TITLE: Differentiation of human induced pluripotent stem cells to neural stem cells
AUTHORS: Malin Andersson, Alexandra Neaverson, Adam Hunter, Ben Newman, Luke Foulser, Andy Day, Minal Patel, Sebastian Gerety

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the steps of differentiating human induced pluripotent stem cells (iPSCs) into cortical linage neural stem cells (NSCs) using a dual-SMAD and Wnt-inhibition method. The cells are cultured in 6 well plates over the course of 14 days, generating approximately 10 million NSCs per well. An immunocytochemistry quality control assay is used to assess the expression of NSC markers including PAX6, FOXG1 and NESTIN. </div></div>

[STEPS]
?. [Passaging iPSC for Neural Induction]
PreparationCoat Falcon 6 well plates with Vitronectin diluted 1:50 in DPBS(-/-) and incubate for at least 2 hours at 37ºC.
?. [Neural Induction (Day 0 - Day 9)]
Preparation: Make up E6 base media. Excess media can be aliquotted into appropriate volume (e.g. 100ml) and stored   for 12 months.Neural Induction Media should be prepared on the day of its first use, then kept at  .
-80 °C
4 °C
?. [Accutase Passage of Neuroepithelial Sheet (Day 10)]
PreparationCoat Falcon 6 well plates with 1:100 vitronectin in DPBS(-/-) and incubate for at least 2 hours at 37ºC. We recommend plating cells for the ICC QC assay at this passage.
?. [Cryopreservation of Neural Stem Cells (Day 14)]
Preparation:Make up neural freeze media by combining NMM (90%) with DMSO (10%). 1ml is needed per 2.5x106 cells.
?. [Immunocytochemistry Quality Control Assay]
The immunocytchemistry assay is used to determine the success of the neural differentiation by estimating the expression of NSC markers. We also use the stem cell marker OCT4 to highlight any non-differentiated cells.In this section, PBS with Calcium and Magnesium is used to prevent cell detachment. Be very gentle when aspirating and pipetting as differentiated cells are prone to peeling. The ICC staining can be done in a non-sterile environment, however formaldehyde should be handled in a fume hood.
?. [Passaging iPSC for Neural Induction]
Make up seeding media by supplementing complete E8 media with rock inhibitor (Y-27632) to a final concentration of 10μM. Make enough seeding media to give 2ml per well to be re-plated into.
?. [Passaging iPSC for Neural Induction]
Aspirate media from iPSC wells and wash with 2ml DPBS(-/-) per well.  Aspirate DPBS(-/-) and add 1ml accutase to each well. Incubate for 3-6 minutes at 37ºC, checking after 3 minutes to see if cells have begun to detach.
?. [Passaging iPSC for Neural Induction]
Add 4ml E8 media per well, pipetting up and down using a stripette to dislodge the cells. Collect all wells into a 50ml Falcon tube.
?. [Neural Induction (Day 0 - Day 9)]
Preparation of Neural Induction Media  ABC1Media ComponentVolumeFinal Concentration2E6 Base Media100ml100%3ITS (100X)2ml2X4SB431542 (10mM)100µl10µM5XAV939 (10mM)20µl2µM6LDN193189 (1mM)10µl100nMReconstitute SB431542 and XAV939 to 10mM using DMSO. Aliquots can be stored at   for up to 6 months. Reconstitute LDN193189 to 1 mM using DMSO. Aliquots can be stored at   until the expiry date listed on the stock vial. Thaw aliquot(s) of E6 Base Media.Add 2ml of Insulin-Transferrin-Selenium (ITS) per 100ml of E6 Base Media. Add 100µl of SB43142, 20µl XAV939 and 10µl LDN193189 per 100ml E6 Base Media. Note: LDN193189 is light sensitive. If possible, work with cabinet lighting turned off and avoid leaving the media in the light for long periods of time. Neural Induction Media should be stored at  for up to 12 days. Bring to room temperature before use. Do not warm NIM in direct sunlight or under bright lighting; if possible keep it in the dark.
ABC1Media ComponentVolumeFinal Concentration2E6 Base Media100ml100%3ITS (100X)2ml2X4SB431542 (10mM)100µl10µM5XAV939 (10mM)20µl2µM6LDN193189 (1mM)10µl100nM
-20 °C
-20 °C
4 °C
?. [Passaging iPSC for Neural Induction]
Cell countDetermine the cell count using an appropriate cell counter. Calculate the volume of cell suspension required to seed 2x106 cells per well of a 6 well plate to be re-plated into (0.2x106 cell per cm2).
?. [Passaging iPSC for Neural Induction]
Aspirate supernatant and re-suspend each cell line in 2ml E8 with rock inhibitor per 2x106 cells.
?. [Passaging iPSC for Neural Induction]
Quickly remove vitronectin from the newly coated plate(s) and transfer 2ml cell suspension into each well. Gently shake plate back and forth and side to side, ensuring that the cells become evenly distributed.
?. [Passaging iPSC for Neural Induction]
Transfer cells into a  5% CO2 tissue culture incubator overnight, gently agitating the plate once more.
37 °C
?. [Accutase Passage of Neuroepithelial Sheet (Day 10)]
Supplement NMM with rock inhibitor (Y-27632)  to a final concentration of 10μM. Make up enough NMM plus rock inhibitor to give 4ml per well to be re-plated into.
?. [Accutase Passage of Neuroepithelial Sheet (Day 10)]
Aspirate NI media from cells and gently wash cells with 2ml DPBS (-/-) per well. Aspirate DPBS and add 1ml accutase to each well. Incubate for 3-6 minutes at 37ºC, checking after 3 minutes to see if cells are detaching. While cells are incubating, prepare wash buffer containing DMEM/F12+Glutamax and DNAse I at a final concentration of 0.03mg/ml. This should be prepared fresh for each cell line to ensure optimum enzyme activity of the DNAse.
?. [Accutase Passage of Neuroepithelial Sheet (Day 10)]
After incubation, add 4ml wash buffer to each well, pipetting up and down using a stripette to dislodge the cells. Collect all wells for each cell line into a 50ml Falcon tube. Mix the cell suspension thoroughly and aliquot into 15ml Falcon tubes of equal volume for centrifugation.
?. [Accutase Passage of Neuroepithelial Sheet (Day 10)]
Centrifuge cells for .
Centrifuge: 200 34, 5 min
?. [Accutase Passage of Neuroepithelial Sheet (Day 10)]
Aspirate the supernatant and resuspend the cells in 4ml NMM per well to be re-plated into. Since this passage is 1:1, this should be the same number of wells per cell line as in the Neural Induction stage.
?. [Accutase Passage of Neuroepithelial Sheet (Day 10)]
Quickly remove the vitronectin from the newly coated plate(s) and transfer 4ml cell suspension into each well. Agitate plate back and forth and side to side to ensure cells become evenly distributed.
?. [Accutase Passage of Neuroepithelial Sheet (Day 10)]
Transfer plate(s) to a  5% CO2 tissue culture incubator overnight, gently agitating plate once more.
37 °C
?. [Neural Cell Maintenance Culture (Day 11 to Day 13)]
Culture the cells for three days, media changing on day 11 and day 13 with 4ml neural maintenance media per well. During this period, the cells should proliferate and reach a morphology of small, round/oval cells. Neural rosette structures should be visible when viewing the cells under the microscope with a 10x or 20x objective.
?. [Neural Induction (Day 0 - Day 9)]
24 hours after plating, the cells should have attached in a uniform sheet across the well. From this point, the cells are prone to peeling. When aspirating, be sure not to touch the cell layer at any point. Media should be dispensed gently on the side of the well, never directly onto the cells.
?. [Neural Induction (Day 0 - Day 9)]
Aspirate off the media from the well and gently wash cells with DPBS(-/-).
?. [Neural Induction (Day 0 - Day 9)]
Aspirate the DPBS(-/-) and gently dispense 2ml of neural induction media onto the side of the well. Return the cells to the incubator. This is day 0 of neural induction.
?. [Neural Induction (Day 0 - Day 9)]
Media change the cells every 24 hours with 2-3ml of neural induction media per well until day 10.
?. [Neural Induction (Day 0 - Day 9)]
The cells should form a neuroepithelial sheet around day 3. Towards the end of the differentiation period the cell proliferation is high and the media may be very yellow when media changing. There is also an increased risk of the neuroepithelial sheet peeling off on later days. If there is excessive peeling it might be necessary to passage the cells ahead of day 10. Neural rosette structures might start to appear in the culture around day 10.
?. [Passaging iPSC for Neural Induction]
Isolate required volume into a 15ml Falcon tube. Centrifuge all cells for .If you are planning RNA-Seq or qPCR analysis of the cell line, isolate the required suspension volume into separate 15ml Falcon tube(s) for making pellets. We recommend a minimum of 2 million cells per cell pellet.
Centrifuge: 200 34, 5 min
?. [Cryopreservation of Neural Stem Cells (Day 14)]
Aspirate the media from your plate(s). Wash gently with 2ml DPBS(-/-) per well. Aspirate DPBS and add 1ml accutase per well. Incubate for   at  , checking after 2 minutes to see if cells are detaching.
37 °C
?. [Cryopreservation of Neural Stem Cells (Day 14)]
Neutralise accutase by adding 4ml DMEM/F12+Glutamax to each well, pipetting up and down with a stripette to break up the cell layer.
?. [Cryopreservation of Neural Stem Cells (Day 14)]
Pass the cells through a 70μm filter, collecting all the cells from one cell line into a 50ml Falcon tube.
?. [Cryopreservation of Neural Stem Cells (Day 14)]
Cell countDetermine the cell count using an appropriate cell counter. NSCs should be frozen at 2.5x106 cells per vial. Isolate the required number of cells, dividing the volume evenly between 15ml Falcon tubes.If you are planning RNA-Seq or qPCR analysis of the cell line, isolate the required suspension volume into separate 15ml Falcon tube(s) for making pellets. We recommend a minimum of 2 million cells per cell pellet.
?. [Accutase Passage of Neuroepithelial Sheet (Day 10)]
Preparation of Neural Maintenance Media (NMM)  ABC1Media ComponentVolumeFinal Concentration2DMEM/F12+Glutamax250ml50%3Neurobasal-A250ml50%4CTS N-2 (100X)2.5ml0.5X5B27 (50X)5ml0.5X6GlutaMAX (100X)2.5ml0.5X7MEM NEAA (100X)2.5ml0.5X8Sodium Pyruvate (100X)2.5ml0.5X92-mercaptoethanol (50mM)0.5ml50µM10Insulin (10mg/ml)125µl2.5µg/mlIf making larger volumes of the media we recommend filtering it using a filter unit.NMM should be stored at  and expires after 14 days.
ABC1Media ComponentVolumeFinal Concentration2DMEM/F12+Glutamax250ml50%3Neurobasal-A250ml50%4CTS N-2 (100X)2.5ml0.5X5B27 (50X)5ml0.5X6GlutaMAX (100X)2.5ml0.5X7MEM NEAA (100X)2.5ml0.5X8Sodium Pyruvate (100X)2.5ml0.5X92-mercaptoethanol (50mM)0.5ml50µM10Insulin (10mg/ml)125µl2.5µg/ml
4 °C
?. [Cryopreservation of Neural Stem Cells (Day 14)]
Spin cells in a centrifuge for .
Centrifuge: 200 34, 5 min
?. [Cryopreservation of Neural Stem Cells (Day 14)]
Remove supernatant from the cells to be cryopreserved. Resuspend in 1ml Neural Freezing Media per 2.5x106 cells, then divide cell suspension between 1.8ml cryovials, adding 1ml to each cryovial. Place the cryovials into a CoolCell or other approriate freezing container, then transfer to a  freezer. Cells should be transferred to LN2 storage after 24 hours.
-80 °C
?. [Cryopreservation of Neural Stem Cells (Day 14)]
To create cell pellets, aspirate supernatant media and re-suspend the cell pellet with DPBS(-/-) and divide between Eppendorf tubes. Centrifuge tubes for 30 seconds to pellet the cells, then aspirate supernatant and snap-freeze. Store snap-frozen cell pellets at  .
-80 °C
?. [Neural Induction (Day 0 - Day 9)]
Preparation of E6 Base Media  ABC1Media ComponentVolumeFinal Concentration2DMEM/F121000ml100%3L-Ascorbic Acid64mg64mg/L4Sodium Chloride1g1g/L5Sodium Bicarbonate543mg543mg/LWeigh out 64mg of L-Ascorbic Acid, 1g sodium chloride, and 543mg sodium bicarbonate and add all to a 50ml Falcon tube. Add mixed chemicals to 500ml of DMEM/F12. Use DMEM/F12 to wash all the residue out of the Falcon tube, ensuring all the powder is retrieved. Add the 500ml DMEM/F12 plus chemical mixture to a 1L filter unit, and add a second 500ml of DMEM/F12 (without chemicals). Filter to create E6 base media.Ensure E6 base media is fully mixed before aliquotting and freezing. E6 base media can be stored at  for up to 12 months.
ABC1Media ComponentVolumeFinal Concentration2DMEM/F121000ml100%3L-Ascorbic Acid64mg64mg/L4Sodium Chloride1g1g/L5Sodium Bicarbonate543mg543mg/L
-80 °C
?. [Passaging iPSC for Neural Induction]
To create cell pellets, aspirate supernatant media and re-suspend the cell pellet with DPBS(-/-) and divide between Eppendorf tubes. Centrifuge tubes for 30 seconds to pellet the cells, then aspirate supernatant and snap-freeze. Store snap-frozen cell pellets at  .
-80 °C
?. [Immunocytochemistry Quality Control Assay]
Fixing the cells using formaldehydeAspirate the media from the cells and replace it with 4% formaldehyde in PBS with Calcium and Magnesium. Incubate for 20 minutes at room temperature.
?. [Immunocytochemistry Quality Control Assay]
Aspirate the formaldehyde and wash the cells twice times with PBS with Calcium and Magnesium, each time for 5 minutes.
?. [Immunocytochemistry Quality Control Assay]
Replace the PBS from the second wash with new PBS. At this point the cells are fixed and it is possible to store the plate at  for up to 7 days before staining the cells. Make sure plates are wrapped in parafilm to prevent evaporation during storage.
4 °C
?. [Immunocytochemistry Quality Control Assay]
Staining the cellsMake up 0.1% Triton X-100 in PBS with Calcium and Magnesium. It can be stored for up to 1 month at  .
4 °C
?. [Immunocytochemistry Quality Control Assay]
Prepare 10% donkey serum (DS) using 0.1% Triton X-100.
?. [Immunocytochemistry Quality Control Assay]
Blocking and permeabilisation step:Aspirate PBS from the cells and add 10% DS. Incubate at room temperature for 2 hours.
?. [Immunocytochemistry Quality Control Assay]
Dilute 10% DS to 1% DS using 0.1% Triton X-100 (for example 1ml 10% DS in 9ml 0.1% Triton X-100).
?. [Immunocytochemistry Quality Control Assay]
Prepare primary antibodies (Table 1): Towards the end of the blocking time, dilute primary antibodies in 1% DS and mix by using a pipette (keep on ice until needed). If using the antibodies suggested in table 1, the anti-OCT4 stain can be co-stained with any of the NSC markers.  ABCDE1  Antibody   Localisation  Dilution    Catalogue Number    Supplier  2Rabbit anti-PAX6NSC nuclear  1:200    60433S    Cell Signalling Technology  3Rabbit anti-FOXG1NSC nuclear  1:2000    ab196868    Abcam  4Rabbit anti-NestinNSC cytoskeletal (intermediate filaments)1:100ab105389Abcam5Mouse anti-OCT4stem cell nuclear  1:100    sc-5279    Santa Cruz
ABCDE1  Antibody   Localisation  Dilution    Catalogue Number    Supplier  2Rabbit anti-PAX6NSC nuclear  1:200    60433S    Cell Signalling Technology  3Rabbit anti-FOXG1NSC nuclear  1:2000    ab196868    Abcam  4Rabbit anti-NestinNSC cytoskeletal (intermediate filaments)1:100ab105389Abcam5Mouse anti-OCT4stem cell nuclear  1:100    sc-5279    Santa Cruz
?. [Immunocytochemistry Quality Control Assay]
Aspirate blocking buffer and add the primary antibodies (50µl per well for a 96 well plate, or 1ml per well for a 6 well plate). Incubate for 1 hour at room temperature.We recommend having a no primary control sample which are only stained with the secondary antibody. Add 1% DS to these wells during this step.
?. [Immunocytochemistry Quality Control Assay]
Prepare secondary antibodies (Table 2) in 1% DS and add DAPI at a dilution of 1:1000. Pipette to mix. Keep on ice in the dark until needed.  ABCD1  Antibody     Dilution    Catalogue Number    Supplier  2  Donkey Anti-Rabbit AF488    1:1000  A21206Thermo Fisher3  Donkey Anti-Mouse AF647    1:1000    A31571  Thermo Fisher4DAPI  1:1000    40043    Biotium
ABCD1  Antibody     Dilution    Catalogue Number    Supplier  2  Donkey Anti-Rabbit AF488    1:1000  A21206Thermo Fisher3  Donkey Anti-Mouse AF647    1:1000    A31571  Thermo Fisher4DAPI  1:1000    40043    Biotium
?. [Immunocytochemistry Quality Control Assay]
Aspirate the primary antibody and wash 3 times with PBS with Calcium and Magnesium, leaving it on for 5 minutes at room temperature each time.
?. [Immunocytochemistry Quality Control Assay]
Aspirate PBS and add the secondary antibodies (50µl per well for a 96 well plate, or 1ml per well for a 6 well plate). Incubate in the dark (wrapped in foil or in a dark box) for 1 hour at room temperature.
?. [Immunocytochemistry Quality Control Assay]
Aspirate the secondary antibody and wash twice times with PBS, leaving it on for 5 minutes at room temperature in the dark each time.
?. [Immunocytochemistry Quality Control Assay]
Replace the PBS from the second wash with new PBS. At this point, the plate can be stored at   in the dark. The fluorescence from the secondary antibodies will decrease with time. Analyse the staining using fluorescence microscopy or a high-content image scanner.
4 °C
A successful assay would show that the NSCs are expressing PAX6, FOXG1 and Nestin. A lack of NSC marker expression or presence of OCT4 expression in the NSC population would indicate that the differentiation from iPSCs to NSCs did not work as expected. If an iPSC control sample has been stained, this sample should be positive for OCT4 and negative for all NSC markers.No staining should be visible in the no primary control wells. If there is, it is likely there is non-specific binding from the secondary antibodies and the staining observed may be false.
?. [Thawing NSCs]
PreparationCoat Falcon 6 well plates with 1:100 vitronectin in DPBS(-/-) and incubate for at least 2 hours at 37ºC.
?. [Passaging NSCs]
PreparationCoat Falcon 6 well plates with 1:100 vitronectin in DPBS(-/-) and incubate for at least 2 hours at 37ºC.
?. [Thawing NSCs]
Partially thaw cells in   water bath. Add 1ml DMEM/F12+Glutamax to the cryovial dropwise and transfer the partially thawed NSC to a fresh falcon tube containing a further 7ml DMEM/F12+Glutamax. Use another 1ml of DMEM/F12+Glutamax to wash the cryovial to ensure all cells are collected.
37 °C
?. [Thawing NSCs]
Centrifuge the cells at 200rcf for 5 minutes.
Centrifuge: 200 34, 5 min
?. [Thawing NSCs]
Aspirate the supernatant and gently re-suspend cell pellet in 4ml of Neural Maintenance Media supplemented with rock inhibitor (final concentration 10μM) and bFGF (final concentration 20ng/ml).
?. [Thawing NSCs]
If cells were frozen at >1.5x106 cells/vial then seed one vial to one well of a 6 well plate. If cell numbers in the frozen vial are unknown it is recommended to count before plating and aim to seed at a density of 0.15-0.25x106 cells/cm2 (1.5-2.5x106 cells/well of a 6 well plate).
?. [Thawing NSCs]
Remove vitronectin from the prepared plate and replace with 4ml of cell suspension at the correct density.
?. [Thawing NSCs]
Gently agitate plate back and forth and side to side to ensure cells do not centre in middle of the vessel and transfer for overnight incubation in humidified incubator at  .
37 °C
?. [Thawing NSCs]
24h after plating, remove spent media and replace with 4ml fresh NMM supplemented with bFGF (final concentration 20ng/ml).
?. [Thawing NSCs]
Media change cells every other day with NMM supplemented with bFGF. The cells should be confluent enough to passage after 4-6 days of culture.
?. [Passaging NSCs]
Dispense Neural Maintenance Media into a falcon tube and supplement with rock inhibitor (final concentration 10μM) and bFGF (final concentration 20ng/ml).
?. [Passaging NSCs]
Aspirate media and wash cells with DPBS(-/-).
?. [Passaging NSCs]
Aspirate DPBS(-/-) and add 1ml Accutase per well and incubate for 2-4 min in humidified incubator at
37 °C
?. [Passaging NSCs]
Add 4ml DMEM/F12+Glutamax per well, gently pipette up and down to dislodge cells and collect into a 50ml falcon tube. NOTE: If cells remain very clumpy despite accutase treatment it is possible to filter them using a 70μm filter into a 50ml falcon tube.
?. [Passaging NSCs]
Determine the cell count using an appropriate cell counter. Calculate the volume of cell suspension required to seed 2x106 cells per well of a 6 well plate (0.2x106 cell per cm2).
?. [Passaging NSCs]
Centrifuge the cells at 200rcf for 5 minutes.
Centrifuge: 200 34, 5 min
?. [Passaging NSCs]
Aspirate supernatant and gently re-suspend the pellet in 4ml of NMM supplemented with rock inhibitor and bFGF per well to be seeded.
?. [Passaging NSCs]
Remove vitronectin from the prepared plate and replace with 4ml of the cell suspension.
?. [Passaging NSCs]
Gently agitate plate back and forth and side to side to ensure cells do not centre in middle of the vessel and transfer for overnight incubation in humidified incubator at  .
37 °C
?. [Passaging NSCs]
24h after plating, remove spent media and replace with 4ml fresh NMM.