Differentiation of RGC Induced Neurons (RGC-iNs)
Devansh Agarwal, Kevin W. Mazo, Karl Wahlin
Abstract
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.
Steps
PSC expansion step
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): 2): Plate 5,000 PSCs into each well of 12-well plates in mTeSR1 in the presence of 5micromolar (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) 2): 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.
Day -1: Priming of stem cells for neural induction
One day prior to neural induction, pre-coat TC plates overnight with 0.1mg/mL poly-L-ornithine hydrobromide (PLO). To do this, add 1mL 2mg/mL PLO (20x) to 19mL cell culture grade H2O, use 1mL per well for 6-well plates, 0.5mL per well for 12 well plates and 0.25mL per well for 24 well plates. Incubate overnight at 37°C.
Pre-treat stem cells by replacing mTeSR1 media with fresh mTeSR1 supplemented with 1µg/mL doxycycline (dox; 1,000x stock) and 100nanomolar (nM) LDN-193189 (10,000x stock).
Day 0: Neural Induction
Wash overnight PLO-coated plates >3 times with culture grade H2O. Let plates dry completely in the back of the TC hood for 1h 0m 0s, then coat with 1% (v/v) Matrigel. For Matrigel coating, use 1mL per well for 6-well plates, 0.5mL per well for 12 well plates and 0.25mL per well for 24 well plates. Incubate for >3h 0m 0s at 37°C.
Prepare Neural Induction Medium (NIM) initiation cocktail with 2µg/mL dox (500x stock), 100nanomolar (nM) LDN (10,000x stock), 1x CultureOne (100x stock) and 5micromolar (µM) blebb.
Incubate the cells with prewarmed Accutase (~ 0h 5m 0s) in the incubator for 0h 12m 0s The volume of Accutase to use is 1/2 the volume that you maintain the cells in (e.g., 1mL per well of a 6 well plate, 0.5mL per well of a 12 well plate and 0.25mL per well of a 24 well plate).
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.
Put the cells into a conical 1.5mL or 5mL tube with 2 times the volume of prewarmed mTeSR1 with 5micromolar (µM) blebb (e.g. 1mL Accutase + 2mL mTeSR1) to quench the Accutase, then pellet the cells for 0h 5m 0s at 80x g,0h 0m 0s.
Aspirate the supernatant and then resuspend the cell pellet in prewarmed NIM + 5micromolar (µM) blebb.
Filter cells with a 40µm cell strainer. Count the cells with a hemocytometer.
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) 2/well; 57.6 cm2 total; 7,000 cells/cm2): Add 403,200 cells into 12mL (67,200 cells per well) of NIM initiation cocktail (NIM + dox, LDN, CultureOne, blebb) in a 15mL conical tube, mix well and distribute across the wells (2mL per well).
To make a 12-well plate (3.5 cm2/well; 42 cm2 total; 7,000 cells/cm2) 2/well; 42 cm2 total; 7,000 cells/cm2): Add 294,000 cells into 12mL (24,500 cells per well) of NIM initiation cocktail in a 15mL conical tube, mix well and distribute across the wells (1mL per well).
To make a 24-well plate (1.9 cm2/well; 45.6 cm2 total; 7,000 cells/cm2): 2/well; 45.6 cm2total; 7,000 cells/cm2): Add 319,200 cells into 12mL (13,300 cells per well) of NIM initiation cocktail in a 15mL conical tube, mix well and distribute across the wells (0.5mL per well).
Day 1: Maintenance
Do nothing.
Day 2: Feed – add 1/3 of media
For 6 well plate : Add 1mL NIM + dox + 1xCultureOne 1µg/mL dox + 1xCultureOne to each well.
For 12 well plate : Add 0.5mL NIM + dox + CultureOne to each well.
For 24 well plate : Add 0.25mL NIM + dox + CultureOne to each well.
Day 3: Maintenance
Do nothing.
Day 4: Feed – exchange 1/3 of media
For 6 well plate : Remove 1mL media and replace with fresh 1mL NIM + dox + 1xCultureOne + NIC ( ). 1µg/mL dox + 1xCultureOne + NIC (10millimolar (mM)).
For 12 well plate : Remove 0.5mL media and replace with fresh 0.5mL NIM + dox + CultureOne + NIC.
For 24 well plate : Remove 0.25mL media and replace with fresh 0.25mL NIM + dox + CultureOne + NIC.
Beyond day 4, plates need to be fed every other day.
Day 6: Feed – exchange 1/3 of media
For 6 well plate : Remove 1mL media and replace with fresh 1mL BrainPhys + B27 (1x) + dox + BDNF ( ) + GDNF ( ) + NIC ( ). 1µg/mL dox + BDNF (50ng/mL) + GDNF (10ng/mL) + NIC (10millimolar (mM)).
For 12 well plate : Remove 0.5mL media and replace with fresh 0.5mL BrainPhys + B27 + dox + BDNF + GDNF + NIC.
For 24 well plate : Remove 0.25mL media and replace with fresh 0.25mL BrainPhys + B27 + dox + BDNF + GDNF + NIC.
Day 8: Feed – exchange 1/3 of media
For 6 well plate : Remove 1mL media and replace with fresh 1 1mL BrainPhys + B27 (1x) + BDNF ( ) + GDNF ( ) + NIC ( ). 50ng/mL) + GDNF (10ng/mL) + NIC (10millimolar (mM)).
For 12 well plate : Remove 0.5mL media and replace with fresh 0.5mL BrainPhys + B27 + BDNF + GDNF + NIC.
For 24 well plate : Remove 0.25mL media and replace with fresh 0.25mL BrainPhys + B27 + BDNF + GDNF + NIC.
For long-term experiments >1 week, continue feeding by 1/3 media exchange every other day with BrainPhys + B27 + BDNF + GDNF + NIC .
Media/Reagent Recipes
Neural Induction medium (NIM medium):
| A | B | C |
|---|---|---|
| Component | Catalog # | Volume |
| DMEM/F12, HEPES | Thermo Fisher Scientific # 11330 | 485 ml |
| N2 supplement (100x) | Thermo Fisher Scientific # 17502048 or recipe below | 5 ml |
| NEAA (non-essential amino acids, 100x) | Thermo Fisher Scientific # 11140 | 5 ml |
| GlutaMAX supplement (100x) | Thermo Fisher Scientific # 35050061 | 5 ml |
| Total 500 ml |
N2 supplement (100X) recipe - If making in-house add the following:
| A | B | C | D | E | F | G |
|---|---|---|---|---|---|---|
| Component | Catalog # | 100 ml | 150 ml | 200 ml | 250 ml | 500 ml |
| Transferrin | Sigma-Aldrich # T0665 | 1 g | 1.5 g | 2 g | 2.5 g | 5.0 g |
| Insulin | Roche # 11376497001 | 50 mg | 75 mg | 100 mg | 125 mg | 250 mg |
| Progesterone | Sigma-Aldrich # P8783 | 63 µg | 94.5 µg | 126 µg | 157 µg | 315 µg |
| Putrescine | Sigma-Aldrich # P5780 | 161 mg | 241.5 mg | 322 mg | 402.5 mg | 805 mg |
| Sodium Selenite | Sigma-Aldrich # S5261 | 50.2 µg   | 75.3 µg | 100.4 µg | 125.5 µg | 251 µg |
| DMEM/F12 | Thermo Fisher Scientific # 11330 | to 100 ml | to 150 ml | to 200 ml | to 250 ml | to 500 ml |
Reagent Stock Dilutions:
Recombinant Human BDNF Protein (Qkine, Cat# Qk050): 50µg/mL stock in 10millimolar (mM) HCl with 0.1% (v/v) BSA; 1,000X; use at 50ng/mL. Store aliquots in -80°C.
Recombinant Human GDNF Protein (Qkine, Cat# Qk051): 10µg/mL stock in cell culture grade H2O with 0.1% (v/v) BSA; 1,000X; use at 10ng/mL. Store aliquots in -80°C.
Doxycycline (dox) (Sigma-Aldrich, Cat# D5207): Make 1mg/mL stock (working concentration is 0.5-2µg/mL) in cell culture grade ddH2O and filter sterilize; 1,000X; use at 1µg/mL. Store 1mL aliquots at -20°C. Protect from light.
LDN-193189 hydrochloride (LDN) (Sigma Cat# SML0559-5MG): 1millimolar (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 = 0.012L or 5mg LDN in 12mL DMSO; Store aliquots in -80°C.
Matrigel (GF reduced) ( ) 1% (v/v)) (Corning, Cat# 354230):
Thaw stock 10ml bottle overnight 4On ice before aliquoting. Always keep matrigel and tubes on ice and never let it come to room temperature or it will gel.
Make 200µL aliquots. Store aliquots in -80°C.
Add 200µL matrigel to 24mL ice-cold DMEM/F12 (~1% (v/v) final).
Add 1mL per well of 6-well plate, 0.5mL per well of 12-well plate or 0.25mL per well of 24-well plate.
Nicotinamide (NIC) (Sigma Cat# 72340): 1Molarity (M) (100x) stock solution (10millimolar (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)
= 6.11g NIC in 50mL 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): 2mg/mL (20x) stock:
Add 500mg PLO to 250mL cell culture ddH2O.
Make 1mL aliquots. Store aliquots in -80°C.
Blebbistatin (blebb) (10millimolar (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 1mg blebbistatin into 342µL DMSO, divide into 20µL aliquots (store at -80°C). Working concentration is 5micromolar (µM).
