May 23, 2023

Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes

Cell reports
DOI
https://dx.doi.org/10.17504/protocols.io.x54v9dj4zg3e/v1
  • Dan Dou1,2,
  • 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: https://dx.doi.org/10.17504/protocols.io.x54v9dj4zg3e/v1
External link: https://doi.org/10.1016/j.celrep.2023.112448
Protocol Citation: Dan Dou, Alexander Boecker, Erika L.F. Holzbaur 2023. Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9dj4zg3e/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 13, 2022
Last Modified: May 31, 2024
Protocol  Integer ID: 71294
Keywords: iPSC, iNeuron, live-imaging, axon, ASAPCRN, imaging of axonal cargo, glutamatergic neuron, excitatory glutamatergic neuron, excitatory glutamatergic neurons for the purpose, transport of axonal cargo, ineuron differentiation from human ipsc, derived neuron, axonal cargo, differentiation of neuron, transfection of ipsc, disk confocal microscopy, neuron, transfect human ipsc, confocal microscopy, derived excitatory, microscopy, ipscs for differentiation, human ipsc, imaging, ipsc, live imaging
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000350
Abstract
Here, we plate, culture, and transfect human iPSC-derived excitatory glutamatergic neurons for the purpose of observing transport of axonal cargoes under spinning disk confocal microscopy. Protocol is largely as previously described (Boecker et al., 2020, 2021; Fernandopulle et al., 2018). For preceding differentiation of neurons, see “Protocol: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons” and “Protocol: iNeuron differentiation from human iPSCs.”
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Materials

Materials

Equipment
35 mm Dish | No. 1.5 Coverslip | 20 mm Glass Diameter | Uncoated
NAME
Coverslip
TYPE
Mattek
BRAND
p35g-1-5-20-c
SKU
https://www.mattek.com/store/p35g-1-5-20-c-case/
LINK


Reagents

  • PLO (CATALOG)
  • Borate buffer (CATALOG)
  • BrainPhys media (CATALOG)
  • NT-3 (CATALOG)
  • BDNF (CATALOG)
  • B-27 supplement (CATALOG)
  • Mouse laminin (CATALOG)
  • 5-Fluoro-2′-deoxyuridine
  • Uridine
Culture and transfection of iPSCderived neurons for live-imaging of axonal cargoes
30m
In advance, prepare 10x PLO stock.


AB
PLO50 mg
0.1M borate buffer50 mL

Note
Store 10x PLO stock at -80 °C .


The day before plating, coat imaging dishes with 1x PLO solution (10x PLO stock diluted in ddH2O).
Note
It is only necessary to fully coat the glass center of the imaging dish.

The day of plating, remove PLO solution from imaging dishes and wash twice with ddH2O.
Add 2 mL of iNeuron culture media.

BrainPhys supplemented with
AB
BDNF10 ng/mL
NT-310 ng/mL
Laminin1 μg/mL
B-27 supplement1x

Place dishes in cell culture incubator for >00:30:00 .
30m
Rapidly thaw cryopreserved iNeurons in 37 °C water bath.
Note
Retrieve vial to tissue culture hood when only a small amount of ice remains visible.

Centrifuge to remove freezing media and resuspend cell pellet in iNeuron culture media.

BrainPhys supplemented with

AB
BDNF10 ng/mL
NT-310 ng/mL
Laminin1 μg/mL
B-27 supplement1x

Count cells and plate 300k neurons per 35 mm imaging dish.



Add cells dropwise to the center area of the dish (so that they sink onto the glass, PLO-coated center).
For Piggybac-delivered NGN2 neurons, include 10 micromolar (µM) 5-Fluoro-2′-deoxyuridine and 10 micromolar (µM) uridine at the time of plating to prevent survival of mitotic cells.
Note
These drugs were removed 24 hours after plating.


Store neurons in cell culture incubator. Perform partial change of iNeuron media twice per week.
On DIV18 (~72:00:00 prior to imaging), transfect iNeurons for imaging.
Note
Transfection conditions may require optimization, but a typical transfection will use 4 µL Lipofectamine Stem and 1 µg of plasmid DNA. Plasmids with the PGK or EF1α promoters express best in iNeurons.




3d