TITLE: DIFFERENTIATION OF i3LMNS (Basic Protocol 7)
AUTHORS: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the rapid and robust differentiation of hiPSCs into lower motor neurons (i</span><span style = "vertical-align:super;">3</span><span>LMNs) via induced expression of the transcription factors NGN2, ISL1, and LHX3 (hNIL) (Mazzoni et al., 2013; Shi et al., 2017). In particular, a donor construct containing these factors under the tetracycline response element (TRE3G) (Gossen & Bujard, 1992), a CAG promoter driving constitutive expression of the reverse tetracycline transactivator (rtTA3G), and an EF-1α promoter driving constitutive expression of selection genes (mCherry for </span><a href="https://www.addgene.org/105841/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene, cat. no. 105841</span></a><span> and SBP-LNGFR and mApple for </span><a href="https://www.addgene.org/105842/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene, cat. no. 105842</span></a><span>) was stably integrated into the safe-harbor </span><span style = "font-style:italic;">CLYBL</span><span> locus via TALENs (Cerbini et al., 2015). Motor neuron differentiation efficiency can vary between iPSC lines, and homozygous insertion into both CLYBL alleles may result in improved efficiency. Reduced differentiation efficiency of mCherry positive clones can occur with off-target integration of the donor construct. Additionally, the mCherry reporter is flanked by loxP sites, permitting excision by transient transfection of Cre recombinase if desired.</span></div></div>

[STEPS]
?. [Day 0]
Prepare a sufficient amount of hNIL-iPSC for differentiation.
Approximately two wells of a 6-well dish at 80 % confluency are sufficient to begin the differentiation of a 10-cm dish, and cell numbers may be scaled by well surface area.Typically, hNIL lines proliferate for longer than hNGN2 lines, and this protocol calls for plating in E8 before switching to IM on d1, so hNIL cells are seeded at lower density on d0.
?. [Day 0]
Prepare cells as for EDTA split (see Basic Protocol 1).
?. [Day 0]
Without aspirating the EDTA, tilt the plate and pipet  DMEM/F12 into each well with a 10-ml pipet and remove the cells from the bottom of the dish. Gently triturate the cells by pipetting the cells up and down two to three times in each well.
3 ml
?. [Day 0]
Count the cells (Phelan & May, 2015).
?. [Day 1]
Gently rock and swirl the plate by hand to resuspend and collect debris.
?. [Day 1]
Add  of IM freshly supplemented with  Y-27632 ROCK inhibitor, doxycycline, and 1:10,000 Compound E from stock.
12 ml
Doxycycline will induce the expression of the hNIL construct and promote the differentiation into motor neurons. Morphological changes should be evident after 24 hr of doxycycline treatment. The induction efficiency can be tested by comparing parallel wells treated with and without doxycycline, or by performing a separate dox-GFP experiment as described in Support Protocol 6.
?. [Day 2]
Observe differentiating cells under an inverted microscope.
Cells should be beginning to spread out and form processes.
?. [Day 2]
If long-term culture is desired immediately, coat plates overnight with PLO, PDL, or PEI (see Basic Protocol 8).
?. [Day 3]
Treat the differentiating cells with Accutase (see Basic Protocol 1). Use  Accutase to digest each 10-cm dish.
3 ml
?. [Day 3]
Tilt the plate and add  PBS to the Accutase and cell solution using a 10-ml pipet.  Gently triturate four to five times to break the cells apart.
6 ml
For best results, pass the cells and solution through a 40-μm cell strainer to ensure fulldissociation. Add 1 ml IM after straining to improve pelleting.
?. [Day 3]
Centrifuge the cells  at , .
0 Room temperature
?. [Day 3]
Aspirate the supernatant.
?. [Day 3]
Count the cells (Phelan & May, 2015).
?. [Day 3]
Cells may be re-plated immediately (see Basic Protocol 8) or frozen for future use (see Basic Protocol 1).
Cells to be frozen should be diluted to a standard cell density (i.e., 1 × 106 cells/ml) in IM containing a final concentration of 10 % DMSO.
?. [Day 0]
Incubate at  for  to  (until colonies begin to detach).
0 Room temperature
?. [Day 0]
Transfer 1.5 × 106 iPSCs per 10 cm dish to be differentiated to a 15-ml conical tube.
?. [Day 0]
Centrifuge  at , .
0 Room temperature
?. [Day 0]
Aspirate the supernatant.
?. [Day 0]
Plate the resuspended cells in  E8 medium supplemented with  ROCK inhibitor on a 10-cm Matrigel-coated dish.
12 ml
?. [Day 1]
Aspirate off the medium.
?. [Day 1]
Wash once with PBS.
?. [Day 3]
Resuspend in  IM.
5 ml