TITLE: Neuronal transdifferentiation from human primary adult fibroblasts
AUTHORS: Ching-Chieh Chou, Judith Frydman

[DESCRIPTION]
This is a protocol for the direct conversion of human primary fibroblasts into neurons using a combination of transcription factor- and small molecule-based approach. The majority of converted neurons showing characteristics of cortical glutamatergic neurons.

[STEPS]
SECTION: Preparation of culture medium and reagents
1. 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)
SECTION: Transduction/induction/selection of human fibroblasts
2. Day -1: Plate human fibroblasts at 200K cells per well of a poly-L-ornithine coated 6-well plate.
SECTION: Transduction/induction/selection of human fibroblasts
2.1. Dilute sterile-filtered 0.01% poly-L-ornithine in PBS at a 1-to-10 ratio.
SECTION: Transduction/induction/selection of human fibroblasts
2.2. Coat the plate and incubate in a 37°C incubator for 2 hr.
SECTION: Transduction/induction/selection of human fibroblasts
2.3. Wash the plate with H2O for 5 min on a shaker and repeat for three times. Airdry the plate before plating the cells.
SECTION: Transduction/induction/selection of human fibroblasts
3. Day 0: Infect cells with 2 mL of viral supernatants per well of a 6-well plate.
SECTION: Transduction/induction/selection of human fibroblasts
3.1. 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.
SECTION: Transduction/induction/selection of human fibroblasts
3.2. Combine these viral supernatants and add cold Fibroblast medium to reach a total of 12 mL for one 6-well plate. 
SECTION: Transduction/induction/selection of human fibroblasts
3.3. Add 4 µg/mL Polybrene to the virus dilutions.
SECTION: Transduction/induction/selection of human fibroblasts
3.4. Remove old Fibroblast medium and incubate cells with virus dilutions for 24 hr.
SECTION: Transduction/induction/selection of human fibroblasts
4. Day 1: Remove virus-containing medium and add fresh Fibroblast medium plus 1 µg/mL doxycycline.
SECTION: Transduction/induction/selection of human fibroblasts
5. Day 2: Change to fresh Fibroblast medium plus doxycycline and puromycin.
SECTION: Transduction/induction/selection of human fibroblasts
6. Day 4: Coat glass coverslips or plates with Vitronectin/rLaminin-521 and replate the transduced cells.
 

SECTION: Transduction/induction/selection of human fibroblasts
6.1. 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.
SECTION: Transduction/induction/selection of human fibroblasts
6.2. 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.
SECTION: Transduction/induction/selection of human fibroblasts
6.3. 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.
SECTION: Transduction/induction/selection of human fibroblasts
6.4. Replate the PSA-NCAM+ cells in Fibroblast medium plus doxycycline. Generally ~50K cells per cm2.
SECTION: Transduction/induction/selection of human fibroblasts
7. 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
SECTION: Transduction/induction/selection of human fibroblasts
8. Change half of the culture medium every 2 to 3 days.
SECTION: Transduction/induction/selection of human fibroblasts
9. 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
SECTION: Transduction/induction/selection of human fibroblasts
10. Change half of the culture medium every 2 to 3 days.
SECTION: Transduction/induction/selection of human fibroblasts
11. 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

 

SECTION: Transduction/induction/selection of human fibroblasts
12. Change half of the culture medium every 3 to 4 days.
SECTION: Transduction/induction/selection of human fibroblasts
13. Neurons will mature on and after 5 weeks in culture and be ready for experiments
SECTION: Transduction/induction/selection of human fibroblasts
14. Neurons can be cultured for about 9 weeks.
SECTION: Transduction/induction/selection of human fibroblasts
2.4. Rinse the cells with sterile 1x PBS to remove all complete medium.
SECTION: Transduction/induction/selection of human fibroblasts
2.5. 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.
SECTION: Transduction/induction/selection of human fibroblasts
2.6. Neutralize trypsin-EDTA activity by adding the complete media.
SECTION: Transduction/induction/selection of human fibroblasts
2.7. Gently pipette to resuspend the cells and transfer the cell-containing medium to a 15 mL conical tube.
SECTION: Transduction/induction/selection of human fibroblasts
2.8. Centrifuge at 200xG for 5 min to pellet the cells. 
SECTION: Transduction/induction/selection of human fibroblasts
2.10. Add fresh culture medium to adjust the cell concentration to 200K cells per well and each well contain 2 mL of culture medium.
SECTION: Transduction/induction/selection of human fibroblasts
2.9. Remove the supernatant. Cell numbers are determined for each sample by a hemocytometer.