Human PSC cells H9 embryonic stem cells (ESCs) (WA09, WiCell, Madison, WI, (http://www.wicell.org) passages 45–55), H7 ESC (WA07, WiCell, passages 41–51), and iPSC2497 (a kind gift from Dr. Fred Gage , passages 30–40) were maintained on Matrigel (BD, San Jose, CA (http://www.bdbiosciences.com)) in mTeSR media (Invitrogen, Carlsbad, CA (http://www.lifetechnologies.com/us/en/home.html)) with 10 ng/ml bFGF (Peprotech, Rocky Hill, NJ (http://www.peprotech.com)), and passaged using Dispase (Stem Cell Technologies, Vancouver, BC, Canada (http://www.stemcell.com)). For differentiation, PSCs were trypsinized and grown as floating aggregates in low adherent flasks in Knockout Serum Replacement (KSR) media (20% knockout serum replacement, Dulbecco's modified Eagle's medium, 2mM l-glutamine, and 10 µM β-mercaptoethanol [all from Invitrogen, Carlsbad, CA (http://www.lifetechnologies.com/)]). Rock inhibitor (Y-27632, 10 µM, Tocris, Bristol, U.K. (http://www.tocris.com)) was added on the first day of differentiation. After 2 weeks of floating culture, cells were transferred to polyornithine (PLO; 15 mg/ml; Sigma, St. Louis, MO (http://www.sigmaaldrich.com)) and fibronectin (FN; 1 mg/ml; Sigma, St. Louis, MO (http://www.sigmaaldrich.com))-coated surfaces. For neural induction, cells were treated with LDN193189 (100 nM, Stemgent, Cambridge, MA (www.stemgent.com)) from day 0 to day 14 and with SB431542 (10 µM, Tocris, Bristol, U.K. (http://www.tocris.com)) from day 0 to day 7 . For MGE induction, cells were treated with IWP2 (5 µM, EMD Millipore, Billerica, MA (http://www.emdmillipore.com/)) from day 0 to day 7, with Smoothened Agonist (SAG) (0.1 µM, EMD Millipore, Billerica, MA (http://www.emdmillipore.com)) from day 0 to day 21, and with FGF8 (100 ng/ml, Peprotech, Rocky Hill, NJ (http://www.peprotech.com)) from day 8 to day 21. After 3 weeks of differentiation, cells were trypsinized and droplets of 10,000 cells per microliter transferred to PLO/FN-coated coverslips in differentiation media (N3 media  with 10 ng/ml Glial cell line-derived neurotrophic factor (GDNF) [Peprotech, Rocky Hill, NJ (http://www.peprotech.com)], 10 ng/ml Brain-derived neurotrophic factor (BDNF) [Peprotech, Rocky Hill, NJ (http://www.peprotech.com)], and 2.5 µM gamma secretase inhibitor, DAPT [Tocris, Bristol, U.K. (http://www.tocris.com)]) for further differentiation and maturation.
For Matrigel two-dimensional migration analysis, MGE cells or control cells (Pax6+ cells without IWP2, SAG, and FGF8 treatment) were trypsinized at day 21 of differentiation and reaggregated in low attachment round-bottomed 96-well plate in differentiation media (10,000 cells/well). MGE spheres or cortical spheres were plated on coverslips coated with 1:100 diluted Matrigel in differentiation media after 25 days of further differentiation and analyzed 5 days after plating. For analysis of migrating cell numbers, total cell numbers that migrated out of the spheres were counted, and then the cells were trypsinized to count the total cell numbers for normalization. For measuring migration distances, ImageJ software was used to assess each cell migration distance between the edge of the sphere and the center of the migrating cell body. Some of the spheres were also fixed for immunocytochemistry analysis.
For Matrigel two-dimensional migration analysis of mouse explant, E13.5 embryos were removed one at a time from anesthetized CD1 dams, brains were isolated, embedded in 8% low gelling temperature agarose (Sigma, St. Louis, MO (http://www.sigmaaldrich.com)), and cut at a thickness of 300 µm on a vibratome in the coronal plane. Both cortical and MGE regions were punched out from these coronal sections using a micropunch (Guide wire and tube assembly, 19 gauge; Inner Diameter 0.027 in., Small Parts, Inc., Miami Lakes, FL (http://www.smallpartsinc.com)) and collected in Neurobasal medium. E13.5 cortex or MGE explants were plated on coverslips coated with1:100 diluted Matrigel, and analyzed the same way as the human spheres. For Matrigel three-dimensional (3D) migration analysis, MGE explants or human MGE spheres were embedded in undiluted 3D matrigel matrix, cultured in differentiation media, and their migration was analyzed 2 days after embedding.