High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons

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

“…Effects on cellular metabolic activity were then assessed using CellTiter Blue as a surrogate readout for viability. A follow‐up toxicity assessment was also performed using an image‐based single cell algorithm based on DAPI stained nuclei [23]. Screening activities using both neuronal populations were robust using this assay format with Z ‐factors = 0.7–0.8 (Fig.…”

“…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]. 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].…”

“…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]. Further western blot analysis demonstrated the progressive expression of synaptophysin with neuronal differentiation and maturation (Fig.…”

“…To examine the electrophysiological properties RMP, AP generating ability, and spiking pattern, we performed whole cell current-clamp recordings on maturing cultures (i.e., 4-6 weeks) using our modified culturing conditions [23]. Patched cells showed fluctuating depolarized RMPs (i.e., À15 to À45 mV), which led us to artificially dial the membrane potential to near normal RMP (À55 to À70 mV).…”