A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

Abstract

Source: Assetta, B.,et al. Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions. J. Vis. Exp. (2020).

This video demonstrates the establishment of a co-culture of human induced neurons (iNs) and induced oligodendrocyte precursor cells (iOPCs). iNs are mixed with iOPCs in a co-culture and then incubated to support the maturation of both iNs into neurons and iOPCs into oligodendrocytes. Neurons form synaptic connections with other neurons and oligodendrocytes, while oligodendrocytes develop myelin sheaths around the neurons' axons.

Protocol

1. Human neuron induction from human pluripotent stem cells Lentivirus preparation (~5 days) Plate ~1 million Human embryonic kidney 293T (HEK293T) cells each T75 flask, to have them ~40% confluent when performing transfection. Transfect them with plasmids expressing tetracycline-inducible neurogenin 2 (Ngn2) and puromycin-resistant gene (PuroR; under the same Tet operator (TetO) promoter control), reverse tetracycline transactivator (rtTA) and the three helper plasmids pRSV-REV, …

Representative Results

Figure 1: Direct Generation of human induced neurons (iNs) from hPSCs. (A) Flow diagram of iN generation. (B) Representative bright field and immunofluorescence images of the starting culture of human pluripotent stem cells (H1) to confirm the pluripotency. Oct4 is shown in red and Sox2 in green. (C) Representative bright field images of iNs at D…

Materials

Accutase	STEMCELL Technologies	7920
B27 supplement	ThermoFisher	17504044
bFGF	ThermoFisher	PHG 0266
cAMP	MilliporeSigma	A9501
Clemastine	MilliporeSigma	SML0445
DMEM/F12 medium	STEMCELL Technologies	36254
DMSO	ThermoFisher	D12345
Doxycycline	MilliporeSigma	D3072
Fetal Bovine Serum	ScienCell	10
H1 human ES cells	WiCell	WA01
Matrigel	Corning	354234
mTeSR plus	STEMCELL Technologies	5825
N2 supplement	ThermoFisher	17502001
Neurobasal A medium	ThermoFisher	10888-022
Non Essential Amino Acids	ThermoFisher	11140-050
PDGF-AA	R&D Systems	221-AA-010
PEI	VWR	71002-812
pMDLg/pRRE	Addgene	12251
Polybrene	MilliporeSigma	TR-1003-G
pRSV-REV	Addgene	12253
Puromycin	ThermoFisher	A1113803
ROCK Inhibitor Y-27632	STEMCELL Technologies	72302
SAG	Tocris	4366
STEMdiff Neural Progenitor Freezing Media	STEMCELL Technologies	5838
STEMdiff SMADi Neural Induction Kit	STEMCELL Technologies	8581
T3 triiodothyronine	MilliporeSigma	T6397
Tempo-iOlogo: Human iPSC-derived OPCs	Tempo BioScience	SKU102
TetO-Ng2-Puro	Addgene	52047
VSV-G	Addgene	12259