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Figure 1. Human iPSC-Derived Forebrain Neuron Precursor Cells Exhibit High-Quality Morphology

Cryopreserved Human iPSC-Derived Forebrain Neuron Precursor Cells were generated from SCTi003-A iPSCs were thawed and plated onto PLO/Laminin-coated plates at 80,000 cells/cm2 in STEMdiff™ Forebrain Neuron Maturation Kit. Neurons were incubated at 37°C and subsequently analyzed by brightfield microscopy at 10x magnification. Forebrain neurons display the expected neuronal morphology with minimal clumping over long-term culture. iPSC = induced pluripotent stem cell

Figure 2. Neurons Matured from Human iPSC-Derived Forebrain Neuron Precursor Cells Consist of a Highly Pure Neuronal Population

Human iPSC-Derived Neuron Precursor Cells generated from SCTi003-A iPSCs were thawed, established in culture, and fixed for immunocytochemistry. (A) Day 14 neurons express high levels of neuronal marker βIII-TUB (red) with low expression of S100B (green). In addition, they display the typical neuronal morphology with healthy neurites and minimal cell clumping. (B) The percentage expression of these markers were quantified. Neuronal marker βIII-TUB was found to be expressed in 95% of neurons, while glial marker S100B was expressed in less than 4% of neurons. Error bars represent standard deviation (n = 2 biological replicates). iPSC = induced pluripotent stem cell

Figure 3. Neurons Matured from Human iPSC-Derived Forebrain Neuron Precursor Cells Express Characteristic Neuronal Markers

Human iPSC-Derived Neuron Precursor Cells generated from SCTi003-A iPSCs were thawed, established in culture, and fixed for immunocytochemistry. Day 24 neurons express neuronal marker MAP2, (A) GABA, (B) forebrain neuron marker FOXG1, and (C) pre-synaptic marker Synapsin. In addition, they display the typical neuronal morphology with healthy neurites and minimal cell clumping. iPSC = induced pluripotent stem cell

Figure 4. Human iPSC-Derived Forebrain Neuron Precursor Cells Express Expected Neuronal Markers

Human iPSC-Derived Forebrain Neuron Precursor Cells were generated from iPS cell line SCTi003-A. The neuron precursors were then matured with STEMdiff™ Forebrain Neuron Maturation Kit. RNA from forebrain neurons was harvested at various time points during maturation (Day 0, Day 14, and Day 21 in maturation media). This RNA from forebrain neurons along with RNA from the parent hPSC control were subsequently sequenced using bulk RNA-seq. The heat map displays expression levels for select genes associated with hPSCs, NPCs or forebrain neuron precursors, and forebrain neurons. iPS = induced pluripotent stem; hPSC = human pluripotent stem cell; NPC = neural progenitor cell

Figure 5. Human iPSC-Derived Forebrain Neuron Precursor Cells Increase Neuronal Activity Over 42 Days in Culture

Human iPSC-Derived Forebrain Neuron Precursor Cells were generated from the hiPSC line SCTi003-A (Catalog #200-0511). The neuron precursors were then matured on a 48-well MEA plate (Axion Biosystems) with STEMdiff™ Forebrain Neuron Maturation Kit (Catalog #08605). Electrical activity from 16 electrodes was measured over time using the Maestro Pro™ MEA system (Axion Biosystems). (A - D) Detected spikes (black lines), single channel bursts (blue lines; a collection of at least 5 spikes, each separated by an ISI of no more than 100 ms), and network bursts (orange boxes; a collection of at least 50 spikes from a minimum of 35% of participating electrodes, each separated by an ISI of no more than 100 ms) were recorded for each timepoint. Neuronal activity can be detected by Day 14 and increases over time throughout the 42-day culture period. (E) Mean firing rate, (F) number of bursts, and (G) synchrony index were all shown to increase over the 42-day culture period. hiPSC = human induced pluripotent stem cell; MEA = microelectrode array; ISI = inter-spike interval