Mar 29, 2024

Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons V.2

Cell reports
DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwj54dlmk/v2
  • Dan Dou1,2,
  • C. Alexander Boecker3,
  • Erika L.F. Holzbaur1,2
  • 1Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, USA;
  • 3Department of Neurology, University Medical Center Goettingen, 37077 Goettingen, Germany
  • Protocols
Dan Dou
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwj54dlmk/v2
External link: https://doi.org/10.1016/j.celrep.2023.112448
Protocol Citation: Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur 2024. Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwj54dlmk/v2Version created by Dan Dou
Manuscript citation:
Pantazis, C.B., Yang, A., Lara, E., McDonough, J.A., Blauwendraat, C., Peng, L., Oguro, H., Zou, J., Sebesta, D., Pratt, G., et al. (2022). A reference induced pluripotent stem cell line for large-scale collaborative studies. BioRxiv 2021.12.15.472643.
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 29, 2024
Last Modified: May 31, 2024
Protocol  Integer ID: 97519
Keywords: iPSC, Differentiation, iNeuron, Piggybac, NGN2, ASAPCRN, inducible ngn2 in human ipsc, glutamatergic neuron, stable expression of ngn2, employing piggybac transfection, ineuron differentiation from human ipsc, excitatory glutamatergic neuron, piggybac transfection, inducible ngn2, ngn2, stable integration of ngn2, mediated stable expression, doxycycline, ineuron differentiation
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000350
Abstract
We adapted a previously-described method (Pantazis et al., 2022) for employing Piggybac transfection to stably express doxycycline-inducible NGN2 in human iPSCs. After stable integration of NGN2, proceed to differentiate iPSCs using protocol “iNeuron differentiation from human iPSCs.”
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549-1145.pdf
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Guidelines
Citations:

  • Pantazis, C.B., Yang, A., Lara, E., McDonough, J.A., Blauwendraat, C., Peng, L., Oguro, H., Zou, J., Sebesta, D., Pratt, G., et al. (2022). A reference induced pluripotent stem cell line for large-scale collaborative studies. BioRxiv 2021.12.15.472643.
Materials

Materials

  • 10 cm cell culture dish
  • 6-well cell culture dish
  • Cryovials

Reagents

  • Growth Factor Reduced (GFR) Matrigel®CorningCatalog #354230
  • Essential 8™ MediumGibco - Thermo Fisher ScientificCatalog #A1517001
  • Accutase® solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #A6964
  • Y-27632 2HClSelleckchemCatalog #S1049
  • Opti-MEM™ I Reduced Serum MediumThermo FisherCatalog #31985070
  • Lipofectamine™ Stem Transfection ReagentThermo Fisher ScientificCatalog #STEM00008
  • PB-TO-hNGN2addgeneCatalog #172115 RRID:Addgene_172115
  • piggyBac™ transposase vector (Transposagen/Hera BioLabs) #SPB-D10
  • KnockOut™ Serum ReplacementThermo FisherCatalog #10828010
  • DMSO (CATALOG)









Safety warnings
Wear proper PPE when transferring cryovials to liquid N2.
Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
3d 6h
Culture iPSCs in a 10 cm dish coated with Growth Factor Reduced Matrigel (Corning) and feed daily with Essential 8 media (ThermoFisher).
Passage iPSCs with warm Accutase into Essential 8 media with 10 micromolar (µM) ROCK inhibitor. Plate 800,000 iPSCs into one Matrigel-coated well of a 6-well plate.
3 - 6 hours after plating, cells should be healthy and attached. Perform transfection using Lipofectamine Stem and a 2:1 ratio of donor plasmid to transposase:

AB
OptiMEM200 μL
PB-TO-hNGN2-puro-BFP plasmid0.75 μg
EF1α-transposase plasmid0.37 μg
Lipofectamine Stem4 μL

Check for transfection efficiency (BFP-labeled cells) on the next day using fluorescence microscopy.
Passage iPSCs with Accutase to a 10 cm dish when cells are confluent enough for splitting.
Note
Continue to feed iPSCs daily with Essential 8 media without ROCK inhibitor, and confirm division of stably-expressing transfected cells (should observe local clusters of BFP-fluorescent cells).

72:00:00 after transfection, select for transfected iPSCs with 0.5 Mass Percent puromycin.

3d
Confirm purity of surviving transfected cells with fluorescence microscopy. When population is pure, withdraw puromycin.
Cryopreserve selected iPSCs with

AB
Essential 8 media70%
Knockout serum replacement20%
DMSO10%
ROCK inhibitor (Supplement)10 µM

Proceed to culture and induction to neuronal fate using doxycycline (see “Protocol: iNeuron differentiation from human iPSCs”).