TITLE: Differentiation of RGC Induced Neurons (RGC-iNs)
AUTHORS: Devansh Agarwal, Karl Wahlin

[DESCRIPTION]
This protocol is designed to create induced retinal ganglion cells neurons (RGC-iNs) using a doxycycline-inducible polycistronic transcription factor cassette containing NEUROG2, ATOH7, ISL1, and POU4F2 and TetO integrated at the CLYBL safe harbor site. The cassette is integrated into the CLYBL safe harbor site using a CRISPR-Cas12a ribonucleoprotein. This process of generating neurons is greatly enhanced by the inclusion of the BMP blocker LDN193189.

[BEFORE_START]
For media/reagent recipes see the last section of the protocol.

[GUIDELINES]
Apart from observation under the microscope, counting, and centrifugation, all steps should be carried out in a sterile biological safety cabinet.

[STEPS]
SECTION: PSC expansion step
1. Grow iPSCs in hypoxia (5 % (v/v) O2/10 % (v/v) CO2) or normoxia (20 % (v/v) O2/5 % (v/v) CO2) at 37 °C.
12-well plate (3.5 cm2): Plate 5,000 iPSCs into each well of 12 well plates in the presence of blebbistatin. Feed daily in mTeSR1 and grow for ~4 more days. Typically, we can get 200,00 – 500,000 cells per well when cells are ready for passaging. 
6-well plate (9.6 cm2): Plate 15,000 iPSCs into each well of 6-well plates in the presence of blebbistatin. Feed daily in mTeSR1 and grow for ~4 more days. Typically, we can get 750,000 – 1,000,000 cells per well when cells are ready for passaging.
 

SECTION: Day -1: Priming of stem cells for neural induction
2. One day prior to neural induction, pre-coat TC plates overnight with 0.1 mg/mL poly-L-ornithine (PLO). Add 1 mL 20x PLO to 19 mL cell culture grade H2O, use 1 mL per well of 1x PLO for 6-well plates (0.5 mL per well for 12 well plates) and incubate coated plate overnight at 37 °C.
 



SECTION: Day 0: Neural Induction
3. Wash overnight PLO-coated plates >3 times with culture grade H2O. Let plates dry in the back of the TC hood for 60 min, then coat with 1 % (v/v) Matrigel (recommended >180 min).
SECTION: Day 0: Neural Induction
4. Prepare Neural Induction Medium Initiation Cocktail (NIM) with 2 µg/mL doxycycline (500x stock), 100 nanomolar (nM) LDN, 1x CultureOne (100x stock) and 5 micromolar (µM) blebbistatin (blebb; 2,000x stock). Place in 37 °C bead bath to warm during the dissociation process.
 

SECTION: Day 0: Neural Induction
5. Aspirate the media from the wells and add Accutase prewarmed for 5 min (the volume of Accutase to use is 1/2 the volume that you maintain the cells in).
SECTION: Day 0: Neural Induction
6. Put the cells with the Accutase back into the incubator for 12 min.
SECTION: Day 0: Neural Induction
7. Gently rinse the wells using a P1000 and pipet up and down 3 times to further break up the cell clumps into single cells.
SECTION: Day 0: Neural Induction
8. Put the cells into a 5 mL tube with 2 times the volume of mTeSR+ 5 micromolar (µM) blebb (e.g. 1 mL Accutase + 2 mL mTeSR) to quench the Accutase, then pellet the cells for 5 min at 80 x g.

SECTION: Day 0: Neural Induction
9. Aspirate the supernatant and resuspend the cell pellet in 1 mL NIM + blebb.
SECTION: Day 0: Neural Induction
10. Filter cells with a 35 µm or 40 µm cell strainer (for e.g., Greiner #542040).
SECTION: Day 0: Neural Induction
11. Count the cells with a hemocytometer.
SECTION: Day 0: Neural Induction
12. To make a 6-well plate (9.6 cm2/well; 57.6 cm2 total; 7,000 cells/cm2): Add 403,200 cells into 12 mL (67,200 cells per well) of NIM initiation cocktail (NIM + doxy, LDN, CultureOne, blebb) in a 15 mL conical tube, mix well and distribute across the wells.
To make a 12-well plate (3.5 cm2/well; 42 cm2 total; 7,000 cells/cm2): Add 294,000 cells into 12 mL (24,500 cells per well) of NIM initiation cocktail in a 15 mL conical tube, mix well and distribute across the wells.
To make a 24-well plate (1.9 cm2/well; 45.6 cm2 total; 7,000 cells/cm2): Add 319,200 cells into 12 mL (13,300 cells per well) of NIM initiation cocktail in a 15 mL conical tube, mix well and distribute across the wells.

SECTION: Day 1: Maintenance
13. Do nothing.
SECTION: Day 2: Feed – exchange 1/3 of media
14. For 12 well plate: Add 0.5 mL NIM + 1 µg/mL doxy + 1xCultureOne to each well.
For 6 well plate: Add 1 mL NIM + 1 µg/mL doxy + 1xCultureOne to each well.
 

SECTION: Day 3: Maintenance
15. Do nothing.
SECTION: Day 4: Feed – exchange 1/3 of media.
16. For 12 well plate: Remove 0.5ml media and replace with fresh 0.5 mL NIM + 1 µg/mL doxy + 1xCultureOne + NIC (10 millimolar (mM)).
For 6 well plate: Remove 1 ml media and replace with fresh 1 mL NIM + 1 µg/mL doxy + 1xCultureOne + NIC (10 millimolar (mM)).

Beyond day 4, plates need to be fed every other day.
SECTION: Day 6: Feed – exchange 1/3 of media.
17. For 12 well plate: Remove 0.5 mL media and replace with fresh 0.5 mL BrainPhys + B27 (50x) + 1 µg/mL doxy + BDNF (50 ng/mL) + GDNF (10 ng/mL) + NIC (10 millimolar (mM)).
For 6 well plate: Remove 1 mL media and replace with fresh 1 mL BrainPhys + B27 (50x) +  1 µg/mL doxy + BDNF (50 ng/mL) + GDNF (10 ng/mL) + NIC (10 millimolar (mM)).
SECTION: Day 8: Feed – exchange 1/3 of media.
18. For 12 well plate: Remove 0.5 mL media and replace with fresh 0.5 mL BrainPhys + B27 (50x) + 1 µg/mL doxy + BDNF (50 ng/mL) + GDNF (10 ng/mL) + NIC (10 millimolar (mM)).
For 6 well plate: Remove 1 mL media and replace with fresh 1 1 mL BrainPhys + B27 (50x) + 1 µg/mL doxy + BDNF (50 ng/mL) + GDNF (10 ng/mL) + NIC (10 millimolar (mM)).
 

SECTION: Media/Reagent Recipes
20.  Neural Induction medium (NIM medium):
     Component:      Catalog #      Volume      cost        DMEM/F12      Life Technologies      495 ml               N2 supplement (100×)      Life technologies # 17504044   or make as below.      5 ml               Culture One supplement (100x)      Life technologies # A3320201      0.5ml ** add this into 50ml tube as needed.                              Total 500 ml               ** filter sterilize with 0.2um PES filter -   N2 not sterile. **                        

SECTION: Media/Reagent Recipes
22. Reagent Stock Dilutions:

Recombinant Human BDNF Protein (Qkine, Cat# Qk050): 50 µg/mL stock in 10 millimolar (mM) HCl with 0.1 % (v/v) BSA; 1,000X; use at 50 ng/mL. Store aliquots in -80 °C.
Recombinant Human GDNF Protein (Qkine, Cat# Qk051): 10 µg/mL stock in cell culture grade H2O with 0.1 % (v/v) BSA; 1,000X; use at 10 ng/mL. Store aliquots in -80 °C.
Doxycycline (doxy) (Sigma-Aldrich, Cat# D5207): Make 1 mg/mL stock (working concentration is 0.5-2 µg/mL) in cell culture grade ddH2O and filter sterilize; 1,000X; use at 1 µg/mL. Store 1 mL aliquots at -20 °C.
LDN-193189 hydrocholoride (BMP blocker - Noggin replacement – 10,000X stock (1 millimolar (mM)); Sigma SML0559-5MG):
g = Molecular Weight (g/mol) * Molarity (M) * Volume (ml)
0.005 g = (406.48) * (0.001 M) * (Volume)
0.005 g = 0.41 * Volume
Volume = 0.012 L or 5 mg LDN in 12 mL DMSO; Store aliquots in -80 °C.
Matrigel (GF reduced) (1 % (v/v)) (Corning, Cat# 354230): 
Thaw stock bottle overnight on ice before aliquoting. Always keep on ice and never let come to room temperature or it will gel.
Make 200 µL aliquots. Store aliquots in -80 °C.
Add 200 µL matrigel to 24 mL ice-cold DMEM/F12 (~1 % (v/v) final).
Add 1 mL per well of 6-well plate or 0.5 mL per well of 12-well plate.
NIC (Nicotinamide) (Sigma #72340): 1 Molarity (M) (100x) stock solution (10 millimolar (mM) working solution). Soluble in water to ~ 1g/10ml. 
g = Molecular Weight (g/mol) * Molarity (M) * Volume (L); g = (122.12)*(1M)*(0.05L)
= 6.11 g NIC in 50 mL of DMEM/F12 (or water); filter sterilize and store at 4 °C.
poly-L-ornithine hydrobromide (PLO) - 20X stock (2 mg/mL; mol wt 30,000-70,000; Sigma P3655-500MG):
Add 500 mg PLO to 250 mL cell culture ddH2O.
Make 1 mL aliquots. Store aliquots in -80 °C.




SECTION: Media/Reagent Recipes
21.  N2 supplement (100X) recipe:
            100 ml      125 ml      200 ml      250 ml ** most practical      500 ml        Transferrin      1g (2x500mg bottles)      1.25 g      2g (4 x 500mg bottles or 2x1g bottles)      2.5 g (5x500mg bottles)      5.0 g (10 x 500mg bottles)        Insulin ** make 25 mg/ml aliquots      50 mg (2 x 25mg/ml aliquots)      62.5 mg      100mg (one bottle)      125 mg (5x 25mg/ml aliquots)      250 mg (10 x 25mg/ml aliquots)        progesterone      63 µg      78.5 µg      126 µg      157 µg      315 µg        putrescine      161 mg      201.5 mg      322 mg      403 mg      806 mg        selenium      50.2 µg  **       62.75 µg      100.4 µg      125.5 µg      251 µg        Neurobasal      to 100ml      to 150ml      to 200ml      to 250ml      to 500ml   
 
SECTION: Day 8: Feed – exchange 1/3 of media.
19. For long-term experiments >1 week, continue feeding by 1/3 media exchange every other day with BrainPhys + B27 (50x) +  1 µg/mL doxy + BDNF (50 ng/mL) + GDNF (10 ng/mL) + NIC (10 millimolar (mM)).