TITLE: Differentiation of human Dopamine Neurons (DaNs) from induced pluripotent stem cells (iPSCs)
AUTHORS: kaitlyn.cramb, Ana Belen Malpartida, Maria Claudia Caiazza, Richard Wade-Martins, Brent Ryan

[DESCRIPTION]
This protocol employs a floor plate-based differentiation method to produce human midbrain dopaminergic neurons from induced pluripotent stem cells. Based on Kriks, S. et al., 2011 with adaptations from Fedele, S. et al., 2017, this protocol incorporates expansion of floor plate progenitors which can be frozen and thawed with limited cell death. By the end of the protocol, tyrosine hydroxylase positive neurons are produced with high efficiency that fire action potentials and produce and release dopamine upon stimulus.

[BEFORE_START]
All growth-factor containing medias should be prepared fresh the day of the feedings. All medias throughout the entirety of the protocol should be filtered using a 0.22μm filter prior to their use to prevent risk of contamination. 

Rock inhibitor should be included at any steps containing a split or replating of cells, and its use should typically be followed by a full feed containing media with no rock inhibitor. 

These cells are particularly temperature sensitive, so media should always be at least at room temperature before use but preferentially briefly warmed to 37⁰C

Cells should be checked daily at least until final replating day. The most success will come from gauging best practices from experience based on how cells look under a brightfield microscope at each stage. 

[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 
2.2.1.  Add 49 μL of Phosphate Buffered Saline (PBS) to one Eppendorf per cell line for cell dilution.
2.2.2. Add 1 μL of Solution 18 to a separate Eppendorf for counting. 
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 (automatically using cell counter)
2.4.1. Dilute cells by adding 1 μL of cell suspension to 49 μL of previously prepared PBS in an Eppendorf. 
2.4.2. Mix thoroughly. 
2.4.3. Take 19 μL of diluted cell mixture and add to Eppendorf containing 1 μL of Solution 18. 
2.4.4. Mix thoroughly and count cells.
2.4.5. Calculate and plate cells based on the following optimal density for Day -1 plating: 1.425 million cells / well of 6-well plate (150 000/ cm2).
 

2.4.6. Transfer cells to a 6-well plate after aspirating the Matrigel, and top up with mTesR media to have 2 mL media total.
SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3. Before starting, check the confluency. The iPSCs should be at least >80% 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.

Days 0 through 20 of the protocol contain no antibiotics and therefore, it is extra pertinent to use the most aseptic technique.

If at any point Day 1 through Day 10 the cells peel, they can be replated by collecting the media and, if any cells remain adhered, using Accutase as described in step 4.3. 
SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3.1. Day prior to preparing differentiation medias
3.1.1. Thaw supplements (KSR, B27 and N2) (ideally in fridge overnight).
3.1.2. Prepare KO DMEM KSR base media and NNB base media (see Materials).
 

SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3.2. Day 0: Full media change
1. Add to KO DMEM KSR base media: 
1:10 000 LDN
1:1000 SB
SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3.3. Day 1 : Full media change
1. Add to KO DMEM KSR base media:
1:10 000 LDN
1:1000 SB
1:1000 SHH
1:5000 FGF8a
1:5000 Puro
SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3.4. Day 2: Half media change
1. Add to KO DMEM KSR base media:
1:10 000 LDN
1:1000 SB
1:1000 SHH
1:5000 FGF8a
1:5000 Puro
SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3.5. Day 3: Full media change
1. Add to KO DMEM KSR base media:
1:10 000 LDN
1:1000 SB
1:1000 SHH
1:5000 FGF8a
1:5000 Puro
SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3.6. Day 4: Half media change 
1. Add to KO DMEM KSR base media:
1:10 000 LDN
1:1000 SB
1:1000 SHH
1:5000 FGF8a
1:5000 Puro
1:3333 CHIR
SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3.7. Day 5 : Full media change 
75% KO DMEM KSR base media
25% NNB base media
1:10 000 LDN
1:1000 SHH
1:5000 FGF8a
1:5000 Puro
1:3333 CHIR
SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3.8. Day 6: Half media change 
75% KO DMEM KSR base media
25% NNB base media
1:10 000 LDN
1:1000 SHH
1:5000 FGF8a
1:5000 Puro
1:3333 CHIR
SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3.9. Day 7: Full media change 
50% KO DMEM KSR base media
50% NNB base media 
1:10 000 LDN
1:3333 CHIR
SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3.10. Day 8: Half media change 
50% KO DMEM KSR base media
50% NNB base media 
1:10 000 LDN
1:3333 CHIR
SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3.11. Day 9: Full media change 
25% KO DMEM KSR base media
75% NNB base media 
1:10 000 LDN
1:3333 CHIR
SECTION: Differentiation of iPSCs into Dopamine Neurons (DaNs)
3.12. Day 10: Full media change (D10 media)
25% KO DMEM KSR base media
75% NNB base media 
1:10 000 LDN
1:3333 CHIR
SECTION: Expansion of NPCs
4. This section is optional. If not desired, continue on to Section: Patterning NPCs into dopaminergic neurons Day 11 to 22+. 

Expansion allows for the freezing and thawing of progenitors midway through the protocol, as well as evidence suggests the final neurons may be more mature (Fedele. S. et al., 2017). It consists of maintaining progenitors cells on Day 10 media (D10; step 3.12) for 1 to 21 days with splits throughout. Therefore, time points are labelled (D10+exp), for instance,  cells in Day 10 media for 4 days are labelled D10+4exp .
SECTION: Expansion of NPCs
4.1. Day 10: Preparation on day prior to first expansion split 
Geltrex 6-well plates as described in step 1.
SECTION: Expansion of NPCs
4.2. Day 10+1: Preparation on the day of the first expansion split.
4.2.1. Pre-warm spinning falcons containing 9mL of KO DMEM base media. 
4.2.2. Prepare D10 media (step 3.12) + ROCKi (1:1000). 
4.2.3. Allow an aliquot of desired volume (1mL per well of a 6-well plate) to reach room temperature. 
 

SECTION: Expansion of NPCs
4.3.  Day 10+1: Splitting Day 10 NPCs
Some lines may survive 1:2, but it is advisable to split cells 2:3, as there will be quite some cell death and the cells need to be fairly confluent to maintain viability and cell identity. If split too harshly, they may differentiate into other cell types. 

4.3.1. Aspirate media and wash each well with 1mL of PBS.
4.3.2. Immediately aspirate PBS and add 1mL of Accutase.
4.3.3. Incubate at 37⁰C for 5 minutes.
4.3.4. Gently collect cells using a 1000 μL pipette and place in pre-warmed spinning falcon.
4.3.5. Spin cells for 5 minutes at 350g. 
4.3.6. While cells are spinning, aspirate Geltrex and replace with 3mL of pre-warmed D10 media  (step 3.12)+ ROCKi (1:1000).
4.3.7. Aspirate media from pelleted cells, re-suspend pellet in 1mL of D10 media (step 3.12) + ROCKi and add dropwise to well of 6-well plate. 
4.3.8. Gently swirl to distribute cells evenly around dish.
SECTION: Expansion of NPCs
4.4. Day 10+2: Maintaining NPCs in Day 10 Expansion Media
4.2.1. Day following expansion split, full feed cells with D10 media (step 3.12). 
4.2.2. Subsequently feed neurons daily with D10 media (also named as D10 expansion media), alternating between half and full feeds.
4.2.3. Passage cells when confluent, generally success is found with a 1:2 split on Day 10+8, Day 10+15 and Day 10+20.
 

SECTION: Expansion of NPCs
4.5. Freezing Day 10+expNPCs
4.5.1. Passage cells as described in step 4.3. 
4.5.2. Resuspend pellet in freezing media containing 90% D10 media (step 3.12) and 10% DMSO. 
4.5.3. Store in liquid nitrogen.
 

SECTION: Patterning NPCs into Dopaminergic Neurons (DaNs)
5. Day 11: Full media change
1. Add to NB base media:
1:3333 CHIR
1:5000 BDNF
1:5000 GDNF
1:10 000 TGFβ3
1:10 000 DAPT
1:1000 Ascorbic Acid
1:200 dCAMP
SECTION: Patterning NPCs into Dopaminergic Neurons (DaNs)
6. Day 12: Half media change 
1. Add to NB base media:
1:3333 CHIR
1:5000 BDNF
1:5000 GDNF
1:10 000 TGFβ3
1:10 000 DAPT
1:1000 Ascorbic Acid
1:200 dCAMP
 

SECTION: Patterning NPCs into Dopaminergic Neurons (DaNs)
7. Day 13 - Day 19: Daily media changes 
Alternate half and full feeds with Final Maturation Media (see Materials).
SECTION: Final replating of Dopamine Neurons (DaNs)
8. Replate the dopamine neurons into final desired dish on Day 20±1 of the protocol (this has been tested to be equally successful on Day 19 and 21). Replating neurons at later stages of their maturation is not recommended and will decrease viability.
SECTION: Final replating of Dopamine Neurons (DaNs)
8.1. One to two days prior to final replating: Dish coating
Choice of coating will affect cell distribution and likelihood of peeling. Geltrex and PLO/Biolaminin are both suitable for replating the neurons. PLO/Biolaminin is thought to reduce peeling and provide a more distributed layer of neurons advantageous for imaging. 

If using Geltrex:
1 day prior to Day 20 replating:
Coat dishes with Geltrex prepared in neurobasal media

If using PLO/Biolaminin:
2 days prior to Day 20 replating:
Coat plates with undiluted PLO

1 day prior to Day 20 replating:
Wash 3x with PBS without letting plates dry. 
Dilute Biolaminin 1:10 in cold, sterile PBS. 
Aspirate and add Biolaminin at same volume as PLO. 
SECTION: Final replating of Dopamine Neurons (DaNs)
8.2. Replating of Day 20 Dopamine Neurons 
 

8.2.1. Wash cells 1x with 1mL PBS and replace with 1 mL Accutase, incubate at 37⁰C for 5 minutes.
8.2.2. Gently collect cells into a pre-warmed spinning falcon.
8.2.3. Spin at 350g for 5 minutes.
8.2.4. Resuspend pellet in 1 mL of pre-warmed Final Maturation Media (see Materials) and count cells using Solution 18.
8.2.5. Replate at final desired concentrations (Table 1). 
 

SECTION: Remove non-neuronal proliferating cell types (Mitomycin C treatment)
9. It is normal to produce a small number of non-neuronal cells throughout this protocol, but ideally these should be at a minimum. We try to reduce the amount remaining in our culture following the final replating of post-mitotic dopamine neurons (previous section) by treating the cells with Mitomycin C on Day 22. 
SECTION: Remove non-neuronal proliferating cell types (Mitomycin C treatment)
9.1. Day 22: Prepare medias
9.1.1. Prepare MitoC media (see Materials), Neurobasal (wash media) and Final Maturation Media (see Materials). 
9.1.2. Pre-warm and filter all three medias. 
SECTION: Remove non-neuronal proliferating cell types (Mitomycin C treatment)
9.2. Day 22: Mitomycin C treatment
9.2.1. Aspirate Final Maturation Media from DaNs.
9.2.2. Add pre-warmed and filtered MitoC media from step 9.1.1.
9.2.3. Incubate at 37⁰C for 1 hour.
9.2.4. Aspirate Mitomycin C and wash 1x with pre-warmed Neurobasal media.
9.2.5. Aspirate Neurobasal media and replace with Final Maturation Media.
SECTION: Maintaining and maturing Dopaminergic Neurons (DaNs) in culture
10. Dopaminergic neurons can be kept in culture for multiple months before obvious demise. The length of survival will depend on feeding schedule, density and various other factors.
SECTION: Maintaining and maturing Dopaminergic Neurons (DaNs) in culture
10.1. Day 24 - Day 25: Full media change
First feed immediately following Mitomycin C treatment should be a full feed with Final Maturation Media (see Materials).
SECTION: Maintaining and maturing Dopaminergic Neurons (DaNs) in culture
10.2. Day 25+: Biweekly Half media changes
10.2.1. From Day 25 onwards, half feed neurons with Final Maturation Media (see Materials) two to three times a week. 
10.2.2. Monitor cells regularly using a brightfield microscope for cell health and contamination.