Differentiating Human Induced Pluripotent Stem Cells into Neurons on Micro-Electrode Arrays

Abstract

Source: Frega, M., et al., Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays. J. Vis. Exp. (2017)

The video demonstrates a method for generating excitatory cortical neurons from human-induced pluripotent stem cells (hiPSCs). Upon doxycycline induction, the hiPSCs overexpress neurogenin-2, a neural-specific transcription factor. By using specific media and co-culturing with astrocytes, this process facilitates the hiPSCs' differentiation into neurons with synaptic connections.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Differentiation of rtTA/Ngn2-positive hiPSCs to Neurons on 6-well MEAs and Glass Coverslips

NOTE: In this protocol, the details are provided for differentiating rtTA/Ngn2-positive hiPSCs on two different substrates, i.e. 6-well MEAs (devices composed of 6 independent wells with 9 recording and 1 reference embedded microelectrodes per well) and glass coverslips in the wells of a 24-well plate. The protocols, however, can ea....

Results

Figure 1: hiPSC Differentiation into Neurons. A. Three time points of hiPSCs differentiation into neurons on coverslips. B. Plating of hiPSCs on MEAs. C. Astrocytes at 100% confluency in T75 flask (note that the cells form a tessellated monolayer). Scale .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
B-27 supplement	Gibco	0080085SA
Ca2+/Mg2+-free HBSS	Gibco	14175-095
0.05 % Trypsin-EDTA	Gibco	25300-054
2.5 % Trypsin	Gibco	15090-046
High-glucose DMEM	Gibco	11965-092
FBS	Sigma-Aldrich	F2442-500ML
Penicillin/Streptomycin	Sigma-Aldrich	P4333
70 µm cell strainer	BD Falcon	352350
DPBS	Gibco	14190-094
Basement membrane matrix	Gibco	A1413201
DMEM/F12	Gibco	11320-074
Cell detachment solution	Sigma-Aldrich	A6964
E8 medium	Gibco	A1517001
ROCK inhibitor	Gibco	A2644501	Alternatively, ROCK inhibitors like thiazovivin can be used.
6-well MEAs	Multi Channel Systems	60-6wellMEA200/30iR-Ti-tcr
Glass coverslips	VWR	631-0899
Poly-L-ornithine	Sigma-Aldrich	P3655-10MG
Laminin	Sigma-Aldrich	L2020-1MG
Doxycyclin	Sigma-Aldrich	D9891-5G
N-2 supplement	Gibco	17502-048
Non-essential amino acids	Sigma-Aldrich	M7145
NT-3, human recombinant	Promokine	C66425
BDNF, human recombinant	Promokine	C66212
Trypsin-EDTA	Gibco	25300-054
Neurobasal medium	Gibco	21103-049
Cytosine β-D-arabinofuranoside	Sigma-Aldrich	C1768-100MG