促炎刺激诱导了神经炎症反应，为球体的适用性提供了原理验证。
我们的方案提供了一种包含多种固有发育的神经外胚层和中胚层来源的细胞类型的 3D 人类大脑细胞模型，为全面研究细胞间中枢神经系统通讯和神经疾病机制提供了一个多功能平台。
