Method Article

Human Pluripotent Stem Cell Differentiation in Neurons Using Engineered Lentiviruses

July 8th, 2025

In This Article

Abstract

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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 process of inducing human pluripotent stem cells to differentiate into neurons by transfecting them with engineered lentiviruses carrying a neurogenic transcription factor gene under the control of tetracycline-responsive element, followed by treatment with a tetracycline derivative and maturation medium.

Protocol

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1. Human neuron induction from human pluripotent stem cells

  1. Lentivirus preparation (~5 days, detailed protocol as described previously)
    1. Plate ~1 million HEK293T cells in 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 lentiviral vector DNA and 6 µg of each of the helper plasmid DNA). Prepare at least three flasks per lentivirus preparation. Use PEI for transfection following ....

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Results

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Differentiation timeline and hESC fluorescence images illustrating neural induction and synapse formation.
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 pluripote.......

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
AccutaseSTEMCELL Technologies7920
B27 supplementThermoFisher17504044
DMEM/F12 mediumSTEMCELL Technologies36254
DMSOThermoFisherD12345
DoxycyclineMilliporeSigmaD3072
H1 human ES cellsWiCellWA01
MatrigelCorning354234
mTeSR plusSTEMCELL Technologies5825
N2 supplementThermoFisher17502001
Neurobasal A mediumThermoFisher10888-022
Non Essential Amino AcidsThermoFisher11140-050
PEIVWR71002-812
pMDLg/pRREAddgene12251
PolybreneMilliporeSigmaTR-1003-G
pRSV-REVAddgene12253
PuromycinThermoFisherA1113803
STEMdiff Neural Progenitor Freezing MediaSTEMCELL Technologies5838
STEMdiff SMADi Neural Induction KitSTEMCELL Technologies8581
Tempo-iOlogo: Human iPSC-derived OPCsTempo BioScienceSKU102
TetO-Ng2-PuroAddgene52047
VSV-GAddgene12259

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Tags

Human Pluripotent Stem CellsNeuron DifferentiationEngineered LentivirusesTetracycline Responsive ElementNeurogenic Transcription FactorViral TransfectionPuromycin SelectionCell Detachment SolutionECM Coated WellsMaturation Medium

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