TITLE: Differentiation of human cortical neurons (CNs) from  induced pluripotent stem cells (iPSCs)
AUTHORS: Quyen Do, Bryan Ng, Richard Wade-Martins

[DESCRIPTION]
This protocol described the production of human cortical neurons from induced pluripotent stem cells. The differentiation protocol of cortical neurons is adapted and modified from Shi, Y. et al., 2012 (pharmacologically directed) and Zhang, Y. et al., 2013 (transcription factor directed), as previously described by Hedegaard et.al., 2020. These neurons exhibited molecular and functional hallmarks of cortical neurons.

[BEFORE_START]
Sterile working techniques are an absolute must to ensure cell viability and vitality. This includes but not limited to filtering of all media to be used with 0.22μm filter, sterilisation of gloves, stripettes, falcons or any materials to be in contact with cells or cell media.

All growth factors should be added fresh on the day of intended use, or within 48 hours. Prior to use media must be warmed preferentially to 37ºC, or room temperature at the very least, as these cells are temperature-sensitive.

[STEPS]
SECTION: Differentiation of iPSCs into Neuronal Progenitor Cells (NPCs)
1. Day -2: Preparing plates for replating
Two days before intending on starting the differentiation (Day -2), add 1 mL/well in a 6-well plate of Geltrex one day prior to replating the iPSCs to begin the differentiation.
 

SECTION: Differentiation of iPSCs into Neuronal Progenitor Cells (NPCs)
2. Day -1: Replating iPSCs for differentiation
Replating iPSCs for differentiation is identical to described in Protocol: Expansion and maintenance of human induced pluripotent stem cells (iPSCs), however, includes a cell counting step.
SECTION: Differentiation of iPSCs into Neuronal Progenitor Cells (NPCs)
2.1. Prepare for splitting 
Follow steps described in steps 6 and 7 of Protocol: Expansion and maintenance of human induced pluripotent stem cells (iPSCs).
SECTION: Differentiation of iPSCs into Neuronal Progenitor Cells (NPCs)
2.2. Prepare for cell counting 
Prepare 99 μl of Phosphate Buffered Saline (PBS) into one Eppendorf per cell line for cell dilution.
SECTION: Differentiation of iPSCs into Neuronal Progenitor Cells (NPCs)
2.3. Replate iPSCs
As described in step 7 of Protocol: Expansion and maintenance of human induced pluripotent stem cells (iPSCs), pausing when cell pellet is suspended in 1 mL of mTesR media (i.e. mTesR plus their accompanying Supplement and 1% Penicilline/Streptomycin) + ROCKi (1:1000) to count cells.
SECTION: Differentiation of iPSCs into Neuronal Progenitor Cells (NPCs)
2.4. Count cells (manually using a haemocytometer)
2.4.1.  Dilute cells by adding 1 μL of cell suspension to 99 μL of previously prepared PBS in an Eppendorf. 
2.4.2. Mix thoroughly. 
2.4.3. Take 10 μL of diluted cell mixture and add to a haemocytometer. 
2.4.4. Using a microscope, focus on the grid lines of the hemocytometer with a 10X objective.
2.4.5. Manually count cells from all 4 all 4 sets of 16 corners of the haemocytometer using a tally counter.
2.4.6. Average cell count from each of the sets of 16 corner squares and multiply by 10,000 (104).
2.4.7. Multiply by 100 to correct for the dilution in step 2.4.1. 
2.4.8. Calculate and plate cells based on the following optimal density for Day -1 plating: 1.5 millions cells per 6 well .

 

2.4.9. Replate about 1.5 to 2 million cells per 6-well suspended in mTesR media+ ROCKi (1:1000) and swirl plate gently in a figure 8 motion.
2.4.10. Add more mTesR media + ROCKi (1:1000) to make up the total volume of 3 mL per well of a 6-well plate.
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
3. Day 0: Differentiation starts when cells reach 90-100% confluency
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
4. Day 1 – Day 12: Daily feed with NIM
Replace medium every day and monitor morphological changes.


 

SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
5. Day 11: Prepare plates for Day 12 passaging
Coat new plates with 15 ug/mL fibronectin and incubate at 37°C overnight.
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
6. Day 12: Splitting cells


SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
6.2. Add 10 mL of NMM to a 15ml falcon (spinning tube) for each line to be passaged and pre-warmed to 37°C.


 

SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
6.3. Add 10% medium volume of pre-warmed Dispase (10 mg/mL, sterilized, in PBS) directly into the well that still has medium in it. 
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
6.4. Incubate at 37°C for 10 min at least.

 

SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
6.5. In the well, break the cell sheet a couple of times with P1000 and keep the big clumps of cells as intact as possible. 
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
6.6. Transfer the cells into 10 mL of NIM in 15-mL falcon tubes. Let the cells settle by gravity.
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
6.7. Remove the supernatant carefully without removing the cells at the bottom.
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
6.8. Add another 10 mL of NIM in 15-mL falcon tubes and repeat steps 6.6 and 6.7. 
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
6.9. Split the cells 1:2 into 15 mg/mL fibronectin-coated plates with NIM and ROCKi (1:1000). 
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
6.10. Allow cells to attach overnight.

 

SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
7. Day 13 : Full media change
1. Add to NMM base medium:
20 ng/ml FGF2 

 

SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
8. Day 15 : Full media change
1. Add to NMM base media:
20 ng/ml FGF2 

 

SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
9. Day 16 : Prepare plates for splitting 
Wells are coated with fibronectin or 100 mg/mL poly-L-ornithine overnight followed by 10 mg/mL laminin for at least 2 hours (up to 24 hours).
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
10. Day 17 : Splitting of NPCs

 

SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
10.1. Repeat the splitting and washing steps as described in step 6. 
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
10.2. Seed the cells on Poly-L-ornithine/Laminin/fibronectin-coated plates previously prepared in step 9  and feed with media as described in step 8.
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
11. Day 18 : Full media change
NMM base media

SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
12. Day 20 – Day 26 : Full media change
Change NMM media every 2 days.
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
13. Day 26 : Freezing 
Cells can be frozen at 5-10 million cells/vial. 
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
13.1. Add 1 mL of Accutase per well to be frozen.
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
13.2. Spin the cells down at 300 g for 5 mins.
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
13.3. Resuspend the cells slightly into clumps in Cryostore 10 or self-made freezing medium (see Materials).
SECTION: Lentivirus packaging
14. This section describes the third-generation lentiviral packaging of plasmids to be used for transduction of NPCs.

Plasmids to be packaged into lentiviruses:
1. FUdeltaGW-rtTA (rtTA; Addgene #19780)
2. pTet-O-Ngn2- puro (Ngn2; Addgene #52047)
3. pLV-TetO-hNGN2-eGFP-Puro (Ngn2-GFP; Addgene #79823)
SECTION: Lentivirus packaging
14.1. Prepare HEK293T cells
14.1.1. Prepare DMEM/F12 medium supplemented with 10% FBS, 2 mM L-glutamine, and 50 U/mL Penicillin-Streptomycin.
14.1.2. Passage cells by single-cell passaging with TrypLE and seed at approximately 150,000 cells/cm2, and at least 24 hours before plasmid transfection.
 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
SECTION: Lentivirus packaging
14.2. Day of transfection
14.2.1 Pre-warm Optimem to room temperature.

14.2.2 Prepare Lipofectamine mix (Tube 1):
14.2.2.1. Add Lipo 3K to Optimem.
14.2.2.2. Flick to mix.
14.2.2.3. Incubate for 5-20 min.

14.2.3. Prepare DNA mix (Tube 2): 
14.2.3.1. Prepare master mix of packaging vectors + P3000 in Optimem.
14.2.3.2. Add transgene-expressing lentiviral vector (e.g. pLEX).
14.2.3.3. Mix by briefly vortexing.

14.2.4. Add DNA mix (Tube 2) to Lipofectamine mix (Tube 1), mix by flicking tube 8 times
14.2.5. Wait ~ 20 minutes
14.2.6. Add DNA/Lipofectamine mixture to cells dropwise, and swirl plate immediately to evenly mix with fresh cell media without penicillin/streptomycin.
                                     Tube   1                   Tube   2                                                              Dish         Cell #    ( x 106)         Media (mL)         Tube 1:   Optimem         Lipo 3K         Optimem         P3000         psPAX2         pMD2G         pAdVantage         Lenti vector            1 x 6 well         2.5          3         150 uL         1.9 uL         150 uL         5 uL         1.6 ug         0.6 ug         0.2 ug         2.4 ug            1 x 10 cm         15         18         900 uL         11 uL         900 uL         30 uL         10 ug         3.4 ug         1.4 ug         14.6 ug            1 x 15 cm         45         36         2.4 mL         30 uL         2.4 mL         80 uL         26.3 ug         9 ug         3.6 ug         39 ug      
 
SECTION: Lentivirus packaging
14.3. Day of transfection +1
Replace media with 3 mL of DMEM/FCS medium + 6uL ViralBoost (Alstem).

 

SECTION: Lentivirus packaging
14.4. Day of transfection +2 : Collecting cell media
Collect all cell media containing live viruses.

 

SECTION: Lentivirus packaging
14.5. Concentrating viruses
This step was adopted from manufacturer’s guideline for the TAKARA Lenti-X concentrator kit.

14.5.1. Lentivirus-containing supernatants are centrifuged briefly at 500g for 10 mins or filtered through a 0.45 μm filter.

 

14.5.2. Transfer clarified supernatant to a sterile container and combine 1 volume of Lenti-X Concentrator with 3 volumes of clarified supernatant. 
14.5.3. Mix by gentle inversion. Larger volumes may be accommodated through the use of larger (i.e., 250 mL or 500 mL) centrifuge tubes.
14.5.4. Incubate mixture at 4°C overnight.
14.5.5. Centrifuge sample at 1,500g for 45 minutes at 4°C. 
14.5.6. Carefully remove supernatant without disturbing the pellet. Residual supernatant can be removed with either a pipette tip or by brief centrifugation at 1,500g.
14.5.7. Gently resuspend the pellet in 1/100th of the original volume using complete DMEM. 

 

14.5.8. Immediately titrate sample into single-use aliquots and stored at -70°C.
SECTION: Differentiation of NPCs into cortical neurons
15. The second part of the differentiation protocol beyond Day 26 was adapted and modified from Zhang, Y. et al., 2013. The protocol employs doxycycline-inducible overexpression system of Ngn2 to direct excitatory cortical neuronal identity. 
SECTION: Differentiation of NPCs into cortical neurons
15.1. Prepare plates for replating
Add 100 μg/mL of Poly-L-ornithinne onto plastic wells and incubate at 37⁰C overnight.
SECTION: Differentiation of NPCs into cortical neurons
15.2. Prepare media for thawing and replating
15.2.1. Pre-warm spinning falcons containing 9 ml of Neurobasal. 
15.2.2. Prepare Day 25 media (i.e. NMM) + ROCKi (1:1000) + 20 ng/mL FGF2.
SECTION: Differentiation of NPCs into cortical neurons
15.3. Thawing and replating of Day 26 NPCs
15.3.1. Thaw cryovial containing Day 26 NPCs in water bath until only a small component remains frozen.
15.3.2. Carefully transfer contents of cryovial to pre-warmed spinning tubes. 
15.3.3.  Centrifuge at 350g for 5 min.
15.3.4.  While spinning, aspirate Matrigel and replace with Day 25 media (i.e. NMM) + ROCKi (1:1000) + 20 ng/ml FGF2.
15.3.5.  Aspirate media from cell pellet in spinning falcon and replace with Day 25 media (NMM) + ROCKi (1:1000) + 20 ng/ml FGF2, slowly and gently resuspending the pellet. 
15.3.6. Transfer an appropriate amount of cell suspension into previously prepared wells and swirl plate gently in a figure 8 motion. 
SECTION: Differentiation of NPCs into cortical neurons
15.4. Transduction of Day 26 NPCs
15.4.1. Add Ngn2 and rTA lentiviruses directly into the well with cell media immediately after depositing the cell suspension.
15.4.2. Replace medium with fresh NMM 8 hours later.

 


SECTION: Differentiation of NPCs into cortical neurons
15.5. Day 28 – Doxycycline induction
The NMM media is replaced with freshly-made Ngn2 medium (see Materials) to induce doxycycline-dependent expression of Ngn2 in successfully transduced neurons.
SECTION: Differentiation of NPCs into cortical neurons
15.6. Day 30 – Antibiotic selection 
Full media change with freshly-made Ngn2 medium (see Materials) supplemented with 1 μg/mL puromycin to select for successfully transduced neurons.
SECTION: Co-culture of iPS-cortical neurons with rat astrocytes
16. Resulting iPS-cortical neurons are co-cultured with commercially available rat astrocytes as previously described in Hedegaard et.al., 2020. This coculture approach enhances both cortical intrinsic capacity to fire action potential and network-dependent synaptic activities. 
SECTION: Co-culture of iPS-cortical neurons with rat astrocytes
16.1. Rat Astrocyte Expansion
16.1.1. Thaw and replate primary rat astrocytes onto uncoated T75 flasks according to manufacture's protocol, which is similar to steps 15.2 and 15.3. The only difference is the medium - use Astrocyte Maintenance Medium (AMM) (see Materials) for rat astrocytes.
16.1.2. Astrocytes are allowed to expand for 2 to 3 weeks till confluence. 
16.1.3. Full media change twice a week.
SECTION: Co-culture of iPS-cortical neurons with rat astrocytes
16.3. Freezing of rat astrocytes
16.3.1. Lift confluent astrocytes using Accutase and centrifuge at 350g for 5 mins.
16.3.2. Remove the supernatant and resuspend cell pellet in AMM.
16.3.3. Freeze the cells at 2-5 million cells/vial in 90% v/v AMM + 10% v/v DMSO as P3 astrocytes.
SECTION: Co-culture of iPS-cortical neurons with rat astrocytes
16.4. Co-culturing of cortical neurons with rat astrocytes
16.4.1. P3 rat astrocytes are thawed and plated onto intended plates at 20,000 cells/cm2 following step 16.1.

 

16.4.2. Day 30 Cortical Neurons (step 15.6.) are lifted with Accutase and replated on top of the adherent astrocyte monolayer at the density of 100,000 cells/cm2. 
16.4.3. Upon centrifugation, cell pellet is resuspended in Ngn2 medium.
16.4.4.2 On Day 32, half media change is performed with fresh Ngn2 medium + 100 nM AraC.
16.4.3. From now on, half media change is done twice per week.
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
3.1. Prepare Neural Induction Medium (NIM; see Materials).
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
3.2. Wash cells once with PBS, and add 2 mL of NIM media to each well.
SECTION: Differentiation of NPCs into Cortical Neurons (CNs)
6.1. Prepare Neural Maintenance Medium (NMM)(see Materials).
SECTION: Co-culture of iPS-cortical neurons with rat astrocytes
16.2. Splitting of Rat Astrocytes
16.2.1. Astrocytes are split into 6 x uncoated T75 flasks. 
16.2.2 Adherent astrocytes are washed with PBS once.
16.2.3. Add 10mL of Accutase per T75 flask and incubate the dish at 37°C for approximately 10-20 mins. 
16.2.4. Check regularly for cell lifting.
16.2.5. Carefully transfer all of the dish content into a pre-warmed falcon.
16.2.6. Centrifuge at 350g for 5 mins.
16.2.7. While spinning, add fresh AMM to the new T75 flasks.
16.2.8. Aspirate media from cell pellet in spinning falcon and replace with AMM, slowly and gently resuspending the pellet.
16.2.9. Transfer an equal amount of cell suspension into previously prepared T75 flasks and swirl plate gently in a figure 8 motion.
16.2.10. Expand again for another 2 to 3 weeks till confluence.