National Human Neural Stem Cell Resource, Centers for Neuroscience and Translational Research, Children's Hospital of Orange County Research Institute, Orange, California.
J Neurosci Res. 2013 Oct;91(10):1247-62. doi: 10.1002/jnr.23245. Epub 2013 Jul 26.
Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials.
开发针对患者特异性、基于人诱导多能干细胞 (iPSC) 的大脑治疗方法的稳健策略需要能够衍生出大量高度定义的神经细胞。iPSC 培养技术的最新进展包括部分至完全消除饲养层、使用定义明确的培养基和单细胞传代。然而，这些技术仍然需要胚状体形成或共培养才能分化为神经干细胞 (NSC)。此外，发表的方法学中没有一种方法在单个培养系统中采用所有的进展。在这里，我们描述了一种使用无饲养层、定义明确的培养系统长期进行单细胞传代的可靠方法，该系统可产生贴壁、贴壁的 PSC，可分化为 NSC。为了提供稳健的质量控制基础，我们设计了一种细胞命名系统，可描述细胞在特定过程阶段的准确基因型和表型。我们证明，该方案允许从所有经过测试的细胞系高效、大规模、符合 cGMP 标准地生产可移植的 NSC。我们还表明，用所描述的方法产生的 iPSC 产生的 NSC 能够形成表达 S100β 的胶质细胞和能够产生重复动作电位的神经元。
