Neuronal transdifferentiation from human primary adult fibroblasts

1. Fibroblast medium @4°C lasts for 1 month. A total of 500 mL:

• 435 ml DMEM (High glucose, GlutaMAX) (Thermo Fisher Scientific)
• 50 ml Fetal bovine serum (FBS) (Thermo Fisher Scientific)
• 5 ml Penicillin-Streptomycin (Thermo Fisher Scientific)
• 5 ml MEM NEAA (100X; Thermo Fisher Scientific)
• 5 ml Sodium pyruvate (100 mM; Thermo Fisher Scientific)
• 0.5 ml beta-mercaptoethanol (55mM; Thermo Fisher Scientific)
• Sterilized by filtration through a 0.22 µm filter

2. Neuronal reprogramming medium @4°C lasts for 1 month. A total of 500 mL:

• 240 mL DMEM/F-12 (Thermo Fisher Scientific)
• 240 mL Neurobasal Medium (Thermo Fisher Scientific)
• 10 mL B-27 Supplement (50X) (Thermo Fisher Scientific)
• 5 mL N-2 Supplement (100X) (Thermo Fisher Scientific)
• 1.25 mL GlutaMAX Supplement (Thermo Fisher Scientific)
• 5 ml Penicillin-Streptomycin (Thermo Fisher Scientific)
• Sterilized by filtration through a 0.22 µm filter

3. Neuronal maturation medium @4°C lasts for 1 month. A total of 500 mL:

• 480 mL BrainPhys Neuronal Medium (STEMCELL Technologies)
• 10 mL B-27 Supplement (50X) (Thermo Fisher Scientific)
• 5 mL N-2 Supplement (100X) (Thermo Fisher Scientific)
• 1.25 mL GlutaMAX Supplement (Thermo Fisher Scientific)
• 5 ml Penicillin-Streptomycin (Thermo Fisher Scientific)
• Sterilized by filtration through a 0.22 µm filter

4. Hexadimethrine Bromide (Polybrene) (Sigma-Aldrich)

5. Forskolin (Sigma-Aldrich)

6. Dorsomorphin (Tocris)

7. SB 431542 (Tocris)

8. XAV939 (Stemgent)

9. Doxycycline (Cayman)

10. Puromycin (Thermo Fisher Scientific)

11. BDNF Recombinant Protein (Peprotech)

12. NT-3 Recombinant Protein (Peprotech)

13. Poly-L-ornithine hydrobromide (Sigma-Aldrich)

14. Human rhLaminin-521 (Thermo Fisher Scientific)

15. Human Vitronectin (Thermo Fisher Scientific)

Day -1 : Plate human fibroblasts at 200K cells per well of a poly-L-ornithine coated 6-well plate.

Dilute sterile-filtered 0.01% poly-L-ornithine in PBS at a 1-to-10 ratio.

Add fresh culture medium to adjust the cell concentration to 200K cells per well and each well contain 2 mL of culture medium.

Coat the plate and incubate in a 37°C incubator for 2 hr.

Wash the plate with H2O for 5 min on a shaker and repeat for three times. Airdry the plate before plating the cells.

Rinse the cells with sterile 1x PBS to remove all complete medium.

Add 0.05% Trypsin-EDTA to cover the bottom of the dish. Incubate at 37°C with 5% CO2 for ~5 minutes. Cells should round up and become dislodged.

Neutralize trypsin-EDTA activity by adding the complete media.

Gently pipette to resuspend the cells and transfer the cell-containing medium to a 15 mL conical tube.

Centrifuge at 200xG for 5 min to pellet the cells.

Remove the supernatant. Cell numbers are determined for each sample by a hemocytometer.

Day 0 : Infect cells with 2 mL of viral supernatants per well of a 6-well plate.

Make virus dilutions by adding each viral supernatant to cold Fibroblast medium. 100 to 400 µL of each viral supernatant (M2rtTA, Ngn2-puro, Ascl1, Brn2, Myt1l) depending on viral titers.

Combine these viral supernatants and add cold Fibroblast medium to reach a total of 12 mL for one 6-well plate.

Add 4 µg/mL Polybrene to the virus dilutions.

Remove old Fibroblast medium and incubate cells with virus dilutions for 24 hr.

Day 1 : Remove virus-containing medium and add fresh Fibroblast medium plus 1 µg/mL doxycycline.

Day 2 : Change to fresh Fibroblast medium plus doxycycline and puromycin.

Day 4 : Coat glass coverslips or plates with Vitronectin/rLaminin-521 and replate the transduced cells.

Coat with vitronectin (1:100 dilution of stock concentration 0.5 mg/mL in PBS) for 1 hr at room temperature. No need to rinse for vitronectin-coating coverslips or plates.

Coat with rhLaminin-521 (1:100 dilution of stock concentration 100 μg/mL in DPBS with Ca²⁺ and Mg²⁺) for 2 hr at 37°C.

In parallel, trypsinize cells by 0.05% Trypsin-EDTA for 5 min at 37°C and perform PSA-NCAM selection using magnetic-based cell sorting.

Replate the PSA-NCAM+ cells in Fibroblast medium plus doxycycline. Generally ~50K cells per cm².

Day 5 : Switch to Neuronal reprogramming medium plus small molecules.

• 5 µM Forskolin
• 2 µM Dorsomorphin
• 10 µM SB 431542
• 2 µM XAV939
• 1 µg/mL Doxycycline

Change half of the culture medium every 2 to 3 days.

Day 12 , Switch to Neuronal reprogramming medium supplemented with small molecules and neurotrophic factors.

• 5 µM Forskolin
• 2 µM Dorsomorphin
• 10 µM SB 431542
• 2 µM XAV939
• 1 µg/mL Doxycycline
• 10 ng/mL BDNF
• 10 ng/mL NT-3

Change half of the culture medium every 2 to 3 days.

Day 20 , switch to Neuronal maturation medium supplemented with small molecules and neurotrophic factors.

• 5 µM Forskolin
• 2 µM Dorsomorphin
• 10 µM SB 431542
• 1 µg/mL Doxycycline
• 10 ng/mL BDNF
• 10 ng/mL NT-3

Change half of the culture medium every 3 to 4 days.

Neurons will mature on and after 5 weeks in culture and be ready for experiments

Neurons can be cultured for about 9 weeks.