Rybachuk Oksana, Kopach Olga, Pivneva Tetyana, Kyryk Vitaliy
Department of Sensory Signaling, Bogomoletz Institute of Physiology, Kyiv, Ukraine.
State Institute of Genetic and Regenerative Medicine, Kyiv, Ukraine.
Bio Protoc. 2019 Feb 20;9(4):e3165. doi: 10.21769/BioProtoc.3165.
For both stem cell research and treatment of the central nervous system disorders, neural stem/progenitor cells (NSPCs) represent an important breakthrough tool. In the expanded stem cell-based therapy use, NSPCs not only provide a powerful cell source for neural cell replacement but a useful model for developmental biology research. Despite numerous approaches were described for isolation of NSPCs from either fetal or adult brain, the main issue remains in extending cell survival following isolation. Here we provide a simple and affordable protocol for making viable NSPCs from the fetal mouse hippocampi, which are capable of maintaining the high viability in a 2D monolayer cell culture or generating 3D neuro-spheroids of cell aggregates. Further, we describe the detailed method for engraftment of embryonic NSPCs onto a host hippocampal tissue for promoting multilinear cell differentiation and maturation within endogenous environment. Our experimental data demonstrate that embryonic NSPCs isolated using this approach show the high viability (above 88%). Within a host tissue, these cells were capable of differentiating to the main neural subpopulations (principal neurons, oligodendrocytes, astroglia). Finally, NSPC-derived neurons demonstrated matured functional properties (electrophysiological activity), becoming functionally integrated into the host hippocampal circuits within a couple of weeks after engraftment.
对于干细胞研究和中枢神经系统疾病的治疗而言，神经干/祖细胞（NSPCs）是一项重要的突破性工具。在基于干细胞疗法的广泛应用中，NSPCs不仅为神经细胞替代提供了强大的细胞来源，还为发育生物学研究提供了有用的模型。尽管已经描述了许多从胎儿或成体大脑中分离NSPCs的方法，但主要问题仍然在于分离后延长细胞存活时间。在这里，我们提供了一种简单且经济实惠的方案，用于从胎鼠海马体中制备有活力的NSPCs，这些细胞能够在二维单层细胞培养中保持高活力，或生成细胞聚集体的三维神经球。此外，我们描述了将胚胎NSPCs移植到宿主海马组织上的详细方法，以促进内源性环境中多系细胞的分化和成熟。我们的实验数据表明，使用这种方法分离的胚胎NSPCs具有高活力（超过88%）。在宿主组织内，这些细胞能够分化为主要的神经亚群（主要神经元、少突胶质细胞、星形胶质细胞）。最后，NSPCs衍生的神经元表现出成熟的功能特性（电生理活性），在移植后几周内功能上整合到宿主海马回路中。
