Aug 19, 2022
Version 1
iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 1 V.1
- erika.laraflores1,
- Andy Qi1,
- Luke Reilly1,
- Marianita Santiana1,
- caroline.pantazis1,
- Michael Ward1,
- Mark Cookson1
- 1National Institutes of Health, NIH Center for Alzheimer's and Related Dementias (CARD)
- Neurodegeneration Method Development Community
- iNDI Protocol Development
External link: https://www.jax.org/jax-mice-and-services/ipsc
Protocol Citation: erika.laraflores, Andy Qi, Luke Reilly, Marianita Santiana, caroline.pantazis, Michael Ward, Mark Cookson 2022. iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 1. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5969ng1b/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: December 15, 2021
Last Modified: August 19, 2022
Protocol Integer ID: 55977
Keywords: iNeurons, NGN2, Piggybac, cortical neuron differentiation, iNDI, Jackson Laboratory, CARD, NIH
Funders Acknowledgement:
National Institute of Aging and National Institute of Neurological Disorders and Stroke
Grant ID: Z01 AG000535
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
KnockOut™ DMEM/F-12Thermo FisherCatalog #12660012
N2 supplement (100x supplement)Gibco, ThermoFisherCatalog #17502048
MEM Non-Essential Amino Acids Solution (100X)Thermo FisherCatalog #11140050
Glutamax (100x)Gibco - Thermo FischerCatalog #35050-061
• Chroman I MedChemExpressCatalog #HY-15392
Doxycycline hydrochlorideSigma AldrichCatalog #D9891
PBS 1xLonzaCatalog #BE17-516F
StemPro™ Accutase™ Cell Dissociation ReagentGibco - Thermo FisherCatalog #A1110501
Poly-L-Ornithine (PLO)Sigma AldrichCatalog #P3655
Borate Buffered SalineSigma AldrichCatalog #08059
BrainPhys™ Neuronal MediumStem Cell TechnologiesCatalog #05790
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
UridineSigma AldrichCatalog #U3003
5-Fluoro-2′-deoxyuridineSigma AldrichCatalog #F0503
N21-MAX Media Supplement (50X)R&D SystemsCatalog #AR008
Bovine Serum AlbuminJackson ImmunoresearchCatalog #001-000-173
Medium Preparation
Medium Preparation
Induction Medium:
For day 0 to day 3
| A | B | C | D | |
| Reagent | Stock | Final concentration | Amount for 50mL of medium | |
| Knock out 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
| A | B | C | D | |
| Reagent | Stock | Final concentration | Amount for 50mL of medium | |
| Knockout 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 |
Neuronal Maturation Medium:
For day 10 to day 28
| A | B | C | D | |
| Reagent | Stock | Final concentration | Amount for 50mL of medium | |
| BrainPhys neuronal medium | ------------ | ------------- | 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 |
Differentiation Protocol
Differentiation Protocol
1h 45m
1h 45m
Day 0
The iPSCs with a stably integrated human NGN2 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 cell 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 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 the culture surface 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 0.5-1 x 106 iPSCs per one well of 6-well plate in 2-3 mL of Induction Medium or 4-6 × 106 per 10-cm dish in 10-12 mL to be differentiated.
Gently rock plate to evenly distribute cells and place in 37 °C incubator.
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.
- Repeat medium exchange with induction medium + Uridine and Fluorodeoxyuridine (FdU) both at 1 micromolar (µM) .
Note
When using PiggyBac system for hNGN2 the culture might have some mitotic cells, and to suppress them, we add to the neuronal maturation medium, Uridine and Fluorodeoxyuridine (FdU) both at 1 micromolar (µM) from day 3 to 28.
Differentiation Protocol
Differentiation Protocol
1h 45m
1h 45m
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.
1h
Plating pre-differentiated neurons day 4
Once cells are confirmed to be healthy, they should be dissociated with Accutase to re-plated onto final dishes for neuronal maturation and experimental manipulation
Prepare fresh Neuronal Maturation Medium + Uridine and Fluorodeoxyuridine (FdU) both at 1 micromolar (µM)
After dissociating cells with Accutase as step 2.4 to 2.9 resuspend cell pellet with Neuronal Maturation Medium + Uridine and Fluorodeoxyuridine (FdU) both at 1 micromolar (µM) and count.
Plate 2 x 106 pre-differentiated neurons onto a PLO-coated 6 well with 3-4 mL of Neuronal Maturation Medium + Uridine and Fluorodeoxyuridine (FdU) both at 1 micromolar (µM) .
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) 10,000 in 100 µL medium/well.
- 96 well plate (imaging) 6 x 104 in 300 µL medium/well.
- 12 well plate (Biochemistry) 0.75 to 0.85 x 104 in 2 mL medium/well.
- 6 well plate (Biochemistry) 2 x 106 in 3-4 mL medium/well
- 10 cm dish 8 to 10 x 106 in 10- 12 mL medium.
After day 4 do half of the medium change every 3-4 days with Neuronal Maturation Medium + Uridine and Fluorodeoxyuridine (FdU) both at 1 micromolar (µM) .