Gabriel Elke, Gopalakrishnan Jay
Center for Molecular Medicine Cologne, University of Cologne.
Center for Molecular Medicine Cologne, University of Cologne; Institute for Biochemistry I, Medical School of University of Cologne;
J Vis Exp. 2017 Apr 14(122):55372. doi: 10.3791/55372.
The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.
能够可靠地模拟复杂人类大脑发育的合适体外模型的有限可用性是一个重大瓶颈，限制了基础脑研究向临床应用的转化。虽然诱导多能干细胞（iPSC）已取代了存在伦理问题的人类胚胎干细胞，但基于iPSC的神经元分化研究在细胞水平上仍只是描述性的，未能充分提供从复杂的三维人类脑组织中可获得的细节。现在，通过应用源自iPSC的三维脑类器官（“培养皿中的大脑”）填补了这一空白，该类器官模拟了复杂人类大脑发育的许多特征。在此，描述了一种生成源自iPSC的三维脑类器官的方法。这些类器官有助于模拟常染色体隐性原发性小头畸形（MCPH），这是一种罕见的人类神经发育障碍。对MCPH中脑畸形的一种广泛接受的解释是，在人类大脑发育早期神经干细胞池的耗竭，这是一种在体外难以重现或证实的发育缺陷。为了研究MCPH，我们从携带中心体蛋白CPAP突变的患者来源的成纤维细胞中生成了iPSC。通过分析小头畸形三维脑类器官的脑室区，我们发现了神经祖细胞的过早分化。这些三维脑类器官是一个强大的体外系统，将有助于模拟由神经毒性化学物质、神经营养性病毒感染或遗传基因突变引起的先天性脑部疾病。
