Apr 04, 2024

Public workspaceGeneration of induced neurons from human induced pluripotent stem cells.

DOI
dx.doi.org/10.17504/protocols.io.e6nvwdbxzlmk/v1
  • 1Rutgers;
  • 2Rutgers University
Prarthana Gowda
Open access
DOI: dx.doi.org/10.17504/protocols.io.e6nvwdbxzlmk/v1
Protocol Citation: Prarthana Gowda, Zhiping Pang, Mahmoud ElAchwah, Sol Diaz de Leon Guerrero 2024. Generation of induced neurons from human induced pluripotent stem cells. . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdbxzlmk/v1
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 20, 2023
Last Modified: April 04, 2024
Protocol Integer ID: 88114
Funders Acknowledgement:
NIH NIMH Assay and Data Generation Center (ADGC) for the Model of iPSC-derived Neurons for NPD (MiNND)
Grant ID: RM1MH133065-01
Abstract



Overview of induced neuron protocol for the generation of excitatory and inhibitory neurons.


Protocol for the generation of Ngn2 (Excitatory) and Ascl1/Dlx2 (Inhibitory) induced neurons from human induced pluripotent stem cells (iPSCs) and co-culture of excitatory and inhibitory neurons on mouse glia (Protocols modified from Wang et al., 2022; Yang et al., 2017; Zhang et al., 2013).

iPSCs are maintained in 35mm plates or 6 well plates in mTESR+ media (https://dx.doi.org/10.17504/protocols.io.ewov1qd5ogr2/v1).

Lentiviral vectors were generated by transfecting HEK293T cells with lentivirus packaging plasmids (pMDLg/pRRE, VsVG and pRSV-REV) with the desired vectors as previously described (Pang et al., 2011) using lipofectamine 3000. The following plasmids were used:  pMDLg/pRRE (Addgene 12251), pRSV-Rev (Addgene #12253), pCMV-VSV-G (Addgene #8454), FUW-M2rtTA (Addgene #20342), FUW-TetO-Ngn2-P2A-puromycin (Addgene #52047), FUW-TetO-Ascl1-T2A-puromycin (Addgene #97329), FUW-TetO-Dlx2-IRES-hygromycin (Addgene #97330). Lentiviral particles were collected in mTESR+ media and stored at -80°C until further use.

Induced neurons are generated by transducing iPSCs with the necessary genes using the lentiviral vectors to induce the expression of different transcription factors with doxycycline: rtTA + Ngn2 for excitatory neurons, rtTA + Ascl1 + Dlx2 for inhibitory neurons. Induced neurons are plated into 96 well plates (18×103 cells) or in 12 well plates (1×106 cells) with mouse glia after 5 days of induction. Primary mouse glia was obtained from postnatal day 0-2 mice cortex kept in DMEM 10% FBS 1% pen/strep, glia is used for plating after the second or third passage (P2-P3). CEPT cocktail was used during iPSC and neuronal plating to enhance cell viability (Chen et al., 2021). Induced neurons are cultured for 30-35 days before analysis.






Materials
ReagentVendorCatalog NumberStock concentrationWorking concentration
Accutaseinnovative cell technologies.incAT-1041x1x
AraC C1768 C1768 8mM2-4 µM
B27Gibco 1750404450x1x
BDNFpepro tech10781-164250µg10ng/mL
CET/Pmedchem express, selleck chem, R&D systems sigma- aldrichHY-15392, S7775, 5284, P8483C (50µM), E (5000µM), P(1x), T (0.7mM)C (50nM), E (5uM), P(1x), T (0.7µM)
DMEMGibco11995-0651x1X
Doxycycline MP biomedicals 1989552mg/mL2µg/mL
dPBSSAFCD85371x1x
FBSr&d systems S11550100%1x
GDNFPepro tech10781-226200µg10ng/mL
glutamaxgibco 35050061100x1x
hygromycinsigma-aldrichH977350mg/mL100µg/mL
matrigelCornig354234
MEMgibco51200-0381x1x
mTESR+Stem cell technology 100-02751x1x + supplement
neurobasal gibco 21103-0491x1x
NT3pepro tech10781-174250µg10ng/mL
penicilin/streptomycin thermo-fisher15070-63100x1x
puromycin sigma-aldrichP88331mg/mL1-2µg/mL
Primocin InvivoGenant-pm-150mg/mL100µg/mL
0.05% trypsin EDTAgibco25300-0541x1x
12 well plate falcon 3530431mL/well4cm^2
6 well plate biofil2304117-074-F2mL/well 9cm^2
96well plate greiner 655090200uL/well0.32cm^2
24well plate biofil230606-076-F1mL/well2cm^2

Media preparation

Neurobasal (neuronal for induction, selection , recovery)- 500mL neurobasal, 10mL B27 (0.5mM), 5mL glutamax supplement
Neurobasal + FBS (neuronal media for maintenance) - 500mL neurobasal, 10mL B27 (0.5mM), 5mL glutamax supplement, 25mL FBS (5%)
mTeSR+ (iPSC maintenance and infection) - 400uL mTeSR, 100mL mTeSR supplement, 1mL Primocin
DMEM - 500mL DMEM, 50mL FBS (10%), 5mL penecillin/streptomycin (1%)

*all media filtered during preparation.











Follow the below protocols for excitatory and inhibitory neuron generation.
Step case

Excitatory neurons
46 steps

Warm up Amount1.5 mL of mTESR+ per cell line
Warm up Amount2.5 mL MEM per cell line
Warm up Amount1 mL of accutase per cell line
Thaw required volumes of NGN2 and RTTA virus on ice
Coat and incubate 12 well plates (1 well per line) with Amount500 µL of Matrigel per well for at least Duration01:00:00

1h
Aspirate old media from confluent iPSC plate. Always use different tips to avoid contamination between the lines.
Wash with Amount500 µL MEM

Add Amount500 µL accutase, incubate at Temperature37 °C and 5% CO2 for Duration00:06:00

6m
Make sure all the cell colonies are suspended, transfer all cell from each well to Amount2 mL of MEM and centrifuge for Duration00:05:00 at 1000rpm at Temperature23 °C .

5m
Add 1:1000 of CET/P (Amount1 µL of CET and Amount1 µL of P per mL of media) to mTESR+, mix well

Add NGN2 and RTTA virus (1:1) to the Amount500 µL mTESR+ with CET/P per line, mix well (discard all tips and tubes in 10% bleach)

Aspirate excess Matrigel from well
Add Amount500 µL of virus+mTESR+ with CET/P to a labelled well.

Aspirate out the supernatant, and resuspend each line of cells will Amount1 mL mTESR+, make sure cells are resuspended well to ensure cells do not form large colonies for better infection.

Use 1:1 dilution of trypan blue to cell to count cells using an automatic cell counter. For a 12 well plate seed ~ 1x10^5 - 1.25x10^5 cells. For a 6 well plate seed ~ 2x10^5 - 2.5x10^5 cells. Add an appropriate volume of cell suspension to the labelled well(s), incubate at Temperature37 °C and 5% CO2
Induction - day 1
Induction - day 1
Warm up Amount1 mL of Neurobasal+B27+Glutamax per well of a 12 well plate.
Prepare induction media
Add 1:1000 of Doxycycline (Amount1 µL ). 2µg/mL stock solution, 2µg/mL working solution
Add 1:1000 CET/P (Amount1 µL of each). 2µg/mL stock solution, 2µg/mL working solution

Remove media from the 12 well plate into 10% bleach (including all tips and tubes)
Add Amount1 mL of induction media (NB+Glut+B27+dox+CET/P) to each well. Incubate at Temperature37 °C and 5% CO2

Selection - day2&3
Selection - day2&3
Prepare selection media (NB+Glut+B27+puro+dox). Warm up Amount1 mL neurobasal+glutamax+B27 per well of a 12 well plate

Add 1:1000 of Doxycycline(Amount1 µL )
Add 1:1000 - 1:500 of puromycin (Amount1 µL -Amount2 µL ) - depending on the number of iPSCs


Aspirate out old media from the 12 well plate
Add Amount1 mL of selection media (NB+Glut+B27+puro+dox) to each well, Incubate at Temperature37 °C and 5% CO2

Recovery- day 4
Recovery- day 4
Take out Amount1 mL of Neurobasal+glutamax+B27 per well of a 12 well plate

Add 1:1000 of Doxycycline (Amount12 µL )

Aspirate out old media from 12 well plate
Add Amount1 mL of recovery media (NB+Glut+b27+dox) to each well, Incubate at 37oC and 5% CO2

Glia plating - day 4
Glia plating - day 4
Coat 96 well plates for sensor (5 wells per line) or high content imaging (8 wells per line) experiments with Amount100 µL matrigel, 12 well plate for RNAseq experiment (1 well per line) with Amount500 µL materiel for Duration01:00:00

1h
Warm up Amount5 mL trypsin, Amount10 mL of NB+B27+Glutmax+5% FBS (plating media), Amount3 mL of NB+B27+Glutmax+5% FBS (resuspension), and Amount5 mL DMEM+10%FBS+1%penecillin-streptomycin

500mL neurobasal, 10mL B27 (0.5mM), 5mL glutamax supplement, 25mL FBS
Select a confluent plate of P2/P3 mouse glia
Aspirate out old media from the glia, wash with Amount5 mL dPBS

Add Amount5 mL of trypsin, incubate for Duration00:05:00 at Temperature37 °C and 5% CO2

5m
Make sure all the cells are lifted and transfer the trypsin suspended cell to Amount5 mL of DMEM+10%FBS+1%p/s, centrifuge for Duration00:05:00 at 1000rpm at Temperature23 °C .

5m
Aspirate out supernatant, and resuspend cells with Amount3 mL NB+B27+Glutmax+5% FBS

Count cell in Amount3 mL suspension, calculate volume for 8x10^3 - 1.2x10^3 cells per well of a 96well plate or 2.5x10^3 - 4x10^3 cells per well of a 12well plate.
(number of glia seeded depends on if the cultures are P2 or P3 and how old the cultures are at the time of plating)
Aspirate excess Matrigel from the wells.
Add the calculated volume of cell suspension required for plating in 100uL per well of 96well plate and Amount750 µL per well of a 12 well plate. Add cell volume to plating media. Mix well, add to well. Incubate at Temperature37 °C and 5% CO2

iN plating - day5
iN plating - day5
5m
Warm up Amount10 mL of Neurobasal+Glutamax+B27+5%FBS (plating media) per 96well plate (Amount100 µL per well) or 12 well plate Amount750 µL per well).
Warm up Amount500 µL of Neurobasal+Glutamax+B27+5%FBS (plating media) per cell line for resuspension.
Warm up Amount500 µL of accutase per well
Warm up Amount3 mL of MEM for each well

Aspirate out old media from excitatory iNs well
Wash with Amount500 µL of MEM

Add Amount500 µL of accutase to each well, Incubate at 37oC and 5% CO2 for Duration00:06:00

6m
Transfer suspended cell into Amount2 mL of MEM in 15mL falcon tube, centrifuge for Duration00:05:00 at 1000rpm at Temperature23 °C

5m
Resuspend each line in Amount500 µL

To count cells, mix Amount10 µL trypan blue with Amount10 µL cell suspension, add 10μl of diluted cells to one sides of the cell counter.

Calculate volume for 12x10^3 cells per well of a 96 well plate or 8.4x10^5 cells per well of a 12 well plate, add calculated volume to Amount500 µL plating media for each well. Make sure to label each well with the condition. Mix well. Incubate at Temperature37 °C and 5% CO2

Excitatory and inhibitory cells are co-cultured for all experiments mentioned above, check inhibitory neuron protocol for more details.
Day6
Day6
Change media to neuronal media - Amount100 µL for a 96 well plate or Amount750 µL of NB+Glut+B27+5%FBS + Factors per well of a 12 well plate
Factors: 1:1000 Doxycyline
1:1000 100ug/mL GDNF 10ng/mL working solution (1:10 dilution in dPBS)
1:1000 100ug/mLBDNF. 10ng/mL working solution
1:1000 100ug/mL NT3. 10ng/mL working solution
1:2000 or 1:4000 8mM AraC 2uM-4uM working solution (based on glia density, only first
feeding- either day6 or day8)
1:500uL of Primocin. 100ug/mL working solution (only first feeding - day6)
Incubate at Temperature37 °C and 5% CO2
Day8
Day8
Add Amount100 µL or Amount750 µL of neuronal media with factors (except for doxycycline and primocin) per well of a 96 well plate and a 12well plate, respectively.
NB+Glut+B27+5%FBS + Factors
Factors: 1:1000 GDNF
1:1000 BDNF
1:1000 NT3
1:2000 or 1:4000 AraC (based on glia density, only first feeding- either day6 or day8)
Incubate at Temperature37 °C and 5% CO2
Day 13, Day18, Day23, Day28, Day 33....
Day 13, Day18, Day23, Day28, Day 33....
Discard half of the old media, replace with Amount100 µL or Amount750 µL media every 5 days (make sure outside wells are not evaporating media faster) per well of a 96well plate and 12 well plate, respectively.
Amount1 mL NB+Glut+B27+5%FBS + Factors
Factors: 1:1000 GDFN
1:1000 BDFN
1:1000 NT3
Incubate at Temperature37 °C and 5% CO2
Maintain cultures for 30-35days
Protocol references
Chen, Y., Tristan, C.A., Chen, L., Jovanovic, V.M., Malley, C., Chu, P.H., Ryu, S., Deng, T., Ormanoglu, P., Tao, D., et al. (2021). A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells (Springer US).

Pang, Z.P., Yang, N., Vierbuchen, T., Ostermeier, A., Fuentes, D.R., Yang, T.Q., Citri, A., Sebastiano, V., Marro, S., Südhof, T.C., et al. (2011). Induction of human neuronal cells by defined transcription factors. Nature 476, 220–223. https://doi.org/10.1038/nature10202.

Wang, L., Mirabella, V.R., Dai, R., Su, X., Xu, R., Jadali, A., Bernabucci, M., Singh, I., Chen, Y., Tian, J., et al. (2022). Analyses of the autism-associated neuroligin-3 R451C mutation in human neurons reveal a gain-of-function synaptic mechanism. Mol. Psychiatry 1–16. https://doi.org/10.1038/s41380-022-01834-x.

Yang, N., Chanda, S., Marro, S., Ng, Y.H., Janas, J.A., Haag, D., Ang, C.E., Tang, Y., Flores, Q., Mall, M., et al. (2017). Generation of pure GABAergic neurons by transcription factor programming. Nat. Methods 14, 621–628. https://doi.org/10.1038/nmeth.4291

Zhang, Y., Pak, C.H., Han, Y., Ahlenius, H., Zhang, Z., Chanda, S., Marro, S., Patzke, C., Acuna, C., Covy, J., et al. (2013). Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron 78, 785–798. https://doi.org/10.1016/j.neuron.2013.05.029.