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

[DESCRIPTION]
This protocol is designed to convert human induced pluripotent stem cells (PSCs) into retinal ganglion cell induced neurons (RGC-iNs) using a doxycycline-inducible polycistronic transcription factor gene cassette containing human NEUROG2, ATOH7, ISL1, and POU4F2. The TetO-driven transgene cassette is integrated into the CLYBL safe harbor site using a CRISPR-Cas12a ribonucleoprotein. The process of generating neurons is greatly enhanced by the inclusion of the BMP blocker LDN-193189.

[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 PSCs in mTeSR1 under 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 PSCs into each well of 12-well plates in mTeSR1 in the presence of 5 micromolar (µM) blebbistatin (blebb; 2,000x stock). Feed daily in mTeSR1 (without blebb) for ~4 more days. Typically, we get ~200,000 – 500,000 cells per well when cells are ready for passaging. 
6-well plate (9.6 cm2): Plate 15,000 PSCs into each well of 6-well plates in mTeSR1 in the presence of blebb. Feed daily in mTeSR1 (without blebb) 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 hydrobromide (PLO). To do this, add 1 mL 2 mg/mL PLO (20x) to 19 mL cell culture grade H2O, use 1 mL per well for 6-well plates, 0.5 mL per well for 12 well plates and 0.25 mL per well for 24 well plates. Incubate overnight at 37 °C.

Pre-treat stem cells by replacing mTeSR1 media with fresh mTeSR1 supplemented with 1 µg/mL doxycycline (dox; 1,000x stock) and 100 nanomolar (nM) LDN-193189 (10,000x stock).
 


SECTION: Day 0: Neural Induction
3. Wash overnight PLO-coated plates >3 times with culture grade H2O. Let plates dry completely in the back of the TC hood for 60 min, then coat with 1 % (v/v) Matrigel. For Matrigel coating, use 1 mL per well for 6-well plates, 0.5 mL per well for 12 well plates and 0.25 mL per well for 24 well plates. Incubate for >180 min at 37 °C.
SECTION: Day 0: Neural Induction
4. Prepare Neural Induction Medium (NIM) initiation cocktail with 2 µg/mL dox (500x stock), 100 nanomolar (nM) LDN (10,000x stock), 1x CultureOne (100x stock) and 5 micromolar (µM) blebb.

 

SECTION: Day 0: Neural Induction
5. Incubate the cells with prewarmed Accutase (~ 5 min) in the incubator for 12 min The volume of Accutase to use is 1/2 the volume that you maintain the cells in (e.g., 1 mL per well of a 6 well plate, 0.5 mL per well of a 12 well plate and 0.25 mL per well of a 24 well plate).
SECTION: Day 0: Neural Induction
6. Gently rinse the wells using a P1000 micropipette and pipet up and down 3 times to further break up the cell clumps into single cells.
SECTION: Day 0: Neural Induction
7. Put the cells into a conical 1.5 mL or 5 mL tube with 2 times the volume of prewarmed mTeSR1 with 5 micromolar (µM) blebb (e.g. 1 mL Accutase + 2 mL mTeSR1) to quench the Accutase, then pellet the cells for 5 min at 80 x g.
SECTION: Day 0: Neural Induction
8. Aspirate the supernatant and then resuspend the cell pellet in prewarmed NIM + 5 micromolar (µM) blebb.
SECTION: Day 0: Neural Induction
9. Filter cells with a 40 µm cell strainer. Count the cells with a hemocytometer.
SECTION: Day 0: Neural Induction
10. Plate cells onto PLO/matrigel-coated plates. 
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 + dox, LDN, CultureOne, blebb) in a 15 mL conical tube, mix well and distribute across the wells (2 mL per well).
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 (1 mL per well).
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 (0.5 mL per well).

SECTION: Day 1: Maintenance
11. Do nothing.
SECTION: Day 2: Feed – add 1/3 of media
12. For 6 well plate: Add 1 mL NIM + 1 µg/mL dox + 1xCultureOne to each well.
For 12 well plate: Add 0.5 mL NIM + dox + CultureOne to each well.
For 24 well plate: Add 0.25 mL NIM + dox + CultureOne to each well.
 

SECTION: Day 3: Maintenance
13. Do nothing.
SECTION: Day 4: Feed – exchange 1/3 of media
14. For 6 well plate: Remove 1 mL media and replace with fresh 1 mL NIM + 1 µg/mL dox + 1xCultureOne + NIC (10 millimolar (mM)).
For 12 well plate: Remove 0.5 mL media and replace with fresh 0.5 mL NIM +  dox + CultureOne + NIC.
For 24 well plate: Remove 0.25 mL media and replace with fresh 0.25 mL NIM + dox + CultureOne + NIC.

Beyond day 4, plates need to be fed every other day.

 


SECTION: Day 6: Feed – exchange 1/3 of media
15. For 6 well plate: Remove 1 mL media and replace with fresh 1 mL BrainPhys + B27 (1x) +  1 µg/mL dox + BDNF (50 ng/mL) + GDNF (10 ng/mL) + NIC (10 millimolar (mM)).
For 12 well plate: Remove 0.5 mL media and replace with fresh 0.5 mL BrainPhys + B27 + dox + BDNF + GDNF + NIC.
For 24 well plate: Remove 0.25 mL media and replace with fresh 0.25 mL BrainPhys + B27 + dox + BDNF + GDNF + NIC.

 


SECTION: Day 8: Feed – exchange 1/3 of media
16. For 6 well plate: Remove 1 mL media and replace with fresh 1 1 mL BrainPhys + B27 (1x) + BDNF (50 ng/mL) + GDNF (10 ng/mL) + NIC (10 millimolar (mM)).
For 12 well plate: Remove 0.5 mL media and replace with fresh 0.5 mL BrainPhys + B27 + BDNF + GDNF + NIC.
For 24 well plate: Remove 0.25 mL media and replace with fresh 0.25 mL BrainPhys + B27 + BDNF + GDNF + NIC.
SECTION: Media/Reagent Recipes
18.  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
20. 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 (dox) (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. Protect from light.

LDN-193189 hydrochloride (LDN) (Sigma Cat# SML0559-5MG): 1 millimolar (mM) (10,000x):
g = Molecular Weight (g/mol) * Molarity (M) * Volume (L)
0.005 g = (406.48 g/mol) * (0.001 M) * (Volume)
0.005 g = 0.41 g/L * 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 10ml bottle overnight on ice before aliquoting. Always keep matrigel and tubes on ice and never let it 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, 0.5 mL per well of 12-well plate or 0.25 mL  per well of 24-well plate.

Nicotinamide (NIC) (Sigma Cat# 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 = (122.12g/mol)*(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) - (Sigma Cat# P3655-500MG; mol wt 30,000-70,000 Da): 2 mg/mL (20x) stock:
Add 500 mg PLO to 250 mL cell culture ddH2O.
Make 1 mL aliquots. Store aliquots in -80 °C.

Blebbistatin (blebb) (10 millimolar (mM) or 2,000x stock, Sigma Cat# B0560-1MG): 
g = Molecular Weight (g/mol) * Molarity (M) * Volume (L)
0.001 g = (292.33 g/mol) * (0.01 M) * (Volume)
0.001 g = 2.92 g/L * Volume
Volume = 0.000342 L = 0.342 mL = 342 µL
Dissolve 1 mg blebbistatin into 342 µL DMSO, divide into 20 µL aliquots (store at -80 °C). Working concentration is 5 micromolar (µM).



SECTION: Media/Reagent Recipes
19.  N2 supplement (100X) recipe -  If making in-house add the following:
            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
17. For long-term experiments >1 week, continue feeding by 1/3 media exchange every other day with BrainPhys + B27 + BDNF + GDNF + NIC.