TITLE: dopaminergic neurons from iPSC
AUTHORS: Rachel Bates

[DESCRIPTION]
This protocol describes our method for the differentiation of human floor plate neural progenitor cells into human midbrain-dopaminergic neurons. This protocol has been developed using a combination of several published protocols.

Adapted from 
 

 


[STEPS]
SECTION: Day 0
1. Human floor plate neuronal progenitor cells (mfNPC) were derived using Smits 2019 protocol. They were maintained for a minimum of 5 passages before being used to generate dopaminergic neurons. 


 

 

SECTION: Day 0
2. mfNPCs were detached using accutase at 37 °C for 3 min .

SECTION: Day 0
3. Re-suspend cells in d0 induction medium and plate 0.1 x 105 cells per well 12 well plate coated with Ultimatrix for 1 hour beforehand.


SECTION: Day 2
4. Change the medium to mfNPC medium supplemented with 0.0001 mg/mL     0.0001 mg/mL   . 



SECTION: Day 4
5. Change the medium to patterning I medium adding 1 mL per well. 

SECTION: Day 6
6. Change medium to patterning II medium with reduced CHIRR adding 1 mL per well. 

SECTION: Day 8
7. Carefully add 1 mL of tissue induction medium to each well. 



SECTION: Day 9
8. Add 2 mL of Tissue induction medium to 12 well plate. 

SECTION: Day 10
10. Change medium to Differentiation medium. 
SECTION: Day 10
11. After day 10 perform a 75% medium  change every 2-3 days. 
SECTION: Day 9
9. Incubate plates at 37 °C .

12. Use neurons after a further 45 days differentiation. 
SECTION: Day 0
1.1. mfNPC maintenance medium

50 % volume                   
50 % volume                   
1:50                                             
1:100                                          
1 % volume                    
1 % volume                                                         
10 micromolar (µM)   
0.5 micromolar (µM)   
250 millimolar (mM)   
200 micromolar (µM)   
3 micromolar (µM)   



SECTION: Day 4
5.1. patterning I medium

50 % volume                   
50 % volume                   
1:50                                             
1:100                                          
1 % volume                    
1 % volume                                                         
0.5 micromolar (µM)   
200 micromolar (µM)   
3 micromolar (µM)   
 0.0001 mg/mL   
  0.0001 mg/mL  
SECTION: Day 6
6.1. Patterning II medium

50 % volume                   
50 % volume                   
1:50                                             
1:100                                          
1 % volume                    
1 % volume                                                         
200 micromolar (µM)   
0.7 micromolar (µM)   
0.0001 mg/mL   
 0.0001 mg/mL  
SECTION: Day 8
7.1. Tissue induction medium


100 % volume                   
1:50                                             
1:100                                          
1 % volume                    
1 % volume                                                         
200 micromolar (µM)   
0.0001 mg/mL   
 0.0001 mg/mL  
0.00025 mg/mL   
0.025 mg/mL   

SECTION: Day 10
10.1. Differentiation medium

100 % volume                   
1:50                                             
1:100                                          
1 % volume                    
1 % volume                                                         
200 micromolar (µM)   
125 micromolar (µM)   
10ng/mL  
10ng/mL