TITLE: DIFFERENTIATION OF i3NEURONS (Basic Protocol 5)
AUTHORS: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the rapid and robust differentiation of cortical neurons from hiPSCs via induced expression of the neurogenin-2 (NGN2) transcription factor (Wang et al., 2017; Zhang et al., 2013). To begin, iPSCs with a stably integrated human or mouse neurogenin-2 transgene under a tetracycline-inducible promoter are exposed to doxycycline in neuronal induction medium (IM). Since iPSCs grow as colonies, they must be single-cell dissociated to a new plate before doxycycline treatment in order to provide the differentiating neurons adequate space to begin producing neuritic extensions.</div><div class = "text-block">Once the cells have been partially differentiated on Matrigel, they are re-plated onto dishes coated with poly-L-ornithine (PLO) for neuronal maturation. After 3 days of doxycycline treatment, the cells are committed to neural differentiation, although at this time they may have only minor neuritic elongations. These neurites are generally well-preserved after dissociation and replating, but the longer neuritic projections present in cells differentiated past 3 days are often damaged during the splitting process. For this reason, differentiated neurons are optimally replated on day 3. At this time, differentiating neurons can also be cryopreserved for use at a later date, enabling curation of large, standardized stocks of partially differentiated neurons for future experiments.</div></div>

[STEPS]
?. [Day 0]
Coat a new 15-cm tissue culture dish to be used for differentiation with  to  of Matrigel solution, tilting to ensure coverage of entire surface area. Place in  incubator for  to  prior to beginning dissociation and re-plating.
7 ml
9 ml
37 °C
Matrigel can be re-used once or twice after 30-min incubation periods (aspirate with serological pipet and store solution at  ). If desired, Matrigel can also be kept on coated plates for 1 week in the incubator. If pursuing this strategy, use 10% to 20% more Matrigel solution than for a 30-min incubation and check plate regularly to ensure complete coverage of the surface area (evaporation and/or non-level incubator shelves can cause parts of the plate to dry). Matrigel incubated at 37°C for more than 1 to 2 total hours should not be re-used.
?. [Day 0]
Prepare  IM with  doxycycline and  Y-27632 ROCK inhibitor(see Table 2). Place in  water or bead bath to warm during the dissociation protocol.
20 ml
37 °C
Doxycycline is light-sensitive, so keep cool ( ) and dark when not in use.
?. [Day 0]
Observe iPSCs under a phase contrast microscope to assess confluency and presence of cell debris. Dish should be dissociated at around 70% to 80% confluency (i.e., 2.5–3 × 107 cells).
?. [Day 0]
Wash plate with PBS. To wash, aspirate medium with an aspirating pipet connected to a vacuum apparatus. Pipet  PBS onto plate by tilting dish and slowly dispensing PBS onto the sidewall of the dish. After dispensing PBS, tilt dish in all directions to spread solution around the plate surface. Aspirate PBS and repeat once more. Observe cells under the microscope to assess clearance of debris. Continue washing until debris are absent (typically one or two washings are needed).
10 ml
Cell debris can increase inaccuracy of downstream cell counting and obstruct pellet formation and resuspension after centrifugation. Debris should be minimal in healthy iPSC cultures.
?. [Day 0]
Split cells with  Accutase (see Basic Protocol 1) and collect dissociated cells in the Accutase and an additional  PBS in 15-ml conical tube.
7 ml
7 ml
?. [Day 0]
Count cells, transfer 2 to 2.5 × 107 iPSCs per 15-cm dish to be differentiated into a 15- or 50-ml conical tube, and centrifuge  at , .Aspirate the supernatant and resuspend cells in  to  of IM with  doxycycline and  Y-27632 ROCK inhibitor.
0 Room temperature
10 ml
12 ml
Add 5% IM to dissociated cells in PBS before centrifugation to improve pelleting.
?. [Day 0]
Aspirate Matrigel solution from coated 15-cm dish.
Alternatively, if the plate was freshly coated, the Matrigel solution may be transferred toa new dish (see step 1).
?. [Day 0]
Gently pipet the cell suspension onto the newly aspirated 15-cm dish. Rinse the 15-ml conical tube with an additional   IM with  doxycycline and  Y-27632 ROCK inhibitor and add to the dish for a total volume of  to . Gently tilt dish in all directions to evenly distribute cells throughout surface area.
8 ml
18 ml
20 ml
Higher cell densities at the time of plating can interfere with robust neuronal differentiation. If more cells are needed for downstream experiments, it is best to increase the number of dishes used rather than the number of cells plated per dish. In general, iPSCs divide one to two times after doxycycline administration and are then post-mitotic. If fewer cells are needed, scale the number of iPSCs plated and volume of medium used according to the surface area of the dish (e.g., 0.8 – 1.0 × 107 cells per 10-cm dish or 1.5 × 106 cells/well of a 6-well plate). If multiple iPSC lines are being simultaneously differentiated, plate the same number of cells for each line to minimize density-dependent differentiation variability.
?. [Day 0]
Observe cells under a microscope to ensure even distribution and high proportion of live cells (identified by light halo around each cell) versus dead cells and debris (darker and smaller than live cells).
?. [Day 0]
Place in  incubator and gently slide dish side-to-side and front-to-back to evenly distribute cells.
37 °C
?. [Day 1]
Check cells under a microscope.
Nascent neuritic extensions should begin to be evident after about 24 hr of doxycycline exposure.
?. [Day 1]
Make  of IM (+ doxycycline, but without ROCK inhibitor; see Table 2) and warm in  water/bead bath for approximately , or until warm to the touch.
20 ml
37 °C
?. [Day 1]
Aspirate old medium, wash one to two times with PBS, and replace with  of  warm IM+ doxycycline.
18 ml
If significant cell debris are noted, wash additional times with PBS prior to adding fresh medium.
?. [Day 2]
Check cells under a microscope. Neuritic extensions should be more evident.
?. [Day 2]
Repeat medium exchange with IM+doxycycline as on day one.
?. [Day 3]
Check cells under a microscope. Neurites should be obvious by this time.
If neurites are not present or cells appear misshapen or otherwise unhealthy, cells should be discarded and the protocol re-attempted with a new batch of d0 iPSCs. Make up new IM medium with fresh doxycycline and ROCK inhibitor.
?. [Day 3]
Once cells are confirmed to be healthy, cells should be dissociated with Accutase and either frozen or re-plated onto final dishes for neuronal maturation and experimental manipulation. Dissociation should follow steps 3 to 8 from Day 0 of this protocol; freezing should be done as described in Basic Protocol 1, and plating for neuronal maturation will be detailed in Basic Protocol 6.
?. [Day 0]
Coat a new 15-cm tissue culture dish to be used for differentiation with  to  of Matrigel solution, tilting to ensure coverage of entire surface area.
7 ml
9 ml
?. [Day 0]
Place in  incubator for  to  prior to beginning dissociation and re-plating.
37 °C
?. [Day 0]
Wash plate with PBS. To wash, aspirate medium with an aspirating pipet connected to a vacuum apparatus.
?. [Day 0]
Pipet  PBS onto plate by tilting dish and slowly dispensing PBS onto the sidewall of the dish.
10 ml
?. [Day 0]
After dispensing PBS, tilt dish in all directions to spread solution around the plate surface.
?. [Day 0]
Aspirate PBS and repeat once more.
?. [Day 0]
Observe cells under the microscope to assess clearance of debris.
?. [Day 0]
Continue washing until debris are absent (typically one or two washings are needed).
?. [Day 0]
Count cells.
?. [Day 0]
Transfer 2 to 2.5 × 107 iPSCs per 15-cm dish to be differentiated into a 15- or 50-ml conical tube.
?. [Day 0]
Centrifuge  at , .
0 Room temperature
?. [Day 0]
Aspirate the supernatant and resuspend cells in  to  of IM with  doxycycline and  Y-27632 ROCK inhibitor.
10 ml
12 ml
?. [Day 0]
Gently pipet the cell suspension onto the newly aspirated 15-cm dish.
?. [Day 0]
Rinse the 15-ml conical tube with an additional  IM with  doxycycline and  Y-27632 ROCK inhibitor and add to the dish for a total volume of  to .
8 ml
18 ml
20 ml
?. [Day 0]
Gently tilt dish in all directions to evenly distribute cells throughout surface area.
?. [Day 1]
Aspirate old medium.
?. [Day 1]
Replace with  of  warm IM+ doxycycline.
18 ml
If significant cell debris are noted, wash additional times with PBS prior to adding fresh medium.
?. [Day 1]
Wash with PBS. (1/2)
?. [Day 1]
Wash with PBS. (2/2)