Jun 07, 2023
Version 1
Differentiation of RGC Induced Neurons (RGC-iNs) V.1
This protocol is a draft, published without a DOI.
- 1Department of Ophthalmology, University of California San Diego, La Jolla, United States
- Devansh Agarwal: Correspondence: d4agarwa@ucsd.edu
- Karl Wahlin: Correspondence: kwahlin@ucsd.edu
Protocol Citation: Devansh Agarwal, Karl Wahlin 2023. Differentiation of RGC Induced Neurons (RGC-iNs). protocols.io https://protocols.io/view/differentiation-of-rgc-induced-neurons-rgc-ins-cu5jwy4n
Manuscript citation:
Devansh Agarwal, Nicholas Dash, Kevin W. Mazo, Manan Chopra, Maria PA. Garcia, Amit Patel, Ryan M. Wong, Cairang Jia, Hope Do, Jie Cheng, Colette Chiang, Shawna L. Jurlina, Mike Perry, Jong Rho, Risa Broyer, Cassidy Lee, Robert N. Weinreb, Cezar Gavrilovici, Nick W. Oesch, Derek S. Welsbie, Karl J. Wahlin. Human Retinal Ganglion Cell Neurons Generated by Synchronous BMP inhibition and Transcription Factor Mediated Reprogramming. 2023 (in press).
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 02, 2023
Last Modified: September 27, 2023
Protocol Integer ID: 82827
Abstract
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.
Guidelines
Apart from observation under the microscope, counting, and centrifugation, all steps should be carried out in a sterile biological safety cabinet.
Materials
Table: Key resources or reagents required.
| A | B | C | |
| REAGENT or RESOURCE | SOURCE | IDENTIFIER | |
| All-trans retinoic acid (ATRA) (for enhancing cell survival) | Sigma-Aldrich | Cat# R2625 | |
| Accutase (single cell passaging of hPSCs) | Sigma-Aldrich | Cat# A6964 | |
| B27 vitamin A (-) (neural supplement) | Thermo Fisher Scientific | Cat# 12587010 | |
| B27 vitamin (neural supplement) | Thermo Fisher Scientific | Cat# 17504044 | |
| BDNF (growth factor for RGC growth and survival) | Qkine | Cat# Qk050 | |
| Blebbistatin (ROCK inhibitor for improving cell survival) | Sigma-Aldrich | Cat# B0560 | |
| BrainPhys Neuronal Medium (basal media for supporting long-term growth of neurons) | StemCell Technologies | Cat# 05790 | |
| CultureOne supplement (for enhancing neural conversion) | Thermo Fisher Scientific | Cat# A3320201 | |
| DMEM (basal media ) | Thermo Fisher Scientific | Cat# 11965 | |
| DMEM/F12 50:50 (basal media) | Thermo Fisher Scientific | Cat# 11330 | |
| Doxycycline hyclate (antibiotic for transgene induction) | Sigma-Aldrich | Cat# D5207 | |
| F12 (basal media) | Thermo Fisher Scientific | Cat# 11765 | |
| GDNF (growth factor for enhancing RGC growth and neuronal survival) | Qkine | Cat# Qk051 | |
| Insulin-Human Recombinant (N2 supplement component) | Roche | Cat# 11376497001 | |
| L-ascorbic acid (N2 supplement component) | Sigma-Aldrich | Cat# A8960 | |
| LDN-193189 (pre-patterning BMP pathway inhibitor) | Sigma-Aldrich | Cat# SML0559 | |
| Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix (cell attachment/differentiation of hPSCs) | Corning | Cat# 354230 | |
| mTeSR1 (maintenance and propagation of hPSCs) | Stem Cell Technologies | Cat# 85850 | |
| N-2 Supplement (neural supplement) | Thermo Fisher Scientific | Cat# 17502048 | |
| NEAA (non-essential amino acids for supporting neuronal growth) | Thermo Fisher Scientific | Cat# 11140 | |
| Nicotinamide (NIC) (vitamin B3 supplement to enhance differentiation) | Sigma-Aldrich | Cat# 72340 | |
| Poly-L-ornithine (PLO) hydrobromide (for neural attachment) | Sigma Aldrich | Cat# P3655 | |
| Sodium selenite (N2 supplement component) | Sigma-Aldrich | Cat# S5261 | |
| Thiazovivin (alternate ROCK inhibitor for cell survival) | LC Labs | Cat# T-9753 | |
| holo Transferrin Human (N2 supplement component) | Sigma-Aldrich | Cat# T0665 | |
| GlutaMAX Supplement (auxiliary energy source for cells) | Thermo Fisher Scientific | Cat# 35050061 |
Before start
For media/reagent recipes see the last section of the protocol.
PSC expansion step
PSC expansion step
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.
Note
For PSC expansion make sure that colonies are not overgrown (70-80 % confluent) and not touching.
Day -1: Priming of stem cells for neural induction
Day -1: Priming of stem cells for neural induction
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 .
Note
For long-term experiments, you need better adhesion of cells so you can dilute 1 mL 20x PLO into 9 mL cell culture grade H2O for a final concentration of 0.2 mg/mL .
Day 0: Neural Induction
Day 0: Neural Induction
Wash overnight PLO-coated plates >3 times with culture grade H2O. Let plates dry in the back of the TC hood for 01:00:00 , then coat with 1 % (v/v) Matrigel (recommended >03:00:00 ).
4h
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.
Note
Throughout the protocol instead of 5 micromolar (µM) blebbistatin, 2 micromolar (µM) thiazovivin (10 millimolar (mM) or 5000x stock) can be used alternatively as a ROCK inhibitor. Doxycycline is light-sensitive, so keep cool (4 °C ) and dark when not in use.
Aspirate the media from the wells and add Accutase prewarmed for 00:05:00 (the volume of Accutase to use is 1/2 the volume that you maintain the cells in).
5m
Put the cells with the Accutase back into the incubator for 00:12:00 .
12m
Gently rinse the wells using a P1000 and pipet up and down 3 times to further break up the cell clumps into single cells.
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 00:05:00 at 80 x g .
5m
Aspirate the supernatant and resuspend the cell pellet in 1 mL NIM + blebb.
Filter cells with a 35 µm or 40 µm cell strainer (for e.g., Greiner #542040).
Count the cells with a hemocytometer.
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.
Day 1: Maintenance
Day 1: Maintenance
Do nothing.
Day 2: Feed – exchange 1/3 of media
Day 2: Feed – exchange 1/3 of media
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.
Note
Do this very carefully by adding media to the sides of the dish. If you are not very careful cells will detach.
Day 3: Maintenance
Day 3: Maintenance
Do nothing.
Day 4: Feed – exchange 1/3 of media.
Day 4: Feed – exchange 1/3 of media.
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.
Day 6: Feed – exchange 1/3 of media.
Day 6: Feed – exchange 1/3 of media.
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) ).
Day 8: Feed – exchange 1/3 of media.
Day 8: Feed – exchange 1/3 of media.
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) ).
Note
Neurons tend to easily dissociate from the dish, so be very careful when aspirating. Take care to aspirate and dissociate by tilting the dish so that the medium accumulates on one side. Then, aspirate/dispense with the pipette directed toward the wall of the dish (i.e., away from the cells at the bottom).
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) ).
Media/Reagent Recipes
Media/Reagent Recipes
Neural Induction medium (NIM medium):
| A | B | C | |
| Component | Catalog # | Volume | |
| DMEM/F12 | Thermo Fisher Scientific # 11330 | 485 ml | |
| N2 supplement (100x) | Thermo Fisher Scientific # 17504044 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:
| A | B | C | D | E | F | |
| Component | 100 ml | 125 ml | 200 ml | 250 ml | 500 ml | |
| Transferrin | 1g | 1.25 g | 2 g | 2.5 g | 5.0 g | |
| Insulin | 50 mg | 62.5 mg | 100 mg | 125 mg | 250 mg | |
| Progesterone | 63 µg | 78.5 µg | 126 µg | 157 µg | 315 µg | |
| Putrescine | 161 mg | 201.5 mg | 322 mg | 403 mg | 806 mg | |
| Sodium Selenite | 50.2 µg | 62.75 µg | 100.4 µg | 125.5 µg | 251 µg | |
| DMEM/F12 | to 100ml | to 150ml | to 200ml | to 250ml | to 500ml |
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 .
Protocol references
1. Fernandopulle, M. S. et al. Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons: Rapid differentiation of iPSCs into neurons. Current Protocols in Cell Biology 79, e51 (2018).
2. Nehme, R. et al. Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission. Cell Reports 23, 2509–2523 (2018).