TITLE: iPSC to Motor Neuron Differentiation Various Protocols
AUTHORS: jwaligor

[DESCRIPTION]
iPSC to Motor Neuron Differentiation Various Protocols

[STEPS]
SECTION: iPSC/MNP Passaging TrypLE Protocol
1. Aspirate media off
SECTION: iPSC/MNP Passaging TrypLE Protocol
2. Rinse with DPBS-/-
SECTION: iPSC/MNP Passaging TrypLE Protocol
3. Add 1X TrypLE, 1ml per 6 well, 2ml per T-25, 3ml per T-75 (can use as low as 0.75X TrypLE according to GESC, dilute in 0.5mM EDTA pH 8)
SECTION: iPSC/MNP Passaging TrypLE Protocol
4. Put in incubator until you see cells detaching (2 minutes to 5 minutes MAX), might have to move flask around to aid in that process
SECTION: iPSC/MNP Passaging TrypLE Protocol
5. When cells detach, take out of the incubator and add 2x the original volume minimum of fresh media in the vessel (to stop enzymatic reactions)
SECTION: iPSC/MNP Passaging TrypLE Protocol
6. Take cells and put them in a 15ml conical tube
SECTION: iPSC/MNP Passaging TrypLE Protocol
7. Spin down at 1200rpm for 5 min (200g/RCF)
SECTION: iPSC/MNP Passaging TrypLE Protocol
8. Take off supernatant, and resuspend cell pellet in the media.
SECTION: iPSC/MNP Passaging TrypLE Protocol
9. Media per well in 6-well is 2ml, 12-well is 1ml, T-25 is 5ml, T-75 is 20 mL
SECTION: iPSC/MNP Passaging TrypLE Protocol
10. IMPORTANT: Add ROCKi once you replate cells. 1ul ROCKi per 1ml of media in the vessel. Remove ROCKi via media change after 24 hrs. (ROCKi is not in the NComm paper, but is suggested by YiHsien and Vijay since it is commonly used to promote survival after single-cell dissociation and is used in the protocol. We have tested with and without and found significantly better differentiation to MNs with ROCKi). Note: RevitaCell is another ROCKi inhibitor (may be better than Y-27632).
SECTION: MNP freezing protocol (to be used during any step of differentiation)
11. Perform single cell dissociation (see above for TryplE protocol - no ROCKi) up until and including the spin down at 200g for 5 min.
SECTION: MNP freezing protocol (to be used during any step of differentiation)
12. Resuspend in 1 mL Media, do the cell count
SECTION: MNP freezing protocol (to be used during any step of differentiation)
13. Spin down again. Aspirate the supernatant, resuspend in 1mL of Cryostor CS10 (or as many ml as vials you want to freeze down, ex: 2ml resuspension = 2 vials frozen down)
SECTION: MNP freezing protocol (to be used during any step of differentiation)
14. Transfer the Cryostor/Cell solution with from the 15ml conical tube to the cryovial (1 ml per cryovial)
SECTION: MNP freezing protocol (to be used during any step of differentiation)
15. Label tube (list that it is MNP, list if it has a library/guide/or no library, list what passage iiPS it came from, list the Step and Day it will be when we thaw, write date, write initials, write estimated cell count)
SECTION: MNP freezing protocol (to be used during any step of differentiation)
16. Place vials on ice for 10 minutes before putting it in a styrofoam box in the -80C Freezer.
17. After 24-48h transfer to permanent storage box in -80C.
SECTION: PDL/Laminin Coating Procedure
18. PDL Instructions
SECTION: PDL/Laminin Coating Procedure
18.1. Prepare a 1:2500 dilution of PDL in molecular grade water, making sure you have enough to coat whatever vessel you are working with (For a quad array: dissolve 2uL of PDL in 5mL of molecular grade water.)
SECTION: PDL/Laminin Coating Procedure
18.2. Add 1mL per quad on the array.
SECTION: PDL/Laminin Coating Procedure
18.3. Incubate at 37 degrees for 1 hour.
SECTION: PDL/Laminin Coating Procedure
18.4. After 1 hour incubation, rinse thrice with molecular grade water as PDL can be toxic to cells.
SECTION: PDL/Laminin Coating Procedure
19. Mouse Laminin Instructions
SECTION: PDL/Laminin Coating Procedure
19.1. Thaw laminin at 2-8C to prevent it from gelling
SECTION: PDL/Laminin Coating Procedure
19.2. Prepare a 1:300 dilution of laminin in molecular grade water, making sure you have enough to coat whatever vessel you are working with (For a quad array: Dissolve 14uL of laminin in 4.2mL of molecular grade water and add 1mL of the solution per quad)
SECTION: PDL/Laminin Coating Procedure
19.3. Incubate at 37 degrees for 4 hours.
SECTION: PDL/Laminin Coating Procedure
19.4. Aspirate laminin before use. Coated vessels can be used immediately without washing.


 

SECTION: Motor Neuron Differentiation Protocol
20. 
 

 

Protocol Overview
To differentiate the iPSC cells, you have to grow them in 5 or 6 different media.
From this point forward, only use the TrypLE protocol above when passaging. As stated in that protocol, add ROCKi EVERY TIME you use TrypLE (1ul/ml media). Remove ROCKi after 24hr via media change unless you are in the later stages of differentiation (Step 4 or Step 5- media changes are less frequent).
 To start the differentiation process, transfer an appropriate amount of cells into your desired vessel using the TrypLE protocol listed above to single-cell dissociate iiPS cells. Use STEP1 media to resuspend and store the cells in a new matrigel coated vessel.

 

SECTION: Motor Neuron Differentiation Protocol
21. Step 1 Media Recipe
 
      Step 1 (6 days) NEP (neuroepithelial cells) inductionStock Conc.Final conc.10 ml dilution50 ml dilutionDMEM/F12 50%4.86ml24.3mlNeural basal medium 50%4.86ml24.3mlN2100X0.5X50ul250ulB2750X0.5X100ul500ulAscorbic acid200 mM0.1 mM5ul25ulGlutaMAX 100X1X100ul500ulP/S .1% 100X1X10ul50ulDMH110mM (5000x)2 uM2 ul10ulSB-43154210mM (5000x)2 uM2 ul10ulCHIR990213mM (1000x)3uM10 ul50ul


 

Leave the STEP1 media on the cells for 6 days, doing media changes everyday. Over the weekend, if you need to and the well is not too crowded, you can double feed the cells and let them go for 2 days. On the last days of STEP1 media, you may start seeing swirls in the clusters of cells (unless density is high).

If you need to split during this time, use the TrypLE protocol (adding the ROCKi) then re-plate in STEP 1 media. Do a media change after 24 hrs to remove ROCKi.

By the end of STEP1, the iPSCs are SOX1+ NEP (neuroepithelial progenitor cells) or the earliest multipotent neural stem cells. You can freeze the NEP during this stage if you want to store them. 

On the end of “Day 5” or beginning of “Day 6”, single-cell dissociate using TrypLE and ROCKi, and re-plate in STEP2 media. Cells should be re-plated in a new matrigel-coated flask/well. Do a media change after 24hr to remove the ROCKi.
SECTION: Motor Neuron Differentiation Protocol
22. Step 2 Media Recipe

     Step 2 (6 days) Induce Oligo2+ MNP Stock Conc.Final conc.10ml dilution50ml dilutionDMEM/F12 50%4.86ml24.3mlNeural basal medium 50%4.86ml24.3mlN2100x0.5X50ul250ulB2750x0.5X100ul500ulAscorbic acid200 mM0.1 mM5ul25ulGlutaMAX100X 1X100ul500ulP/S 0.1% 100X1X10ul50ulDMH110mM (5000x)2 uM2 ul10ulSB-43154210mM (5000x)2 uM2 ul10ulCHIR990213mM (3000x)1uM3.3 ul16.5ulPur10mM (20000x)0.5uM0.5 ul2.5ulRetinoic acid1mM (1000x)0.1 uM1 ul5ul

Do a media change everyday or double feed if you cannot come in over the weekend. At the end of this step, the cells are Olig2+/NKX2- Motor Neuron Progenitors (MNPs).

If you need to split during this time, use the TrypLE protocol (adding the ROCKi) then re-plate in STEP 2 media. Do a media change after 24 hrs to remove ROCKi.

At the end of Day 5 or beginning of Day6, if the cell number is high enough, you can go straight into Step 4. Alternatively, you can proliferate the MNPs in Step 3 media to generate higher cell numbers. Passage with TrypLE/ROCKi after Step2 and re-plate in Step 3 media into matrigel coated flasks/wells. Do a media change after 24hr to remove ROCKi. 
SECTION: Motor Neuron Differentiation Protocol
23. Step 3 (MNP expansion) Media Recipe

     Step 3 (MNP Expansion)Stock Conc.Final conc.10ml dilution50ml dilutionDMEM/F12 50%4.86 ml24.3mlNeural basal medium 50%4.86 ml24.3mlN2100X0.5X50ul250ulB2750X0.5X100ul500ulAscorbic acid200 mM0.1 mM5ul25ulGlutaMAX100X1X100500ulP/S 0.1% 1X10ul50ulDMH110mM (5000x)2 uM2 ul10ulSB-43154210mM (5000x)2 uM2 ul10ulCHIR990213mM (1000x)3uM10 ul50ulPur10 mM (20000x)0.5uM0.5 ul2.5ulVPA (valproic acid)1 M (2000x)0.5mM5 ul25ulRetinoic acid1mM (1000x)0.1 uM1 ul5ul

 The cells can be left in this media for a MAXIMUM of 5 days (the smallest amount of time in step 3 is ideal, so we jackpot less). Media change daily or double feed over the weekend. After this step, the cells will be motor neuron progenitors. At this point, we can expand and freeze many vials down.

After this step, the cells should be single cell dissociated using TrypLE/ROCKi and moved to Step 4 media in a new matrigel flask/well. Remove ROCKi after 24 hrs.
SECTION: Motor Neuron Differentiation Protocol
24. Step 4 Media Recipe
 
     Step 4 (6 days) – induce MNX1+ MNsStock Conc.Final conc10 ml dilution50 ml dilutionDMEM/F12 50%4.86 ml24.3mlNeural basal medium 50%4.86 ml24.3mlN2 0.5X50ul250ulB27 0.5X100ul500ulAscorbic acid200 mM0.1 mM5ul25ulGlutaMAX 1X100ul500ulP/S 0.1% 1X10ul50ulPur10 mM (100000x)0.1uM0.1 ul0.5ulRetinoic acid1mM (1000x)0.5 uM5 ul25ul

While the cells are on STEP 4 media,  they will mature into MNX1+ motor neurons. Reduce media changes to full media changes every other day.

At the end of Day 5 or beginning of Day 6, single-cell dissociate the cells with TrypLE/ROCKi and move to a PDL/Laminin coated vessel in Step 5 media. 

 

 
SECTION: Motor Neuron Differentiation Protocol
25. Step 5 Media Recipe
 
     Step 5 (10days) – maturation into CHAT+ MNStock Conc.Final conc10 ml dilution50ml dilutionDMEM/F12   4.83ml24.3mlNeural basal medium   4.82ml24.3mlN2   50ul250ulB27   100ul500ulAscorbic acid200 mM  5ul25ulGlutaMAX   100ul500ulPur10 mM (100,000x)0.1uM0.1 ul0.5ulRetinoic acid1mM (1000x)0.5 uM5 ul25ulCompound E1mM (10,000x)0.1 uM1 ul5ul

 At step 5, perform half medium change every other day. No full medium change at this step.
 

 

At high density, and early days of Step 5, growth factors are not required. For plating densities on Raft Arrays, lower densities, or longer times, Step 6 media (which includes cAMP and Growth Factors) should be used to maintain neuron survival.  For studying neurodegenerative models, the paper removes the neurotrophic support.

For long term motor neuron growth, do a media change on late stage Step 5 cells and put them on Step 6 media. Yi Hsien had noted that the best time to move neurons was around Step 5 Day 4 or Day 5.
SECTION: Motor Neuron Differentiation Protocol
26. Step 6 Media Recipe
 
     Step 6 (Long term culture)Stock Conc.Final Conc.10 ml dilution50 ml dilutionNeurobasal medium 9867ul49,335ulN2 1X100ul500ulcAMP 50uM0.1 uM20ul100ulILGF/BDNF/CNTF      100ng/ul10ng/ml each1ul each5ul eachRetinoic acid1mM (1000x)1 uM10ul50ul