TITLE: iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 2
AUTHORS: Erika Lara Flores, Andy Qi, Luke Reilly, Marianita Santiana, Michael Ward, Mark Cookson

[DESCRIPTION]
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.

[STEPS]
SECTION: Medium Preparation
1. Induction Medium: 
For day 0 to day 3
 
     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
 
     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
 
     Reagent      Stock      Final concentration      Amount for 50mL of medium        BrainPhys neuronal medium      ------------      -------------      49 mL        B27+ supplement      50X      1X      1 mL        GDNF      10 µg/mL      10 ng/mL      0.05 mL        BDNF      10 µg/mL      10 ng/mL      0.05 mL        NT-3      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   
 

SECTION: Differentiation Protocol
2. 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:

SECTION: Differentiation Protocol
2.1. 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 30 min to 60 min  .
 


SECTION: Differentiation Protocol
2.2. Prepare Induction Medium and place in 37 °C water or bead bath to warm during dissociation.


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

SECTION: Differentiation Protocol
2.4. Aspirate culture medium and wash with PBS 1X.

SECTION: Differentiation Protocol
2.5. Aspirate PBS and add half of culture volume of Accutase
SECTION: Differentiation Protocol
2.6. Transfer to 37 °C  incubator for10 min  

 


SECTION: Differentiation Protocol
2.7. When Incubation is ready, tilt the plate and pipet the accutase solution two to three times up and down to singularize as single cells.


SECTION: Differentiation Protocol
2.8. 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.


SECTION: Differentiation Protocol
2.9. Centrifuge 5 min  at 200 - 300 x g at Room temperature  



 

SECTION: Differentiation Protocol
2.10. Aspirate supernatant and resuspend cell pellet with Induction Medium.

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

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

SECTION: Differentiation Protocol
3. Day 1
Check cells under the microscope, nascent neuritic extensions should begin to be evident after 24 h of doxyxycline exposure.

SECTION: Differentiation Protocol
3.1. Prepare Induction Medium but without Chroman I and warm it.

SECTION: Differentiation Protocol
3.2. Aspirate medium, wash once with PBS 1X and replace with warm induction medium.

SECTION: Differentiation Protocol
4. Day 2
Check cells under the microscope,  neuritic extensions should be more evident.

SECTION: Differentiation Protocol
4.1. Repeat medium change with induction medium as on day 1.

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





SECTION: Differentiation Protocol
6. Day 4
Check cells under a microscope. Pre-differentiated neurons are ready to be re-plated.




SECTION: Differentiation Protocol
6.1. 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 60 min  to   .
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.


 

SECTION: Differentiation Protocol
7. 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
SECTION: Differentiation Protocol
7.1. Prepare fresh Neuronal Maturation Medium for day 4 as referred above. 
SECTION: Differentiation Protocol
7.2. After dissociating cells with Accutase as step 2.4 to 2.9 resuspend cell pellet with Neuronal Maturation Medium for day 4 and count.



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

 

SECTION: Differentiation Protocol
7.5. After day 4 do half of the medium change every  3-4 days with Neuronal Maturation Medium.

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