TITLE: Generation of induced neurons from human induced pluripotent stem cells. 
AUTHORS: Prarthana Gowda, Zhiping Pang, Mahmoud ElAchwah, Sol Diaz de Leon Guerrero

[DESCRIPTION]



 


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. 







[STEPS]
SECTION: Infection of iPSCs - day 0
1. Follow the below protocols for excitatory and inhibitory neuron generation. 
SECTION: Day 13, Day18, Day23, Day28, Day 33....
46. Discard half of the old media, replace with 100 µL or  750 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. 
1 mL NB+Glut+B27+5%FBS + Factors
Factors: 1:1000 GDFN
               1:1000 BDFN
               1:1000 NT3
 Incubate at 37 °C and 5% CO2
SECTION: Day8
45. Add 100 µLor 750 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 37 °C and 5% CO2
SECTION: Day6
44. Change media to neuronal media - 100 µLfor a 96 well plate or 750 µLof  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 37 °C and 5% CO2 
SECTION: iN plating - day5
42. 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 500 µL plating media for each well. Make sure to label each well with the condition. Mix well. Incubate at 37 °C and 5% CO2

SECTION: iN plating - day5
41. To count cells, mix 10 µL trypan blue with 10 µL cell suspension, add 10μl of diluted cells to one sides of the cell counter.

SECTION: iN plating - day5
39. Transfer suspended cell into 2 mL of MEM in 15mL falcon tube, centrifuge for 5 min at 1000rpm at 23 °C

SECTION: iN plating - day5
38. Add 500 µL of accutase to each well, Incubate at 37oC and 5% CO2 for 6 min

SECTION: iN plating - day5
37. Wash with 500 µL of MEM

SECTION: iN plating - day5
36. Aspirate out old media from excitatory iNs well 
SECTION: iN plating - day5
35. Warm up 10 mLof Neurobasal+Glutamax+B27+5%FBS (plating media) per 96well plate (100 µL per well) or 12 well plate 750 µL per well).  
Warm up 500 µL of Neurobasal+Glutamax+B27+5%FBS (plating media) per cell line for resuspension. 
Warm up 500 µL of accutase per well 
Warm up 3 mL of MEM for each well

SECTION: Glia plating - day 4
34. Add the calculated volume of cell suspension required for plating in 100uL per well of 96well plate and   750 µL  per well of a 12 well plate. Add cell volume to plating media. Mix well, add to well. Incubate at 37 °C and 5% CO2

SECTION: Glia plating - day 4
32. Count cell in 3 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) 
SECTION: Glia plating - day 4
33. Aspirate excess Matrigel from the wells.
SECTION: Glia plating - day 4
31. Aspirate out supernatant, and resuspend cells with 3 mL NB+B27+Glutmax+5% FBS

SECTION: Glia plating - day 4
30. Make sure all the cells are lifted and transfer the trypsin suspended cell to 5 mL of DMEM+10%FBS+1%p/s, centrifuge for 5 min at 1000rpm at 23 °C. 

SECTION: Glia plating - day 4
29. Add 5 mL of trypsin, incubate for 5 min at 37 °C and 5% CO2

SECTION: Glia plating - day 4
28. Aspirate out old media from the glia, wash with 5 mL dPBS

SECTION: Glia plating - day 4
27. Select a confluent plate of P2/P3 mouse glia
SECTION: Glia plating - day 4
26. Warm up 5 mL trypsin, 10 mL of NB+B27+Glutmax+5% FBS (plating media), 3 mL of NB+B27+Glutmax+5% FBS (resuspension), and 5 mL DMEM+10%FBS+1%penecillin-streptomycin

500mL neurobasal, 10mL B27 (0.5mM), 5mL glutamax supplement, 25mL FBS
SECTION: Glia plating - day 4
25. Coat 96 well plates for sensor (5 wells per line) or high content imaging (8 wells per line) experiments with 100 µL matrigel, 12 well plate for RNAseq experiment (1 well per line) with 500 µL materiel for 60 min

SECTION: Recovery- day 4
24. Add 1 mL of recovery media (NB+Glut+b27+dox) to each well, Incubate at 37oC and 5% CO2

SECTION: Recovery- day 4
23. Aspirate out old media from 12 well plate 
SECTION: Recovery- day 4
22. Add 1:1000 of Doxycycline (12 µL)

SECTION: Recovery- day 4
21. Take out 1 mL of Neurobasal+glutamax+B27 per well of a 12 well plate 

SECTION: Selection - day2&amp;3
20. Add 1 mL of selection media (NB+Glut+B27+puro+dox) to each well, Incubate at 37 °C  and 5% CO2

SECTION: Selection - day2&amp;3
19. Aspirate out old media from the 12 well plate 
SECTION: Selection - day2&amp;3
18. Add 1:1000 of Doxycycline(1 µL)
Add 1:1000 - 1:500 of puromycin (1 µL-2 µL) - depending on the number of iPSCs


SECTION: Selection - day2&amp;3
17.  Prepare selection media (NB+Glut+B27+puro+dox). Warm up 1 mL neurobasal+glutamax+B27 per well of a 12 well plate 

SECTION: Induction - day 1
16. Add 1 mL of induction media (NB+Glut+B27+dox+CET/P) to each well. Incubate at 37 °C and 5% CO2

SECTION: Induction - day 1
15. Remove media from the 12 well plate into 10% bleach (including all tips and tubes)
SECTION: Induction - day 1
14. Prepare induction media
Add 1:1000 of Doxycycline (1 µL).          2µg/mL stock solution, 2µg/mL working solution 
Add 1:1000 CET/P (1 µL of each).           2µg/mL stock solution, 2µg/mL working solution

SECTION: Induction - day 1
13. Warm up 1 mL of Neurobasal+B27+Glutamax per well of a 12 well plate. 
SECTION: Infection of iPSCs - day 0
10. Add 500 µL of virus+mTESR+ with CET/P to a labelled well. 

SECTION: Infection of iPSCs - day 0
7. Add 1:1000 of CET/P (1 µL of CET and 1 µL of P per mL of media) to mTESR+, mix well

SECTION: Infection of iPSCs - day 0
8. Add NGN2 and RTTA virus (1:1) to the 500 µL mTESR+ with CET/P per line, mix well (discard all tips and tubes in 10% bleach) 

SECTION: Infection of iPSCs - day 0
11. Aspirate out the supernatant, and resuspend each line of cells will 1 mL mTESR+, make sure cells are resuspended well to ensure cells do not form large colonies for better infection. 

SECTION: Infection of iPSCs - day 0
6. Make sure all the cell colonies are suspended, transfer all cell from each well to 2 mL of MEM and centrifuge for 5 min at 1000rpm at 23 °C. 

SECTION: Infection of iPSCs - day 0
5. Add 500 µLaccutase, incubate at 37 °C and 5% CO2 for 6 min 

SECTION: Infection of iPSCs - day 0
4. Wash with 500 µL MEM

SECTION: Infection of iPSCs - day 0
3. Aspirate old media from confluent iPSC plate. Always use different tips to avoid contamination between the lines. 
SECTION: Day 13, Day18, Day23, Day28, Day 33....
47. Maintain cultures for 30-35days 
SECTION: Infection of iPSCs - day 0
2. Warm up 1.5 mL of mTESR+ per cell line
Warm up 2.5 mL MEM per cell line 
Warm up 1 mLof 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 500 µL of Matrigel per well for at least 60 min

SECTION: Infection of iPSCs - day 0
2. Warm up 2 mL of mTESR+ per cell line 
Warm up 4 mL MEM for each line
Thaw required volumes of Asc1 virus, Dlx2, and of RTTA virus on ice
Coat and incubate 6 well plates with 1 mL of Matrigel per well for 60 min at 37 °C

SECTION: Infection of iPSCs - day 0
3. Aspirate old media from confluent iPSC plate. Always use different tips to avoid contamination between the lines. 
SECTION: Infection of iPSCs - day 0
4. Wash with 1 mL MEM
SECTION: Infection of iPSCs - day 0
5. Add 1 mLaccutase, incubate at 37 °C and 5% CO2 for 6 min 
SECTION: Infection of iPSCs - day 0
6. Make sure all the cell colonies are suspended, transfer all cell to 3 mL of MEM and centrifuge for 5 min at 1000rpm at 23 °C. 

SECTION: Infection of iPSCs - day 0
11. Aspirate out the supernatant, and resuspend each line of cells will 1 mL mTESR+, make sure cells are resuspended well to ensure cells do not form large colonies for better infection. 

SECTION: Infection of iPSCs - day 0
8. Add DLX2, Asl1, and RTTA virus (1:1:1.5)to the 1 mL mTESR+ (per well), mix well (all tips and tubes discarded into 10% beach)

SECTION: Infection of iPSCs - day 0
7. Add 1:1000 of CET/P (1 µL of CET and 1 µL of P per mL of media) to mTESR+ media, mix well
SECTION: Infection of iPSCs - day 0
10. Add 1 mL of virus+mTESR+ media with CET/P to a labelled well
SECTION: Infection of iPSCs - day 0
12. Use 1:1 dilution of trypan blue to cell to count cells using an automatic cell counter. 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 37 °C and 5% CO2 

SECTION: Day1 - Induction
13. Warm up 2 mL of Neurobasal+B27+Glutamax per well of a 6 well plate

SECTION: Day1 - Induction
14. Prepare induction media
Add 1:1000 of Doxycycline (2 µL)
Add 1:1000 CET/P (2 µL of each)

SECTION: Day1 - Induction
15. Remove old media from the 6 well plate into 10% bleach
SECTION: Day1 - Induction
16. Add 2 mL of induction media (NB+Glut+B27+dox+CET/P) to each well. Incubate at 37 °C and 5% CO2

SECTION: Day2&amp;3 - Selection
17. Warm up 2 mL neurobasal+glutamax+B27 per well of a 6well plate 

SECTION: Day2&amp;3 - Selection
18. Add 1:1000 of Doxycycline(2 µL)
Add 1:1000 - 1:500 of puromycin (2 µL-4 µL(depending on number of iPSCs)
Add 1:1000 or 1:500 of hygromycin (2 µL-4 µL) (depending on number of iPSCs)

SECTION: Day2&amp;3 - Selection
19. Aspirate out old media from the 6 well plate 
SECTION: Day2&amp;3 - Selection
20. Add 2 mL of selection media (NB+Glut+B27+puro+dox) to each well, Incubate at 37 °C and 5% CO2

SECTION: Day4 - Recovery
21. Take out 2 mL of Neurobasal+glutamax+B27 per well of a 6 well plate

SECTION: Day4 - Recovery
22. Add 1:1000 of Doxycycline (2 µL)

SECTION: Day4 - Recovery
23. Aspirate out old media from 6 well plate 
SECTION: Day4 - Recovery
24. Add 2 mL of recovery media (NB+Glut+b27+dox) to each well, Incubate at 37oC and 5% CO2

SECTION: Day4 - Glia plating
27. Select a confluent plate of P2/P3 mouse glia
SECTION: Day4 - Glia plating
28. Aspirate out old media from the glia, wash with 5 mL dPBS

SECTION: Day4 - Glia plating
29. Add 5 mL of trypsin, incubate for 5 min at 37 °C and 5% CO2

SECTION: Day4 - Glia plating
30. Make sure all the cells are lifted and transfer the trypsin suspended cell to 5mL of DMEM+10%FBS+1%p/s, centrifuge for 5 min at 1000rpm at 23 °C. 

SECTION: Day4 - Glia plating
31. Aspirate out supernatant, and resuspend cells with 3 mL NB+B27+Glutmax+5% FBS

SECTION: Day4 - Glia plating
33. Aspirate excess Matrigel from the plate.
SECTION: Day4 - Glia plating
32. Count cell in 3 mL suspension, calculate volume for 8x10^3 - 12x10^3 cells per well of a 96 well plate, 2.5x10^4 - 4x10^4 per well of a 12 well plate 

SECTION: Day4 - Glia plating
34. Add the calculate volume of cell suspension required for plating in 100uL per well of 96well plate and   750 µL  per well of a 12 well plate. Add cell volume to plating media. Add to well, mix well. Incubate at 37 °C and 5% CO2

SECTION: Day4 - Glia plating
25. Add 100 µL of Matrigel per well of a 96well plate for sensor (5 wells per line) and high content imaging (8 wells per line) experiments, or 500uL per well of a 12 well plate for RNAseq experiments (1 well per line), incubate  at 37 °C  and 5% CO2 for at least 60 min, preferably 120 min

SECTION: Day4 - Glia plating
26. Warm up 5 mL trypsin, 10 mL of NB+B27+Glutmax+5% FBS, 3 mL of NB+B27+Glutmax+5% FBS for resuspension, and 5 mL  DMEM+10%FBS+1%p/s
SECTION: Glia plating
25. 
SECTION: Day5 - iN plating
35. Warm up 10 mLof Neurobasal+Glutamax+B27+5%FBS (plating media) per 96well plate (100 µL per well) or 12 well plate 750 µL per well).  
Warm up 500 µL of Neurobasal+Glutamax+B27+5%FBS (resuspension) per well 
Warm up 1 mL of accutase per well
Thaw 4 mL of MEM for each well

SECTION: Day5 - iN plating
37. Aspirate out old media from iN 6 well plate 
SECTION: Day5 - iN plating
38. Wash with 1 mL of MEM

SECTION: Day5 - iN plating
39. Add 1 mL of accutase, Incubate at 37oC and 5% CO2 for 6 min

SECTION: Day5 - iN plating
40. Transfer suspended cell into 3 mL of MEM in 15mL falcon tube, centrifuge for 5 min at 1000rpm at 23 °C

SECTION: Day5 - iN plating
42. To count cells, mix 10 µL trypan blue with 10 µL cell suspension, add 10μl of trypan blue cells to the cell counter slide. Calculate volume for 6x10^3 cells per well of a 96well plates or 3.6x10^5 cell per well of a 12 well plate, add calculated volume to each well. Mix well. Incubate at 37 °C and 5% CO2
SECTION: Day6
44. Change media to neuronal media from each well- 100 µLper well of a 96 well plates and 750 µL per well of a 12 well plate  NB+Glut+B27+5%FBS + Factors
Factors: 1:1000 Doxycyline 
               1:1000 GDNF       100µg/mL stock solution, 10ng/mL working solution (1:10 dilution in dPBS)
               1:1000 BDNF.       100µgmL stock solution, 10ng/mL working solution 
               1:1000 NT3.         100µg/mL stock solution, 10ng/mL working solution 
               1:2000 or 1:4000 AraC          2-4uM working solution, (based on glia density, only first feeding- either day6 or day8) 
                1:500 Primocin       50µg/mL stock solution, 100µg/mL working solution (only first feeding - day6)
 Incubate at 37 °C and 5% CO2.  
SECTION: Day8
45. Add new 100 µL or 750 µL of new neuronal media with factors (except for doxycycline and primocin) - total volume per well  200 µL   for a 96 well plate and 1.5 mL for a 12 well plate. 
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 37 °C and 5% CO2
SECTION: Day13, 18, 23, 28, 33...
46. Remove half of the old media and replace 100 µLor 750 µL with new neuronal media with factors (except for doxycycline, primocin, and AraC) per well of a 96well plate and a 12 well plate, respectively. 
NB+Glut+B27+5%FBS + Factors
Factors: 1:1000 GDFN
               1:1000 BDFN
               1:1000 NT3
 Incubate at 37 °C and 5% CO2
SECTION: Day13, 18, 23, 28, 33...
47. Maintain cultures for 30-35days 
SECTION: iN plating - day5
40. Resuspend each line in 500 µL

SECTION: Day5 - iN plating
43. Excitatory and inhibitory cells are co-cultured for all experiments, check excitatory neuron protocol for more details. 
SECTION: iN plating - day5
43. Excitatory and inhibitory cells are co-cultured for all experiments mentioned above, check inhibitory neuron protocol for more details. 
SECTION: Day5 - iN plating
41. Resuspend in 500uL of Neurobasal+Glutamax+B27+5%FBS with CET/P and Doxycycline 
SECTION: Day5 - iN plating
36. Add 1:1000 cet/p and Doxycyline to Neurobasal+Glutamax+B27+5%FBS
SECTION: Infection of iPSCs - day 0
12. 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 37 °C and 5% CO2 
SECTION: Infection of iPSCs - day 0
9. Aspirate excess Matrigel from well
SECTION: Infection of iPSCs - day 0
9. Aspirate excess Matrigel from well