Sep 27, 2023
Version 2
Differentiation of RGC Induced Neurons (RGC-iNs) V.2
- Devansh Agarwal1,2,
- Kevin W. Mazo3,
- Karl Wahlin2
- 1Department of Bioengineering, University of California San Diego, La Jolla, United States;
- 2Department of Ophthalmology, University of California San Diego, La Jolla, United States;
- 3Department of Biological Sciences, University of California San Diego, La Jolla, United States
- Devansh Agarwal: Correspondence: d4agarwa@ucsd.edu;
- Kevin W. Mazo: Correspondence: kewima99@gmail.com;
- Karl Wahlin: Correspondence: kwahlin@ucsd.edu
Protocol Citation: Devansh Agarwal, Kevin W. Mazo, Karl Wahlin 2023. Differentiation of RGC Induced Neurons (RGC-iNs). protocols.io https://dx.doi.org/10.17504/protocols.io.14egn2pqzg5d/v2Version created by Devansh Agarwal
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. npj Regen Med 8, 55 (2023). https://doi.org/10.1038/s41536-023-00327-x
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: September 26, 2023
Last Modified: September 27, 2023
Protocol Integer ID: 88422
Funders Acknowledgement:
National Institutes of Health (NIH)
Grant ID: K99/R00EY024648
National Institutes of Health (NIH)
Grant ID: R01EY031318
National Institutes of Health (NIH)
Grant ID: R21EY031122
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.
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 | |
| 40µm Cell Strainer, EASYstrainer (for generating single cell suspension during plating) | Greiner | Cat# 542040 | |
| Accutase (single cell passaging of hPSCs) | Sigma-Aldrich | Cat# A6964 | |
| 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 | |
| Progesterone (N2 supplement component) | Sigma-Aldrich | Cat# P8783 | |
| Putrescine dihydrochloride (N2 supplement component) | Sigma-Aldrich | Cat# P5780 | |
| 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 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.
Note
For PSC expansion make sure that colonies are not overgrown (70-80 % confluent) and not touching.
Alternatively, 2 micromolar (µM) thiazovivin (10 mM or 5000x stock) can be used instead of blebb as a ROCK inhibitor.
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 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).
Note
For long-term experiments, you need better adhesion of cells so you can use a higher concentration of PLO. To do this, dilute 1 mL 2 mg/mL PLO (20x) into 9 mL cell culture grade H2O for a final concentration of 0.2 mg/mL .
1d
Day 0: Neural Induction
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 01:00:00 , 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 >03:00:00 at 37 °C .
4h
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.
Note
Dox is light-sensitive, so keep aliquots cool after thawing and dark when not in use (up to ~1 month at 4 °C ).
In addition, it is critical to prevent the drying of matrigel-coated dishes after the matrigel has been added.
Incubate the cells with prewarmed Accutase (~ 00:05:00 ) in the incubator for 00:12:00 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).
17m
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 1.5 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 00:05:00 at 80 x g .
5m
Aspirate the supernatant and then resuspend the cell pellet in prewarmed NIM + 5 micromolar (µM) blebb.
2m
Filter cells with a 40 µm cell strainer. Count the cells with a hemocytometer.
5m
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).
5m
Day 1: Maintenance
Day 1: Maintenance
Do nothing.
Day 2: Feed – add 1/3 of media
Day 2: Feed – add 1/3 of media
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.
Note
Do this very carefully by adding media to the sides of the dish. If you are not very careful cells will detach.
5m
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 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.
Note
This is the last day to add CultureOne.
For all subsequent exchanges:
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).
5m
Day 6: Feed – exchange 1/3 of media
Day 6: Feed – exchange 1/3 of media
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.
Note
This is the last day to add dox.
5m
Day 8: Feed – exchange 1/3 of media
Day 8: Feed – exchange 1/3 of media
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.
5m
For long-term experiments >1 week, continue feeding by 1/3 media exchange every other day with BrainPhys + B27 + BDNF + GDNF + NIC.
5m
Media/Reagent Recipes
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): 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 (wet) 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) .
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).