TITLE: Generation of functional induced DA neurons (iDANs) from dermal fibroblasts of adult donors
AUTHORS: Janelle Drouin-Ouellet

[DESCRIPTION]
This describes the steps for direct DA neurons reprogramming of patient’s fibroblasts.

[GUIDELINES]
Notes
It is recommended to prepare the ENM and LNM fresh. Both these media can be kept up to one month at 4°C.
The manipulations described in this protocol must be done in a biological safety cabinet, under sterile conditions. Wearing protection including gloves and a labcoat when handling the cells is highly recommended.
Regular testing for mycoplasma contamination is highly recommended, as it could affect the efficiency of conversion as well as cell’s growth and viability rate.
The choice of multi-well plate format depends on the desired neuronal yield. While the conversion efficiency varies depending on the line, a neuronal conversion of 10% of TAU+ cells and 3% of TH+ cells should be expected with this protocol. 
Note that the neuronal markers Tuj1, MAP2, the dopaminergic marker TH as well as neuritic extensions appear early in the reprogramming process but do not necessarily mean a full neuronal conversion. Long-term cultures of the cells are needed for appearance of electrophysiologically functional neuronal properties. 

[STEPS]
SECTION: 1. Seeding of fibroblasts for conversion to induced DA neurons
1. Thaw a vial containing fibroblasts in a water bath at 37 °C, until only a small piece of frozen congelation medium is remaining (See Notes 2 and 3).
SECTION: 1. Seeding of fibroblasts for conversion to induced DA neurons
2. Quickly transfer the content of the vial to a 15 ml tube containing at least 4 mL. Centrifuge 400 x g, 5 min.
SECTION: 1. Seeding of fibroblasts for conversion to induced DA neurons
3. Discard the supernatant and resuspend the cells in 1 mL. 
SECTION: 1. Seeding of fibroblasts for conversion to induced DA neurons
4. Transfer the cell suspension to a T75 flask containing 9 mL and put the flask in the incubator at 37 °C in 5 %.
SECTION: 1. Seeding of fibroblasts for conversion to induced DA neurons
5. The next day, do a full medium change with fresh MEF medium.
SECTION: 1. Seeding of fibroblasts for conversion to induced DA neurons
6. Expand the fibroblasts until they have reached about 95% confluency. Change the medium twice a week
SECTION: 2. Plating the cells for reprogramming
7. Coat the multi-well plate with 0.1 % to each well. Incubate   37 °C. (See Note 4).
SECTION: 2. Plating the cells for reprogramming
8. The following day, vacuum the medium of the T75 flask containing the cells. Wash once with DPBS, then dissociate the cells with 1 mL at 37 °C for about 3 min, or until all the cells are detached and dissociated.
SECTION: 2. Plating the cells for reprogramming
9. Add 3 mL to neutralize the trypsin, then transfer the full 4 mL to a 15 ml tube. Flush out the cells a second time with an extra 3 mL, then transfer to the same tube.
SECTION: 2. Plating the cells for reprogramming
10. Spin the cells at 400 x g, 5 min, then discard the supernatant. Resuspend the cells in 1 mL.
SECTION: 2. Plating the cells for reprogramming
11. Count the cells with a cell counter. To ensure a good conversion efficiency, the overall viability should not be lower than 90%. 
SECTION: 2. Plating the cells for reprogramming
12. Prepare a cell suspension in the appropriate volume to plate 25,000 cells/cm2. The total volume will depend on the number of wells to be plated. 
SECTION: 2. Plating the cells for reprogramming
13. Vacuum the gelatin solution from the wells, then wash each well twice with DPBS. 
SECTION: 2. Plating the cells for reprogramming
14. Quickly, to prevent the gelatin from drying, mix the cell suspension and immediately transfer the appropriate volume to each well. Fill the remaining empty wells with sterile water to prevent evaporation during the conversion. Incubate the cells 2880 min.
SECTION: 3. Fibroblast’s freezing
15. Centrifuge the remaining cell suspension (from  ) at 400 x g, 5 min.
SECTION: 3. Fibroblast’s freezing
16. Meanwhile, prepare the required volume of freezing medium to obtain a final cell concentration of about 500,000 cells/ml. 
SECTION: 3. Fibroblast’s freezing
17. Resuspend the cells in freezing medium and quickly distribute 1 mL to each cryogenic tube.
SECTION: 3. Fibroblast’s freezing
18. Transfer the cryovials in a controlled-rate freezing container and store at -80 °C. Transfer the cryogenic tubes at -140 °C or in a liquid nitrogen tank the following day for long-term storage. 
SECTION: 4. Viral transduction (Day 0)
19. 
 

SECTION: 4. Viral transduction (Day 0)
20. Thaw at -140 Room temperature the vials containing the lentiviral vectors containing the 6 transgenes (Ascl1, Lmx1a, Lmx1b, FoxA2, Otx2 and Nurr1) and the 2 shRNA sequences against REST that will be used for the DA neural conversion.
SECTION: 4. Viral transduction (Day 0)
21. Prepare the necessary volume of MEF medium to a disposable sterile tube or bottle. 
SECTION: 4. Viral transduction (Day 0)
22. Calculate the volume of each virus required for a transduction at multiplicity of infection (MOI) of 5 for each vector. Use the following formula to do so: 

   
SECTION: 4. Viral transduction (Day 0)
23. Add the appropriate volume of each virus to the MEF medium prepared in  . Mix by inverting the tube.

SECTION: 4. Viral transduction (Day 0)
24. Vacuum the medium contained in each well. Add the appropriate volume of the virus-containing medium to each well. Incubate 3 min at 37 °C in 5 %.
SECTION: 4. Viral transduction (Day 0)
25. The next day (on day 1), replace the medium contained in each well with fresh, virus-free MEF medium, and incubate for 2880 min. 

SECTION: Cell maintenance during reprogramming
26. On day 3, change the full MEF medium of each well for early neuronal medium (ENM).
SECTION: Cell maintenance during reprogramming
27. Every 2nd-3rd day, change half of the early neuronal medium in each well until day 18.
SECTION: Cell maintenance during reprogramming
28. From day 18 until the experimental endpoint is reached, change half of the medium with late neuronal medium (LNM) every 2nd-3rd days (See Note 4).