Accelerated Maturation of Non‐cycling Human Induced Pluripotent Stem Cell‐derived neurons for High Content Screening

Abstract

Human induced pluripotent stem cell-derived (iPSC) neurons are increasingly being used as a platform for neurological disease modeling, high throughput/content screening and drug discovery. However, the differentiation and maturation of iPSC-derived neurons is not standardized and varies in methodology (i.e., timing) limiting their usefulness in automated drug screening platforms. To generate mature iPS-derived neurons for high content screening, we derived neuronal progenitor cells (NPCs) from the normal iPSC control lines, 7545-5b and 7753, using monolayer derivation methods. We differentiated these NPCs under environmental oxygen (21%) conditions and discerned that mature neuronal markers were predominantly expressed by 12 weeks post-differentiation. When we differentiated and matured 7753- and 7545-derived neuronal cultures under physiological (5%) oxygen levels, however, iPS neuronal maturation was markedly accelerated with increased gene and protein expression of mature neuronal markers (i.e., calretinin, calbindin, NeuN) being seen at 6 weeks post differentiation. Further modifications of the underlying extracellular matrix (i.e., poly-D-ornithine, laminin), and medium composition and transition shifted the expression of mature neuronal markers to 4 weeks post-differentiation. This coincided with the cessation of the neurogenesis burst which occurred between weeks 2 and 3 post-differentiation. qPCR microarray and western blot data as well as single cell analyses showed that 4 week iPS neuronal cultures were non-cycling with decreased expression of cyclin D1 and Ki67. Further optimization of cell seeding density and incubations ameliorated edge effects associated with prolonged cell culturing. Taken together, we have developed conditions for the derivation of a mature iPSC-derived neurons in 4 weeks, which are amenable to high throughput and high content screening applications.