**3.1. Neuronal differentiation and characterization**

To generate neurons from the hESCs, the cells were taken through a four-step differentiation protocol as outlined in **Figure 1A** and described previously [20, 37, 38]. The neurons

**3.2. Ketamine-induced apoptosis**

ketamine-induced cell death.

peutic target.

**3.3. Alterations in intracellular calcium levels**

treated cells. \*P < 0.05 vs. control. n = 3 for each group.

TUNEL staining was used to assess cell death in hESC-derived neurons following ketamine exposure by labeling breaks in the DNA. The cells were exposed to 100 μM ketamine or control conditions for 24 h. The number of TUNEL-positive cells was significantly increased when compared to control treated cells following ketamine exposure (**Figure 2**). These findings confirm many of the previously published animal studies which have shown increased neuronal cell death following exposure of the developing brain to ketamine [39]. We focused on the investigation of the effect of ketamine on the intracellular calcium level, mitochondrial signaling, and microRNA expression in order to understand the mechanisms governing

Ketamine Induces Neuroapoptosis in Stem Cell–Derived Developing Human Neurons Possibly…

http://dx.doi.org/10.5772/intechopen.72939

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Following exposure of 2-week-old hESC-derived neurons to 100 μM ketamine or control conditions for 24 h, the intracellular calcium levels in the cells were assessed using Fluo-4AM fluorescence. The intracellular calcium levels were significantly elevated in the ketaminetreated cells when compared to control treatment (**Figure 3**). Calcium is a critical ion in the body and is fundamental in proper neuronal functioning. In neurons, calcium is crucial in synaptic activity and plasticity, cell signaling, neurotransmitter release and is involved in nearly every aspect of the cell cycle [24]. The careful balance of intracellular calcium levels is crucial to cell survival. Calcium homeostasis dysregulation, in particular calcium overload in the cell, has been linked to many different neurodegenerative diseases [25]. The findings from this study suggest that disrupted intracellular calcium homeostasis may also be linked to ketamine-induced cell death in developing neurons which could prove to be a novel thera-

The mitochondria are extremely important organelles involved in many cellular processes including energy production, cell signaling, and apoptosis [40–46]. Given that ketamine may

**Figure 2.** Exposure to 100 μM ketamine for 24 h induced significant cell death in the hESC-derived neurons. Ketamine induced an increase in the number of TUNEL-positive cells indicating significant cell death when compared to control-

**3.4. Ketamine induces neuronal apoptosis via mitochondrial pathway**

**Figure 1.** Differentiation protocol and confocal images of immunolabeled human embryonic stem cell (hESC)-derived neurons. (A) Neurons were derived from hESCs through a four step differentiation protocol. (B) Neurons were stained with antibodies against the neuron-specific proteins, microtubule-associated protein 2 (MAP2) and β-tubulin III to assess the differentiation efficiency, and doublecortin to confirm the immaturity of the neurons [37].

exhibited a characteristic neuronal morphology with small cell bodies and long projections. The neurons also formed extensive, interconnected networks over time. The cells displayed a very distinct morphology at each stage of the differentiation protocol. The hESCs formed tight colonies when cultured on a feeder layer and the EBs formed three-dimensional aggregates when suspended in culture media. At the neural rosette stage, the NSCs formed tightly packed arrangements with a characteristic design and were bordered by several additional cell types. Once mechanically separated from the surrounding non-NSCs and digested, the NSCs separated from one another and spread out on the matrigel-coated dishes. At this point, the cells proliferated extensively.

The neurons were immunostained after 2 weeks in differentiation media and expressed the neuron-specific markers MAP2 and β-tubulin III and formed synapses as assessed by the positive staining of Synapsin I. Based upon the immunostaining, the differentiation protocol was 90–95% efficient in the generation of neurons. In an attempt to better gauge the maturity level of the hESC-derived neurons, the cells were also immunostained for doublecortin, a marker of immature/migrating neurons. In assessing the staining results, most of the neurons in culture (90–95%) were positive for this marker of immature neurons (**Figure 1B**) which suggests that this model system is a valuable representation of developing human neurons.
