Gibco™Advanced DMEM/F-12

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3.1. ESC dissociation and plating: 1. Warm 0.25% trypsin and LIF-2i medium to 37°C in water bath. 2. In a Biosafety cabinet, add gelatin to completely cover bottom of 60mm plate for new passage (SeeNote 3). Let the plate sit approximately 20 minutes; in the meantime, proceed to step 3. 3. Aspirate media from ESC plate (cells at ~80% confluency) and wash one time with 5 mL PBS. 4. Add 500 µL trypsin; incubate at RT or in 37°C incubator for 1–2 minutes. Agitate the plate to break up cell clumps and confirm via microscope that cells have rounded and detached from the surface of the plate. 5. Add 1 mL LIF-2i medium to plate and transfer to 2 mL microcentrifuge tube. 6. Pipet in microcentrifuge tube to dissociate cell clumps into single-cells, taking care not to introduce air bubbles into the medium. 7. Centrifuge at RT for 2.5 minutes at 1400 rpm. 8. While cells are in centrifuge, aspirate gelatin from the new 60mm plate; let plate sit at least 5 minutes to dry. 9. Remove supernatant from cell suspension by aspirating or pouring; avoid disturbing the pellet. 10. Re-suspend in 1 mL LIF-2i medium. 11. Split the cells 1:10 for re-plating in approximately 2 days, or 1: 50 for re-plating in approximately 4 days. (See Note 4) 3.2. ES cell differentiation 1. Warm Ectodermal differentiation medium in 37°C water bath. 2. Aspirate LIF-2i media from plate of ~80% confluent ESCs; wash three times with 5 mL PBS (SeeNote 5). 3. Add 500 µL of 0.25% Trypsin-EDTA. Incubate at RT or at 37°C for 1–2 minutes. Agitate plate to break up cell clumps and confirm via microscopy that cells have rounded and detached from the surface of the plate. 4. Add 1 mL warmed Ectodermal differentiation medium to plate; transfer cells to a 2 mL microcentrifuge tube. 5. Dissociate cell clumps into single cells by pipetting up and down with a P1000 tip. Centrifuge for 2.5 minutes at 1400 rpm to pellet the cells. 6. Remove supernatant by pipetting or aspirating; avoid disturbing the pellet. Re-suspend in 1 mL Ectodermal differentiation medium. 7. To a cell-strainer-top test tube, forcefully pipet 1 mL fresh Ectodermal differentiation medium. This will prime the strainer. Then pipet 1 mL ES cell suspension into the cell strainer, followed by 1 mL fresh Ectodermal differentiation medium, for a total of 3 mL in the tube. (See Note 6) 8. With a P1000 tip, pipet the cell suspension to mix. Use a hemacytometer to determine cell concentration. 9. For two 96-well plates, dilute the appropriate volume of cell suspension in 22 mL fresh ectodermal differentiation medium. The desired final concentration is 30,000 cells per mL. Invert to mix. (SeeNote 7) 10. Transfer the cell suspension to a reservoir. Using a multichannel pipette, dispense 100 uL cell suspension per well into two 96-well plates. 11. Centrifuge the plates at RT for 5 minutes at 800 rpm. 12. Store the plates in a 37°C incubator with 5.0% CO2. 3.3. Day 1: Addition of Matrigel 1. Add 440 µL Matrigel to 10.56 mL Ectodermal differentiation medium for a total of 11 mL complete medium. It is important to keep the Matrigel on ice or at 4°C immediately prior to mixing, and to add Matrigel to ice-cold medium, as Matrigel will become gelatinous at temperatures above 15°C. After mixing, hold the medium up to a light source to confirm that the Matrigel has been fully incorporated. 2. Warm Ectodermal differentiation medium with Matrigel in 37°C water bath. 3. Remove 50 µL of medium from each well using a multichannel pipette, making sure to hold the pipette at an angle so as not to disturb the cell aggregate at the bottom of each well. 4. To each well, add 50 µL of Ectodermal differentiation medium plus Matrigel and pipette 4–6 times to mix. (See Note 8) 3.4. Day 3: Addition of BMP4 and SB-431542 1. For each experimental condition, prepare an appropriate amount of Ectodermal differentiation medium. For two 96-well plates, 6 mL of medium is typically adequate. 2. For 6 mL medium, add 3 µL BMP4 and 3 µL SB-431542 (0.5 µL per 1 mL). This results in a 5X concentration of BMP4 and SB. 3. To each well, add 25 µL of Ectodermal differentiation medium plus BMP/SB. Note there is a final volume of 125 uL and a final concentration of 10 ng/mL BMP4 and 1 uM SB-431542 per well. (SeeNote 9) 3.5. Day 4.5: Addition of FGF and LDN-193189 1. For each experimental conditional, prepare an appropriate amount of Ectodermal differentiation medium. For two 96-well plates, 6 mL of medium is typically adequate. 2. For 6 mL, add 4.5 µL of FGF-2 (0.75 µL per 1 mL) and 3.6 µL of LDN-193189 (0.6 µL per 1 mL). This results in FGF-2 and LDN-193189 at a 6X concentration. 3. To each well, add 25 µL of Ectodermal differentiation medium plus FGF/LDN. Note there is a final volume of 150 uL and a final concentration of 25 ng/mL FGF-2 and 1 µM LDN-193189 per well. (See Note 10) 3.6. Day 8: Transfer to long-term culture 1. Cut the end of a P1000 pipette tip and transfer the aggregates from each well into a 15 mL conical tube (See Note 11). Separate different experimental conditions into different 15 mL conical tubes if necessary. 2. Let the aggregates settle to the bottom of the tube; then aspirate the media, taking care not to disturb the aggregates. 3. Wash the aggregates in 5 mL DMEM/F12 (pre-warmed to 37° C). 4. Repeat steps 2 and 3 at least 3 times. 5. Remove any excess DMEM/F12 and then re-suspend the aggregates in Maturation medium containing 1% Matrigel. 6. Using a wide-mouth P1000, transfer the aggregates to a 100 mm bacterial dish. Suspend in a sufficient amount of Maturation medium to account for medium spreading out across the surface of the place. 7. Pipet 1–3 aggregates in 1 mL medium into the wells of a 24-well plate. Alternatively, single aggregates may be transferred to a new 96-well plate (suspend in 150–200 µL Maturation medium containing Matrigel). By this time point, aggregates in the bacterial plate should be visible without use of a microscope. 8. Beginning on day 10 and continuing every other day (day 12, 14, 16, etc.), replace half of the medium in each well with Maturation medium that does not contain Matrigel. For aggregates plated in 96-well plates, replace half of the medium every day beginning on day 9. Aggregates can be incubated for up to 22 days in 24-well plates, or 12 days in 96-well plates. This results in a total optimal culture period of 30 days (in 24-well plates) or 20 days (in 96-well plates). Aggregates can be fixed at any point for imaging as a cryosection or as a wholemount. After the prescribed durations, further culture will likely require changes in culture medium or format.

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