An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Abstract

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This coculture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

Choline Ameliorates Disease Phenotypes in Human iPSC Models of Rett Syndrome

“…The generation of iPSCs from RTT patient fibroblasts has been described previously in Su et al (2015). The fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS).…”

“…The neural induction of iPSCs and the subsequent differentiation of the neural progenitor cells to neurons have been previously described in Su et al (2015). Briefly, when the iPSCs cultures reached approximate confluence of 20 %, the mTeSR TM 1 medium was removed and replaced with neural induction media containing DMEM/F12 and neurobasal media in 1:1 ratio, 19 N2 supplement, 19 B27 supplement, 19 GlutaMAX, 19 penicillin/streptomycin, 5 lg/mL BSA, 4 lM CHIR99021 (Cellagentech), 3 lM SB431542 (Cellagentech), 0.1 lM Compound E (c-Secretase Inhibitor XXI, EMD Chemicals Inc.), and 10 ng/mL human LIF (Millipore).…”

“…Half of the media was replaced every 3-4 days during maintenance. In the electrophysiological studies, hNPCs were co-cultured with primary rat cortical neurons and allowed to differentiate, as reported in Su et al (2015). For cells cultured with choline, choline chloride (Sigma-Aldrich) was dissolved in phosphate-buffered saline (PBS) to make a stock solution of 1 mM.…”

“…As primary neurons have been shown to facilitate the maturation of iPSC-derived neurons (Weick et al 2011;Verpelli et al 2013;Su et al 2015), we cultured iPSC-derived hNPCs together with primary rat cortical neurons. To identify iPSC-differentiated neurons among the co-culture system, we selectively expressed a fluorescent protein, tdTomato, behind a neuron-specific Synapsin 1 (Syn1) promoter in the hNPCs.…”

Neuropilin 2 Signaling Is Involved in Cell Positioning of Adult-born Neurons through Glycogen Synthase Kinase-3β (GSK3β)

“…Hippocampal neurons were isolated from the hippocampi of embryonic rats from timed-mated Sprague-Dawley as described previously (39,40). The following primary antibodies were used for Western blotting analysis: rabbit anti-phospho-GSK3␤ (Ser-9) (Cell Signaling Technology, 1:1000), mouse antiTubulin (Sigma, 1:10000), and rabbit anti-GSK3␤ (Cell Signaling Technology, 1:1000).…”