iPSC to Motor Neuron Differentiation Various Protocols

Aspirate media off

Rinse with DPBS-/-

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)

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

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)

Take cells and put them in a 15ml conical tube

Spin down at 1200rpm for 5 min (200g/RCF)

Take off supernatant, and resuspend cell pellet in the media.

Media per well in 6-well is 2ml, 12-well is 1ml, T-25 is 5ml, T-75 is 20 mL

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).

Perform single cell dissociation (see above for TryplE protocol - no ROCKi) up until and including the spin down at 200g for 5 min.

Resuspend in 1 mL Media, do the cell count

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)

Transfer the Cryostor/Cell solution with from the 15ml conical tube to the cryovial (1 ml per cryovial)

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)

Place vials on ice for 10 minutes before putting it in a styrofoam box in the -80C Freezer.

After 24-48h transfer to permanent storage box in -80C.

PDL Instructions

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.)

Add 1mL per quad on the array.

Incubate at 37 degrees for 1 hour.

After 1 hour incubation, rinse thrice with molecular grade water as PDL can be toxic to cells.

Mouse Laminin Instructions

Thaw laminin at 2-8C to prevent it from gelling

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)

Incubate at 37 degrees for 4 hours.

Aspirate laminin before use. Coated vessels can be used immediately without washing.

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.

Step 1 Media Recipe

A	B	C	D	E
Step 1 (6 days) NEP (neuroepithelial cells) induction	Stock Conc.	Final conc.	10 ml dilution	50 ml dilution
DMEM/F12		50%	4.86ml	24.3ml
Neural basal medium		50%	4.86ml	24.3ml
N2	100X	0.5X	50ul	250ul
B27	50X	0.5X	100ul	500ul
Ascorbic acid	200 mM	0.1 mM	5ul	25ul
GlutaMAX	100X	1X	100ul	500ul
P/S .1%	100X	1X	10ul	50ul
DMH1	10mM (5000x)	2 uM	2 ul	10ul
SB-431542	10mM (5000x)	2 uM	2 ul	10ul
CHIR99021	3mM (1000x)	3uM	10 ul	50ul

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.

Step 2 Media Recipe

A	B	C	D	E
Step 2 (6 days) Induce Oligo2+ MNP	Stock Conc.	Final conc.	10ml dilution	50ml dilution
DMEM/F12		50%	4.86ml	24.3ml
Neural basal medium		50%	4.86ml	24.3ml
N2	100x	0.5X	50ul	250ul
B27	50x	0.5X	100ul	500ul
Ascorbic acid	200 mM	0.1 mM	5ul	25ul
GlutaMAX	100X	1X	100ul	500ul
P/S 0.1%	100X	1X	10ul	50ul
DMH1	10mM (5000x)	2 uM	2 ul	10ul
SB-431542	10mM (5000x)	2 uM	2 ul	10ul
CHIR99021	3mM (3000x)	1uM	3.3 ul	16.5ul
Pur	10mM (20000x)	0.5uM	0.5 ul	2.5ul
Retinoic acid	1mM (1000x)	0.1 uM	1 ul	5ul

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.

Step 3 (MNP expansion) Media Recipe

A	B	C	D	E
Step 3 (MNP Expansion)	Stock Conc.	Final conc.	10ml dilution	50ml dilution
DMEM/F12		50%	4.86 ml	24.3ml
Neural basal medium		50%	4.86 ml	24.3ml
N2	100X	0.5X	50ul	250ul
B27	50X	0.5X	100ul	500ul
Ascorbic acid	200 mM	0.1 mM	5ul	25ul
GlutaMAX	100X	1X	100	500ul
P/S 0.1%		1X	10ul	50ul
DMH1	10mM (5000x)	2 uM	2 ul	10ul
SB-431542	10mM (5000x)	2 uM	2 ul	10ul
CHIR99021	3mM (1000x)	3uM	10 ul	50ul
Pur	10 mM (20000x)	0.5uM	0.5 ul	2.5ul
VPA (valproic acid)	1 M (2000x)	0.5mM	5 ul	25ul
Retinoic acid	1mM (1000x)	0.1 uM	1 ul	5ul

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.

Step 4 Media Recipe

A	B	C	D	E
Step 4 (6 days) – induce MNX1+ MNs	Stock Conc.	Final conc	10 ml dilution	50 ml dilution
DMEM/F12		50%	4.86 ml	24.3ml
Neural basal medium		50%	4.86 ml	24.3ml
N2		0.5X	50ul	250ul
B27		0.5X	100ul	500ul
Ascorbic acid	200 mM	0.1 mM	5ul	25ul
GlutaMAX		1X	100ul	500ul
P/S 0.1%		1X	10ul	50ul
Pur	10 mM (100000x)	0.1uM	0.1 ul	0.5ul
Retinoic acid	1mM (1000x)	0.5 uM	5 ul	25ul

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.

Step 5 Media Recipe

A	B	C	D	E
Step 5 (10days) – maturation into CHAT+ MN	Stock Conc.	Final conc	10 ml dilution	50ml dilution
DMEM/F12			4.83ml	24.3ml
Neural basal medium			4.82ml	24.3ml
N2			50ul	250ul
B27			100ul	500ul
Ascorbic acid	200 mM		5ul	25ul
GlutaMAX			100ul	500ul
Pur	10 mM (100,000x)	0.1uM	0.1 ul	0.5ul
Retinoic acid	1mM (1000x)	0.5 uM	5 ul	25ul
Compound E	1mM (10,000x)	0.1 uM	1 ul	5ul

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.

Step 6 Media Recipe

A	B	C	D	E
Step 6 (Long term culture)	Stock Conc.	Final Conc.	10 ml dilution	50 ml dilution
Neurobasal medium			9867ul	49,335ul
N2		1X	100ul	500ul
cAMP	50uM	0.1 uM	20ul	100ul
ILGF/BDNF/CNTF	100ng/ul	10ng/ml each	1ul each	5ul each
Retinoic acid	1mM (1000x)	1 uM	10ul	50ul