May 23, 2023

Public workspaceiNeuron differentiation from human iPSCs

Cell reports
DOI
dx.doi.org/10.17504/protocols.io.261ge348yl47/v1
  • 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
Dan Dou
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DOI: dx.doi.org/10.17504/protocols.io.261ge348yl47/v1
External link: https://doi.org/10.1016/j.celrep.2023.112448
Protocol Citation: Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur 2023. iNeuron differentiation from human iPSCs. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge348yl47/v1
Manuscript citation:
Boecker, C.A., and Holzbaur, E.L.F. (2021). Hyperactive LRRK2 kinase impairs the trafficking of axonal autophagosomes. Autophagy 00, 1–3. Boecker, C.A., Olenick, M.A., Gallagher, E.R., Ward, M.E., and Holzbaur, E.L.F. (2020). ToolBox: Live Imaging of intracellular organelle transport in induced pluripotent stem cell‐derived neurons. Traffic 21, 138–155. Fernandopulle, M.S., Prestil, R., Grunseich, C., Wang, C., Gan, L., and Ward, M.E. (2018). Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons. Curr. Protoc. Cell Biol. 79, e51.
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: October 12, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 71230
Keywords: iPSC, Differentiation, iNeuron, NGN2, i3 Neuron, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000350
Abstract
We adapted a previously-described method (Boecker et al., 2020, 2021; Fernandopulle et al., 2018) for differentiating iPSCs stably expressing mNGN2 at a safe-harbor locus into human excitatory glutamatergic neurons. Pre-i 3Neuron iPSCs (human iPSCs with an integrated doxycycline-inducible mNGN2 transgene in the AAVS1 safe-harbor locus) were a gift from M. Ward (National Institutes of Health, Maryland).
Attachments
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548-1144.pdf
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Guidelines
Citations:

  • Boecker, C.A., and Holzbaur, E.L.F. (2021). Hyperactive LRRK2 kinase impairs the trafficking of axonal autophagosomes. Autophagy 00, 1–3.
  • Boecker, C.A., Olenick, M.A., Gallagher, E.R., Ward, M.E., and Holzbaur, E.L.F. (2020). ToolBox: Live Imaging of intracellular organelle transport in induced pluripotent stem cell‐derived neurons. Traffic 21, 138–155.
  • Fernandopulle, M.S., Prestil, R., Grunseich, C., Wang, C., Gan, L., and Ward, M.E. (2018). Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons. Curr. Protoc. Cell Biol. 79, e51.
Materials

Materials

  • 10 cm cell culture dish
  • 15 cm cell culture dish
  • Cryovials


Reagents

  • ReagentGrowth Factor Reduced (GFR) Matrigel®CorningCatalog #354230
  • ReagentEssential 8™ MediumGibco, ThermoFisherCatalog #A1517001
  • ReagentACCUTASE™Stemcell TechnologiesCatalog #07920
  • ReagentDMEM/F-12, HEPESThermo Fisher ScientificCatalog #11330032
  • ReagentN-2 Supplement (100X)Thermo FisherCatalog #17502048
  • ReagentMEM Non-Essential Amino Acids Solution (100X)Thermo FisherCatalog #11140050
  • ReagentGlutaMAX™ SupplementThermo FisherCatalog #35050061
  • ReagentDoxycycline hydrochlorideSigma AldrichCatalog #D9891
  • ReagentY-27632SelleckchemCatalog #S1049
  • ReagentFetal bovine serum for cell culture (tetracycline-free)TakaraCatalog #631107
  • DMSO (CATALOG)
  • ReagentBrainPhys™ Neuronal Medium 500 mL Stemcell TechnologiesCatalog #5790
  • ReagentAnimal-Free Recombinant Human NT-3peprotechCatalog #AF-450-03
  • ReagentRecombinant Human/Murine/Rat BDNFpeprotechCatalog #450-02
  • ReagentB-27 SupplementGibco - Thermo FischerCatalog #17504044


Safety warnings
Wear proper PPE when transferring cryovials to liquid N2.
iNeuron differentiation from human iPSCs
iNeuron differentiation from human iPSCs
1w
1w
Culture pre-iNeuron iPSCs in a 10 cm dish coated with Growth Factor Reduced Matrigel in Essential 8 media, fed daily.
Note
Pre-iNeuron iPSCs should either have doxycycline-inducible NGN2 present in the safe-harbor AAVS1 locus (“i3Neurons”) or should stably express doxycycline-inducible NGN2 following piggybac transfection (see protocol: “Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons”). Before performing differentiation, iPSCs should be tested for mycoplasma, and cytogenetic analysis of G-banded metaphase cells should be performed to confirm a normal karyotype.

Passage iPSCs using warm Accutase and plate 5.5 x 106 cells onto a Matrigelcoated 15 cm dish, in Induction Media (DMEM/F12 supplemented with 1% N2- supplement [GIBCO], 1% NEAA [GIBCO], and 1% GlutaMAX [GIBCO], and containing Concentration2 μg/ml doxycycline and Amount10 μm ROCK inhibitor).
Note
DMEM/F12 supplemented with N2-supplement, NEAA and GlutaMAX can be kept at Temperature4 °C for 2-3 months. Doxycycline and ROCK inhibitor should always be added fresh.

Pipetting
After Duration24:00:00 , replace all media with fresh Induction Media, containing Concentration2 μg/ml doxycycline but no ROCK inhibitor.

1d
Pipetting
Replace again with the same media after Duration24:00:00 (Duration48:00:00 after plating).

3d
Pipetting
72 hours after plating, dissociate cells with warm Accutase.

Count cells in freezing media.


Freezing media

AB
BrainPhys70%
FBS20%
DMSO10%
BDNF10 ng/mL
NT-310 ng/mL
B-27 supplement1x

Pipetting
Imaging
Freeze down cells in a Mr. Frosty container placed in a Temperature-80 °C freezer DurationOvernight .

Overnight
On the following day, transfer cryopreserved neurons to liquid nitrogen storage.
Pipetting