Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

Summary

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer’s disease.

Abstract

In Alzheimer’s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

Introduction

Oligodendrocyte lineage cells—including oligodendrocyte precursor cells (OPCs), myelinating oligodendrocytes, and transitional types in between—constitute a major group of human brain cells¹ that actively participate in many critical functions for the proper operation and maintenance of our central nervous system throughout neural development and aging^2,3,4. While oligodendrocytes are well known for producing myelin to facilitate neuronal activity transmission and support axonal health in white matter, OPCs are abundant (~5%) in gray matter....

Protocol

1. Human neuron induction from human pluripotent stem cells

1. Lentivirus preparation (~5 days, detailed protocol as described previously¹⁶)
   1. Plate ~1 million HEK293T cells each T75 flask, to have them ~40% confluent when performing transfection. Transfect them with plasmids expressing tetracycline-inducible Ngn2 and puromycin-resistant gene (PuroR; under the same TetO promoter control), rtTA and the three helper plasmids pRSV-REV, pMDLg/pRRE, and VSV-G (12 µg of lentivira.......

Discussion

In addition to the physical and metabolic support to stabilize the synapse structures and to facilitate the saltatory signal conduction by myelination, oligodendrocyte lineage cells can shape neuronal activity pattern via rapid and dynamic cross-talks with neurons^5,6,7. While in AD pathology the oligodendroglial responses were initially regarded as merely secondary to inflammation and oxidative stresses, there is now promising e.......

Materials

Name	Company	Catalog Number	Comments
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