Extracellular cues accelerate neurogenesis of induced pluripotent stem cell derived neurons

Abstract

Neurogenesis is a complex process encompassing neuronal progenitor cell expansion/proliferation and differentiation, followed by neuron maturation. In vivo models are most commonly used to study neurogenesis; however, human induced pluripotent stem cell-derived (iPSC) neurons are increasingly used to establish cellular models of human neurological processes. Unfortunately, the differentiation and maturation of iPSC-derived neurons varies in methodology, is asynchronous, and has restricted experimental utility because of extended differentiation/maturation times. To accelerate and standardize iPS neuronal maturation, we differentiated and matured feeder layer-free iPSC-derived neuronal cultures under physiological oxygen levels (5%), and modified the underlying extracellular matrix and medium composition. Our results demonstrate that calretinin gene expression occurred earlier under our optimized iPS conditions and the corresponding neurogenesis burst associated with proliferative expansion occurred more synchronously, reliably emerging two and three weeks after differentiation. As expected, the expression of mature neuronal markers (i.e., NeuN+/Calbindin+) started at 4-weeks post-differentiation. qPCR microarray, western blot and single cell analyses using high content imaging indicated that 4-week iPS neuronal cultures were non-cycling with decreased expression of cyclin D1 and Ki67. Our data demonstrate that extracellular cues influence the kinetics of neurogenesis models and that feeder layer-free iPSC-derived neurogenesis can be reproducibly miniaturized.

Pharmacological profiling identifies divergent chemosensitivities of differentiating and maturingiPSC‐derived human cortical neuron populations

“…Neuronal progenitor cell culturing and maintenance was carried out as described using the 7753 NPC line [22,23]. Briefly, NPCs were maintained on Matrigel™‐coated dishes in complete NPC medium [i.e., DMEM‐F12 supplemented with Glutamax (Gibco, Gaithersburg, MD, USA), 2% B27 without vitamin A (Gibco) and 20 ng·mL −1 thermostable recombinant human Fibroblast Growth Factor (Gibco)] in 5% CO 2 and environmental O 2 at 37 °C.…”

“…In previous studies [22], we established neuronal culturing conditions that resulted in the expression of neuronal maturation markers such as NeuN, calbindin, tau, and calretinin at 4 weeks post-differentiation while in asynchronous culture. Subsequent analyses of Ki67 expression in 4-week maturing cultures, in contrast to 2-week (i.e., differentiating cultures) suggested that these neuronal cultures were no longer engaged in the cell cycle [22,23].…”

“…In previous studies [22], we established neuronal culturing conditions that resulted in the expression of neuronal maturation markers such as NeuN, calbindin, tau, and calretinin at 4 weeks post-differentiation while in asynchronous culture. Subsequent analyses of Ki67 expression in 4-week maturing cultures, in contrast to 2-week (i.e., differentiating cultures) suggested that these neuronal cultures were no longer engaged in the cell cycle [22,23]. Subsequent addition of astrocyteconditioned medium, which contains soluble factors such as ECM components [24], enhanced the percentage of NeuN expressing cells at 4-week postdifferentiation and indicated that culturing conditions resulted in mature neurons [23].…”

“…Neuronal progenitor cell culturing and maintenance was carried out as described using the 7753 NPC line [22,23].…”

“…iPSC-derived neuronal cultures received twice weekly 50% medium changes until experimentation at 2-and 4-weeks post-seeding. Two-week cultures are representative of progenitor cells that are actively undergoing cell cycle processes and maturation, while four-week cultures are representative of differentiated and matured neuronal cells that are minimally active in cell cycle processes [22,23].…”