Jun 14, 2023

iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 2

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvj3owblk5/v1
iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 2
  • 1National Institutes of Health, NIH Center for Alzheimer's and Related Dementias (CARD)
  • NIH Center for Alzheimer's and Related Dementias
Erika Lara Flores
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvj3owblk5/v1
Protocol Citation: Erika Lara Flores, Andy Qi, Luke Reilly, Marianita Santiana, Michael Ward, Mark Cookson 2023. iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 2. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj3owblk5/v1
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: June 14, 2023
Last Modified: August 08, 2023
Protocol  Integer ID: 83415
Keywords: ngn2 differentiation of human ipsc, human neurogenin, induced pluripotent stem cell, ngn2 differentiation, developing new therapeutic target, pluripotent stem cell, neurodegenerative disorder, new therapeutic target, diverse types of neurodegenerative disorder, inducible promoter, cortical neuron marker, transcription factor, high percentages of cortical neuron marker, neuron, cortical neurons version, ngn2, cellular mechanism
Abstract
Induced pluripotent stem cell (iPSC)-derived neurons are an important tool for studying diverse types of neurodegenerative disorders, including Alzheimer’s Disease, Parkinson’s disease, and related dementias. Understanding the molecular and cellular mechanisms associated with these diseases is an important step in developing new therapeutic targets. Here we describe a robust differentiation protocol in which we expressed the human neurogenin 2 (NGN2) transcription factor under a tetracycline-inducible promoter as previously described (Fernandopulle et al. 2018), with several modifications and using a PiggyBac system for delivery. This differentiation protocol yields high percentages of cortical neuron markers.
Materials
Reagents
Matrigel hESC-Qualified Matrix, LDEV-freeCorningCatalog #354277
DMEM/F-12, HEPESThermo Fisher ScientificCatalog #11330032
N2 supplement (100x supplement)Gibco - Thermo Fisher ScientificCatalog #17502048
MEM Non-Essential Amino Acids Solution (100X)Thermo FisherCatalog #11140050
Glutamax (100x)Gibco - Thermo Fisher ScientificCatalog #35050-061
• Chroman I MedChemExpressCatalog #HY-15392
DPBS no calcium no magnesiumGibco - Thermo Fisher ScientificCatalog #14190250
StemPro™ Accutase™ Cell Dissociation ReagentGibco - Thermo Fisher ScientificCatalog #A1110501
Poly-L-Ornithine (PLO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3655
Borate Buffered SalineMerck MilliporeSigma (Sigma-Aldrich)Catalog #08059
BrainPhys™ Neuronal MediumSTEMCELL Technologies Inc.Catalog #05790
N21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
Recombinant human GDNFpeprotechCatalog #450-10
Recombinant Human/Murine/Rat BDNFpeprotechCatalog #450-02
Recombinant Human NT-3peprotechCatalog #450-03
Cultrex 3-D Culture Matrix Laminin IR&D SystemsCatalog #3446-005-01
Quality Bio Cell Culture Grade WaterQuality BiologicalCatalog #118-162-101CS
5-Fluoro-2′-deoxyuridineMerck MilliporeSigma (Sigma-Aldrich)Catalog #F0503
UridineMerck MilliporeSigma (Sigma-Aldrich)Catalog #U3003
Bovine Serum AlbuminJackson ImmunoResearch Laboratories, Inc.Catalog #001-000-173
Doxycycline hyclateMerck MilliporeSigma (Sigma-Aldrich)Catalog #D9891




Medium Preparation
Induction Medium:
For day 0 to day 3
ABCD
Reagent Stock Final concentration Amount for 50mL of medium
DMEM/F12 --------- ------- 48.5 mL
N2 supplement 100X 1X 0.5 mL
Non-essential amino acids (NEAA ) 100X 1X 0.5 mL
Glutamax 100X 1X 0.5 mL
Doxycycline 2mg/mL 2µg/mL 0.05 mL
Chroman I 50 µM 50 nM 0.05 mL
Neuronal Maturation Medium:
For day 4 and 7
ABCD
Reagent Stock Final concentration Amount for 50mL of medium
DMEM/F12 --------- -------    24mL
Brainphys --------- --------- 24mL
N21MAX 50X 1X 1mL
  GDNF (in 0.1%BSA/PBS) 10 µg/mL 10 ng/mL   0.05 mL 
  BDNF (in 0.1%BSA/PBS) 10 µg/mL 10 ng/mL   0.05 mL 
  NT-3 (in 0.1%BSA/PBS) 10 µg/mL 10 ng/mL   0.05 mL 
  Laminin    6 mg/mL    1 µg/mL    0.01 mL 
  Doxycycline    2mg/mL    2µg/mL    0.05 mL 
5-Fluoro-2′-deoxyuridine 1 mM 1 µM0.05 mL
Uridine 1 mM 1 µM0.05 mL
Neuronal Maturation Medium:
For day 10 to day 28
ABCD
Reagent Stock Final concentration Amount for 50mL of medium
BrainPhys ------------ ------------- 49 mL
N21MAX 50X 1X 1 mL
GDNF (in 0.1%BSA/PBS) 10 µg/mL 10 ng/mL 0.05 mL
BDNF (in 0.1%BSA/PBS) 10 µg/mL 10 ng/mL 0.05 mL
NT-3 (in 0.1%BSA/PBS) 10 µg/mL 10 ng/mL 0.05 mL
Laminin 6 mg/mL 1 µg/mL 0.01 mL
Doxycycline 2mg/mL 2µg/mL 0.05 mL
5-Fluoro-2′-deoxyuridine 1 mM 1 µM 0.05 mL
Uridine 1 mM 1 µM 0.05 mL

Differentiation Protocol
1h 45m
Day 0
The iPSCs with a stably integrated human NGN2 (plasmid Addgene #198397) using PiggyBac system under a tetracycline-inducible promoter were exposed to doxycycline as follows:

Coat a well of 6 well plate or 10cm dish to be used for differentiation with 1 mL or 4 mL respectively of Matrigel solution, tilting to ensure coverage of entire surface area. Place in 37 °C incubator for 00:30:00 to 01:00:00 .
Note
Overnight incubation gives better results



1h 30m
Prepare Induction Medium and place in 37 °C water or bead bath to warm during dissociation.


Observe iPSCs under a phase contrast microscope to assess confluency and presence of cells debris. Dish should be dissociated at ~70% to 80% confluency.

Aspirate culture medium and wash with PBS 1X.
Aspirate PBS and add half of culture volume of Accutase
Transfer to 37 °C incubator for00:10:00

Note
The time can vary by cell line and density (the optimal density is 70-80% and time can go up to 20 minutes) and the goal to use accutase is singularize as single cells.


10m
When Incubation is ready, tilt the plate and pipet the accutase solution two to three times up and down to singularize as single cells.


Quench the Accutase adding half of the culture volume of PBS. Transfer to a new conical tube and rinse with more PBS the culture surface, combine with the cell solution in the tube.


Centrifuge 00:05:00 at 200 - 300 x g at Room temperature



Note
While centrifuge, aspirate Matrigel solution from plates and add Induction Medium.

5m
Aspirate supernatant and resuspend cell pellet with Induction Medium.

Count cells, Gently transfer 25,000 iPSCs per cm2 (i.e. For a 6 well plate, seed ~2.5 x 105 cells/well).

Gently rock plate to evenly distribute cells and leave it at Room temperature for 00:10:00 to let the cells settle down then place in 37 °C incubator.

10m
Day 1
Check cells under the microscope, nascent neuritic extensions should begin to be evident after 24 h of doxyxycline exposure.

Prepare Induction Medium but without Chroman I and warm it.

Aspirate medium, wash once with PBS 1X and replace with warm induction medium.

Day 2
Check cells under the microscope, neuritic extensions should be more evident.

Repeat medium change with induction medium as on day 1.

Day 3
Check cells under a microscope. Neurites should be obvious by this time.
  1. Repeat medium change with induction media as on day 1.





Day 4
Check cells under a microscope. Pre-differentiated neurons are ready to be re-plated.




Coating dishes
Freshly prepared poly-L-ornithine (PLO), at final concentration at 0.1 mg/mL :
  • Using Sodium Borate Buffer pH 8.2, make a 1 mg/mL stock PLO solution.
  • To prepare working solution dilute to a 0.1 mg/mL with cell culture water then filter through a 0.22µm sterile filter and it is ready to use.
  • Add half of the culture volume of PLO working solution to dishes and Place in 37 °C incubator for 01:00:00 to Overnight .
  • Aspirate PLO working solution from the dishes.
  • Wash dishes with cell culture water three times.
  • Let dry completely in a culture hood.
  • Dishes are ready to use.


Note
Overnight incubation gives better results.


1h
Plating pre-differentiated neurons day 4
Once cells are confirmed to be healthy, they should be dissociated with Accutase and either frozen or re-plated onto final dishes for neuronal maturation and experimental manipulation
Prepare fresh Neuronal Maturation Medium for day 4 as referred above.
After dissociating cells with Accutase as step 2.4 to 2.9 resuspend cell pellet with Neuronal Maturation Medium for day 4 and count.



Plate 1.5 x 106 pre-differentiated neurons onto a PLO-coated 6 well with 3-4 mL of Neuronal Maturation Medium.

Note
The number of pre-differentiated neurons to be re-plated varies depending of the final assay but it can be as follows:
  • 384 well plate (imaging) 7,000 to 9,000 in 100 µL medium/well.
  • 96 well plate (imaging) 4x 104 in 300 µL medium/well.
  • 12 well plate (Biochemistry) 7x 105 in 2 mL medium/well.
  • 6 well plate (Biochemistry) 1.5 x 106 in 4 mL medium/well
  • 10 cm dish 8 x 106 in 10- 12 mL medium.

Gently rock plate to evenly distribute cells and leave it at Room temperature for 00:10:00 to let the cells settle down then place in 37 °C incubator.
10m
After day 4 do half of the medium change every 3-4 days with Neuronal Maturation Medium.