TITLE: Differentiation of human medium spiny neurons (MSNs) from induced pluripotent stem cells (iPSCs)
AUTHORS: Quyen  Do, Nora Bengoa-Vergniory, Richard Wade-Martins

[DESCRIPTION]
This protocol generates human medium spiny neurons (MSNs) from induced human pluripotent stem cells. Incorporating key findings from Telzehkin et.al., 2016 and Arber et.al., 2016, this protocol produces MSNs following the in vivo developmental trajectory of MSN via the subpallium and subsequently, the lateral ganglionic eminence. These neurons expresses canonical markers of striatal projection neurons including glutamic acid decarboxylase as well as co-expression of the dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32) and the Coup-TFI interacting protein 2 (CTIP2). They are also functionally active demonstrated by presence of intrinsic voltage-dependent sodium and potassium currents as well as the capacity to fire action potential upon current stimulation.

[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 0.22 μm 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.

Cells should be regularly checked under brightfield microscope for monitoring of normal growth and identification of potential contamination. 

Frozen supplement in large quantity (e.g. B27, penicillin/Streptomycin) should be ideally thawed overnight in the fridge before use.

[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: 1.2 millions cells per 6 well (125 000/cm2) .

 

2.4.9. Transfer 1.2 -1.5 millions cells / well to a 6-well plate after aspirating the Matrigel, and top up to have 2 mL media total.
SECTION: Differentiation of iPSCs into Medium Spiny Neurons (MSNs)
3. Before starting, check the confluency. The iPSCs should be at least >75% confluent to start, otherwise feed the cells and wait another day. If in doubt, more confluent is better.

Thaw growth factors at room temperature and make every media fresh daily and filter immediately before use.

SECTION: Differentiation of iPSCs into Medium Spiny Neurons (MSNs)
3.1. Day -1: Prepare differentiation media
3.1.1. Thaw supplements (B27, N2, L-glutamin and penicillin/streptomycin) (ideally in fridge overnight).
3.1.2. Prepare Striatal neuronal induction base medium (sNIM)(see Materials).
SECTION: Differentiation of iPSCs into Medium Spiny Neurons (MSNs)
3.2. Day 0 - Day 3
1. Add to sNIM base media:
200 nM LDN (1:5000)
10 μM SB (1:1000)
4 μM XAV (1:5000)

Media is changed daily, 3 mL / well of a 6-well plate.
SECTION: Differentiation of iPSCs into Medium Spiny Neurons (MSNs)
3.3. Day 3: Prepare for splitting   
Add 1 mL of Geltrex to each well of a 6-well plate and leave at 4°C overnight.

 

SECTION: Differentiation of iPSCs into Medium Spiny Neurons (MSNs)
3.4. Day 4: Prepare for splitting
3.4.1. Pre-warm spinning falcons containing 9 mL of KO DMEM basal medium. 
3.4.2. Prepare Day 4 (D4) media (see Materials).
3.4.3. Thaw 1 mL per well of a 6-well plate and allow it to reach room temperature. 
SECTION: Differentiation of iPSCs into Medium Spiny Neurons (MSNs)
3.5. Day 4: 1:2 splitting of Neuronal Progenitor Cells (NPCs)
3.5.1. Add 3 μL of 10 μM ROCKi directly into each well of the 6-well plate containing cells and incubate at 37⁰C for 1 hour.
3.5.2. Aspirate media and wash each well with 1 mL of PBS.
3.5.3. Immediately aspirate PBS and add 1 mL of Accutase.
3.5.4. Incubate at 37⁰C for 5 minutes.
3.5.5. Gently collect cells using a 1000 μL pipette and place in pre-warmed spinning falcon.
3.5.6. Spin cells for 5 minutes at 350g.
3.5.7. While cells are spinning, aspirate Geltrex and replace with 2 mL of pre-warmed D4 media + 10 μM ROCKi (1:1000).
3.5.8. Aspirate media from pelleted cells, re-suspend pellet in 1 mL of D4 media + 10 μM ROCKi (1:1000) and add dropwise to each well of 6-well plate. 
3.5.9. Gently swirl to distribute cells evenly around dish.
SECTION: Differentiation of iPSCs into Medium Spiny Neurons (MSNs)
3.6. Day 5 - Day 8: Daily full media change
1. Add to sNIM base media:
100 nM LDN (1:10000)
10 μM SB (1:1000)
4 μM XAV (1:5000)
 

SECTION: Differentiation of iPSCs into Medium Spiny Neurons (MSNs)
3.7. Day 8: 1:2 splitting of NPCs
3.7.1. Prepare Day 8 (D8) media (see Materials).
3.7.2. Split cells by single-cell passaging as described in step 7 of Protocol: Expansion and maintenance of human induced pluripotent stem cells (iPSCs), but use D8 media instead of iMM media. 

 

SECTION: Differentiation of iPSCs into Medium Spiny Neurons (MSNs)
3.8. Day 9 -  Day 11: Daily full media change
1. Add to sNIM base media:
100 nM LDN (1:10000)
4 μM XAV (1:5000)
SECTION: Differentiation of iPSCs into Medium Spiny Neurons (MSNs)
3.9. Day 12 - Day 15: Daily full media change
1. Add to sNIM base media:
100 nM LDN (1:10000)
4 μM XAV (1:5000)
25 ng/ml  Activin A (1:1000 )
SECTION: Differentiation of iPSCs into Medium Spiny Neurons (MSNs)
3.10. Day 16: Freezing Day 16 MSN precursors
3.10.1.  Passage cells as described in step 3.5.3 to 3.5.6
3.10.2. Resuspend pellet in freezing media Cryostore 10.
3.10.3. Store in cryovial in liquid nitrogen.

 


SECTION: Maturation of MSN precursors into functionally active post-mitotic MSNs
4. Thawing of Day 16 MSN precursors

 

SECTION: Maturation of MSN precursors into functionally active post-mitotic MSNs
4.1. Prepare plates for replating
4.1.1. Add 10 or 100 μg/mL of Poly-D-lysine onto plastic wells and glass coverslips, respectively and incubate at 37⁰C overnight.

 

4.1.2. Wash plenty with PBS, at least 3 times.
4.1.3. Add Matrigel and incubate at 37⁰C for at least 1 hour.
SECTION: Maturation of MSN precursors into functionally active post-mitotic MSNs
4.2. Prepare media for thawing and replating
4.2.1. Pre-warm spinning falcons containing 9 mL of Neurobasal. 
4.2.2. Prepare D16 media (see Materials) + ROCKi (1:1000) and allow it to reach room temperature. 
SECTION: Maturation of MSN precursors into functionally active post-mitotic MSNs
4.3. Thawing and replating of Day 16 MSN precursors
4.3.1. Thaw cryovial containing Day 16 MSN precursors in water bath until only a small component remains frozen.
4.3.2. Carefully transfer contents of cryovial to pre-warmed spinning tubes. 
4.3.3. Centrifuge at 350g for 5 min.
4.3.4. While spinning, aspirate Matrigel and replace with D16 media + 10 μM ROCKi. 
4.3.5. Aspirate media from cell pellet in spinning falcon and replace with D16 media + 10 μM ROCKi, slowly and gently resuspending the pellet. 
4.3.6. Transfer an appropriate amount of cell suspension into previously prepared wells and swirl plate gently in a figure 8 motion. 
5. Further differentiation and maturation of MSN precursors

Maturation media from this stage onwards is adopted and modified from the SynaptojuiceTM recipe reported in Telezhkin et. al., 2016.
5.1. Day 16: Full media change
Add 3 mL of D16 media + 10 μM ROCKi (1:1000) to each well. 
5.2. Day 18: Half media change 
1. Add to sMM1 base media:
100 ng/ml BDNF (1:10000)
200 μM Ascorbic acid (1:1000)
10 μM DAPT (1:10000)
2 μM PD0332991 (1:500)
0.6 mM CaCl2 (1:1000)
1 μM LM22A4 (1:1000)
200 nM cAMP (1:500)
3 μM CHIR (1:3333)
300 μM GABA (1:1000)
25 ng/ml Activin A (1:1000)
200 nM AraC (1:10000)
5.3. Day 20: Half media change 
1. Add to sMM1 base media: 
100 ng/ml BDNF (1:10000)
200 μM Ascorbic acid (1:1000)
10 μM DAPT (1:10000)
2 μM PD0332991 (1:500)
0.6 mM CaCl2 (1:1000)
1 μM LM22A4 (1:1000)
200 nM cAMP (1:500)
3 μM CHIR (1:3333)
300 μM GABA (1:1000)
25 ng/ml Activin A (1:1000)
5.4. Day 22 : Half media change
1. Add to sMM1 base media: 
100 ng/ml BDNF (1:10000)
200 μM Ascorbic acid (1:1000)
10 μM DAPT (1:10000)
2 μM PD0332991 (1:500)
0.6 mM CaCl2 (1:1000)
1 μM LM22A4 (1:1000)
200 nM cAMP (1:500)
3 μM CHIR (1:3333)
300 μM GABA (1:1000)
25 ng/ml Activin A (1:1000)
5.5. Day 24: Full media change
1. Add to sMM2 base media:
100 ng/ml BDNF (1:10000)
200 μM Ascorbic acid (1:1000)
2 μM PD0332991 (1:500)
0.3 mM CaCl2 (1:2000)
1 μM LM22A4 (1:1000)
3 μM CHIR (1:3333)
5.6. Day 26: Half media change 
1. Add to sMM2 base media: 
100 ng/ml BDNF (1:10000)
200 μM Ascorbic acid (1:1000)
2 μM PD0332991 (1:500)
0.3 mM CaCl2 (1:2000)
1 μM LM22A4 (1:1000)
3 μM CHIR (1:3333)

From now on media change is spaced out 3 times a week at half feed till relevant experimental timepoints.