TITLE: 2D differentiation of NPCs to neurons &amp; astrocytes 
AUTHORS: Jessie Buth

[DESCRIPTION]
Protocol to differentiate neural progenitor cells to mature neurons and astrocytes in 2D culture. 

[STEPS]
SECTION: Thawing Neural Progenitor Cells (NPCs)
3. See protocol for thawing hematopoietic progenitor cells (HPCs).
     https://www.protocols.io/view/thawing-frozen-hematopoietic-stem-cells-hpcs-dddt226n
 
It is the same procedure for NPCs, except:
the plates are coated with 0.5 mg/mL standard matrigel
the cells are maintained in NPC base +EGF+FGF+10uM ROCKi (ROCKi is removed 24hr after thawing) 
the cells are plated at 250-300K/well in a 6w plate
SECTION: Expansion of NPCs
5. d1 after passaging NPCs (see STEP 3)
Perform a full media change and replace with NPC maintenance media without ROCKi (NPC base +EGF+FGF)
          NPC+EGF+FGF (10mL):
          10mL NPC base media
          1uL    20ng/mL EGF (stock 200ug/mL)
          2uL    20ng/mL FGF (stock 100ug/mL)

d3
Perform a half media change. Remove 900uL/well, and add a fresh 1mL/well NPC base +EGF+FGF. 

d5
Perform a half media change. Remove 900uL/well, and add a fresh 1mL/well NPC base +EGF+FGF.

d6-7
Passage to new dishes coated with 0.5mg/mL standard matrigel in NPC base +EGF+FGF. 
        for 6w plates -> 1 million cells/well in 2mL NPC base +EGF+FGF
        for 5cm cellBIND dishes -> 2.4 million cells/dish in 6mL NPC base +EGF+FGF

 

SECTION: Differentiation of NPCs to Neurons
6. d1 after passaging NPCs (see STEP 3)
Perform a full media change and replace with neuron differentiation media (NPC base +BDNF+GDNF+cAMP)
          NPC+BDNF+GDNF+cAMP (10mL):
          10mL NPC base media
          1uL    20 ng/mL BDNF (stock 200 ug/mL)
	  2uL   20 ng/mL GDNF (stock 100 ug/mL)
	  1uL    1 uM cAMP (stock 10mM)
d3 
Perform a half media change. Remove 900uL/well, add a fresh 1mL/well NPC base +BDNF+GDNF+cAMP. 

d5
Perform a half media change. Remove 900uL/well, add a fresh 1mL/well NPC base +BDNF+GDNF+cAMP.

d7 [differentiation passage 1 - no ROCKi during this step]
Passage to new dishes coated with 0.5mg/mL standard matrigel in NPC base +BDNF+GDNF+cAMP. 
        for 6w plates -> 1 million cells/well in 2mL media 
        for 5cm cellBIND -> 2.4 million cells/dish in 6mL media
        for 10cm cellBIND dishes -> 5.8 million cells/dish in 12mL media

d9
Perform a full media change and replace with neuron differentiation media (NPC base +BDNF+GDNF+cAMP) to remove any dead cells. 

d11
Perform a half media change. Remove 900uL/well, add a fresh 1mL/well NPC base +BDNF+GDNF+cAMP. 

d13 [differentiation passage 2 - no ROCKi during this step]
Passage to new dishes coated with 0.5mg/mL standard matrigel in NPC base +BDNF+GDNF+cAMP. 
        for 6w plates -> 400K cells/well in 2mL media
        for 5cm cellBIND -> 1 million cells/dish in 6mL media
        for 10cm cellBIND dishes -> 2.5 million cells/dish in 12mL media

d16+
Perform a half media change every 3-4 days until 90-95% confluent. When the cells are 90-95% confluent, passage at the same density as d13 and continue with half media changes every 3-4 days until 90-95% confluent again. 
        for 5cm cellBIND -> remove ~2-2.5mL (depending how much media evaporated, leave enough media 
            to cover the bottom), and add a fresh 3mL/well NPC base +BDNF+GDNF+cAMP. 
        for 10cm cellBIND -> remove ~4.5-5mL (depending how much media evaporated, leave enough media 
            to cover the bottom), and add a fresh 6mL/well NPC base +BDNF+GDNF+cAMP. 

 

~d42
To enrich for neurons, around d42 (or close to this date when the dishes are 95% confluent again), treat the cells with 5ug/mL Mitomycin C for 1hour at 37C. 
After 1hr, perform 3x washes with 6mL PBS, and then replace with 6mL (5cm dishes) or 12mL (10cm dish) of fresh NPC base +BDNF+GDNF+cAMP. 

 

SECTION: Media Recipe
2. NPC Base Media (500mL):
240mL 	50% DMEM/F12 (Hyclone VWR Cat # SH30023.02) 
240mL	50% Neurobasal (FisherSci Cat # 21103-049)
2.5mL	0.5X N2 (100X; Invitrogen Cat # 17502048) 
5mL         0.5X B27 without VA (50X; Invitrogen, 12587010) 
5mL 	1X Glutamax (100x  Invitrogen Cat # 35050061) 
5mL 	1X NEAA (Invitrogen Cat # 11140050)
*250 uL	5 ug/mL Human Insulin Solution (250mg per 500mL) (Sigma Cat # I9278-5ML)
1mL	        1X Primocin (stock 500X, InvivoGen ant-pm-2)
50uL	1 μg/mL Heparin (10 mg/ml stock Sigma H3419-100KU)

 

For NPC maintenance:
+20 ng/mL bFGF (stock 100 ug/mL): 10uL/50mL NPC base
+20 ng/mL EGF (stock 200 ug/mL): 5uL/50mL NPC base

For Neuron differentiation:
d0-d80+
	+20 ng/mL BDNF (stock 200 ug/mL): 5uL/50mL NPC base
	+20 ng/mL GDNF (stock 100 ug/mL): 10uL/50mL NPC base
	+1 uM cAMP (stock 10mM): 5uL/50mL NPC base

~d42 (can be d42-d49)
	1hr pulse with 5 ug/mL Mitomycin C ready made solution (stock 10 mg/mL Sigma Cat# M5353) 
	  ^^ this is enrich for neurons, dividing cells die off or stop proliferating over the next 4-7 days

For Astrocyte differentiation:
d0-d14
	+10 ng/mL hLIF (stock 100 ug/mL): 5uL/50mL
	+10 ng/mL EGF (stock 200 ug/mL): 2.5uL/50mL

d14-d80+
	+10 ng/mL hLIF (stock 100 ug/mL): 5uL/50mL
	+10 ng/mL CNTF (stock 100 ug/mL): 5uL/50mL

SECTION: Differentiation of NPCs to Astrocytes
7. d1 after passaging NPCs (see STEP 3)
Perform a full media change and replace with astrocyte differentiation media #1 (NPC base +hLIF+EGF)
          NPC+hLIF+EGF (10mL):
          10mL NPC base media
          1uL    10 ng/mL hLIF (stock 100 ug/mL)
	  0.5uL 10 ng/mL EGF (stock 200 ug/mL)

d3
Perform a half media change. Remove 900uL/well, and add a fresh 1mL/well NPC base +hLIF+EGF. 

d5
Perform a half media change. Remove 900uL/well, and add a fresh 1mL/well NPC base +hLIF+EGF.

d6-7 [differentiation passage 1 - no ROCKi during this step]
Passage to new dishes coated with 0.5mg/mL standard matrigel in NPC base +hLIF+EGF. 
        for 6w plates -> 1 million cells/well in 2mL media
        for 5cm cellBIND -> 2.4 million cells/dish in 6mL media
        for 10cm cellBIND dishes -> 5.8 million cells/dish in 12mL media

~d9
Perform a full media change and replace with neuron differentiation media (NPC base +hLIF+EGF) to remove any dead cells. 

~d11
Perform a half media change. Remove 900uL/well, and add a fresh 1mL/well NPC base +hLIF+EGF. 

~d13-14 [differentiation passage 2 - no ROCKi during this step]
Passage to new dishes coated with 0.5mg/mL standard matrigel in NPC base +hLIF+CNTF.
        for 6w plates -> 400K cells/well in 2mL media
        for 5cm cellBIND -> 1 million cells/dish in 6mL media
        for 10cm cellBIND dishes -> 2.5 million cells/dish in 12mL media
          NPC+hLIF+CNTF (10mL):
          10mL NPC base media
          1uL    10 ng/mL hLIF (stock 100 ug/mL)
	  1uL    10 ng/mL CNTF (stock 100 ug/mL)

~d16+
Perform a half media change every 3-4 days with NPC base +hLIF+CNTF until 95-100% confluent. 
When the cells are 95-100% confluent, passage at the same density as ~d13 and continue with half media changes with NPC base +hLIF+CNTF every 3-4 days until 95-100% confluent again. 
        for 5cm cellBIND -> remove ~2-2.5mL (depending how much media evaporated, leave enough media 
            to cover the bottom), and add a fresh 3mL/well NPC base +hLIF+CNTF.
        for 10cm cellBIND -> remove ~4.5-5mL (depending how much media evaporated, leave enough media 
            to cover the bottom), and add a fresh 6mL/well NPC base +hLIF+CNTF. 

 

SECTION: Passaging NPCs (every 6-7 days - volumes listed at for 1w of a 6w plate)
4. Before starting, coat 6-well plates with 0.5 mg/mL standard matrigel (Set aside for later)

 

To coat plates:
Thaw an aliquot of matrigel at4 °Coron ice
Dilute with the appropriate amount of ice cold DMEM/F12
          6.5 mLfor 6.5-wells = 3.25 mg matrigel
Aliquot 1 mL in a cold 6-well plate
Shake/tilt the plate until the matrigel evenly coats the bottom
Incubate 37 °Cfor 30 min
After incubating, aspirate the matrigel
Add 2 mLDMEM/F12
Place the plate in the incubator at 37 °C
 

SECTION: Passaging NPCs (every 6-7 days - volumes listed at for 1w of a 6w plate)
4.1. Pre-treat the cells with 10 micromolar (µM) ROCK inhibitor (ROCKi)

Pick up the media in the well to a 15mL conical tube.
Add 10 micromolar (µM) ROCKi (2 µL) and mix well.
Gently return the media to the dish.
Incubate the cells with ROCKi for30 min - 60 minat 37 °C

SECTION: Passaging NPCs (every 6-7 days - volumes listed at for 1w of a 6w plate)
4.2. Lift the cells with Accutase.

Aspirate the media.
Wash with 2mL/well PBS (without Ca/Mg).
Aspirate the PBS.
Add 1mL/well Accutase.
Incubate 5 minutes at 37C.
Pick up the cells in accutase with a glass pipette and blow the cells off the dish.
Transfer the cells to a 15mL conical tube.
Add 6mL NPC base media to the dish and blow the remaining cells off the dish.
Transfer the remaining cells to the 15mL conical tube.
Pipette up/down 3-5x in the conical tube to break apart clumps. To break apart clumps, pipette the cells up, and then press the pipette against the side of the tube and blow out quickly. 
Pick up the entire cell mixture and filter through a 100um cell strainer over a 50mL conical tube to remove any remaining large clumps.
Transfer the cell mixture back to the 15mL conical tube.
Centrifuge at 180g for 5 minutes room temperature.
Aspirate the media.
Close the conical tube lid and tap the pellet to loosen the cell pellet.
Resuspend in 2-3mL of NPC base media.
Pipette up/down 3-5x or until the cell solution is cloudy. 
SECTION: Passaging NPCs (every 6-7 days - volumes listed at for 1w of a 6w plate)
4.3. Count the cells and aliquot to new plates.

Count 10uL cells + 10uL trypan blue with a hemocytometer. 
Determine the #uL for 1 million cells.
Add 1 million cells/well in 2 mLNPC+EGF+FGF+ROCKi in new 0.5 mg/mL matrigel coated plates.
             NPC+EGF+FGF+ROCKI (10mL):
             10mL NPC base media
             1uL    20ng/mL EGF (stock 200ug/mL)
             2uL    20ng/mL FGF (stock 100ug/mL)
             10uL  10uM ROCKi (stock 10mM)
Gently place the plate in the incubator and shake evenly (4x forward/back, 4x side-to-side, 4x forward/back).

 

SECTION: Overview
1. Typical workflow:

d(-14) thaw the NPCs (in ROCKi)
maintain in +EGF+FGF

d(-7) passage (in ROCKi)
maintain in +EGF+FGF

d0 passage (in ROCKi)

d1 full media change to start differentiation to neurons/astrocytes

d7 passage (without ROCKi)

d14 passage (without ROCKi)

d42 Mitomycin C treatment (for neurons only)

~d60-100 replate neurons/astrocytes for experiments