**2.2. Neuronal characterization by immunofluorescence staining**

hESC-derived neurons cultured on matrigel-coated, glass coverslips for 2 weeks were fixed for 30 min in 1% paraformaldehyde at room temperature. The cells were then washed with phosphate-buffered saline (PBS). Next, the cells were incubated for 15 min in 0.5% Triton X-100 (Sigma-Aldrich) in PBS. The cells were washed once again with PBS and blocked for 20 min with 10% donkey serum at room temperature. The cells were then incubated with the primary antibodies [microtubule-associated protein 2 (MAP2), β-tubulin III or doublecortin (Abcam, Cambridge, MA)] for 1 h in a humidified, 37°C incubator. The cells were washed with PBS and incubated for 45 min with Alexa Fluor 488 or 594 donkey anti-mouse or rabbit immunoglobulin G (Invitrogen) secondary antibodies at 37°C. The cell nuclei were stained with Hoechst 33342 (Invitrogen). The coverslips were then mounted onto glass slides and imaged using a laser-scanning confocal microscope (Nikon Eclipse TE2000-U, Nikon Inc., Melville, NY).

#### **2.3. Ketamine exposure**

This chapter will outline: (1) the use of hESCs as a model system to study anesthetic-induced neurotoxicity in the developing human brain, (2) the protocols involved in dissecting the mechanisms behind anesthetic-induced developmental neurotoxicity. We have focused on the role of deregulated intracellular calcium, mitochondrial signaling (e.g., mitochondrial membrane potential, cytochrome c release from mitochondria into cytosol, and mitochondrial fission), and microR-NAs, and (3) a brief discussion on the possible role of intracellular calcium levels, mitochondrial signaling, and altered microRNA profiles in ketamine-induced developmental neurotoxicity.

hESCs (H1 cell line, WiCell Research Institute Inc., Madison, WI) were cultured on a feeder layer of mouse embryonic fibroblasts (MEFs) that were mitotically inactivated using mitomycin C (Sigma-Aldrich, St. Louis, MO) in hESC media containing DMEM/ F12 supplemented with, 1% nonessential amino acids, 20% Knockout serum (Gibco), 1 mM L-glutamine, 4 ng/mL human recombinant basic fibroblast growth factor (bFGF, Invitrogen), 1% penicillin–streptomycin (Invitrogen) and 0.1 mM β-mercaptoethanol (Sigma-Aldrich). The media was changed daily and the cells were mechanically passaged every 5–6 days. For the studies described in this chapter, hESCs at passage 55–70 were used. To generate neurons, the hESCs were taken through a four-step differentiation protocol. The hESCs were dissociated using the protease Dispase (1.5 unit/mL, Invitrogen). The hESCs were then cultured in ultra-low attachment six-well plates (Corning Inc.,

once per day and spherical embryoid bodies (EBs) were present 24 h after dispase digestion. Five days after digestion, EBs were moved to neural induction media containing

mL heparin (Sigma) and 5 ng/mL bFGF. On day 9, the EBs were plated to matrigel-coated 35-mm dishes. Rosette-like structures were present within 5 days of the EBs plating down. The rosettes were manually separated from the surrounding cells using a serological pipette approximately 2 days after the morphology was clearly visible. The rosette cells were then transferred to matrigel-coated culture dishes and cultured in neural expansion media containing DMEM/F12 supplemented with 2% B27 without vitamin A, 1% N2 (Invitrogen), 1% nonessential amino acids, 20 ng/mL bFGF and 1 mg/mL heparin. The neural stem cells (NSCs) were passaged enzymatically every 5 days. NSCs were cultured in 60-mm matrigel-coated dishes (500,000 cells/dish) in neuron differentiation media containing neurobasal media (Gibco) supplemented with 2% B27, 100 ng/mL ascorbic acid (Sigma-Aldrich), 0.1 μM cyclic adenosine monophosphate, 10 ng/mL brain-derived neurotrophic factor, 10 ng/mL insulin-like growth factor 1 (PeproTech Inc., Rocky Hill, NJ) and 10 ng/mL glial cell-derived neurotrophic factor. The media was changed every other day. Following 2 weeks of culture, the cells displayed clear neuronal morphology and were

/5% CO2

, 37°C). The media was changed

(Invitrogen), 1% nonessential amino acids, 1 mg/

**2. Materials and methods**

424 Mitochondrial Diseases

**2.1. Stem cell culture and neuronal differentiation**

Corning, NY) in a normoxic incubator (20% O2

DMEM/F12 supplemented with 1% N2

used for the studies.

While the brain concentration of ketamine in humans during the induction and maintenance of anesthesia is not well understood, reports have found that the peak blood levels of ketamine could be as high as 103 μM. The levels required to maintain anesthesia are typically in the range of 10–20 μM. hESC-derived neurons were exposed to 6 h of 20 μM ketamine or neurobasal media as a vehicle-control in a 5% CO2 incubator with normoxic conditions only for the microRNA studies. For all remaining studies, the cells were exposed to 24 h of 20 or 100 μM ketamine or neurobasal media as the vehicle control in the same incubator.
