Neural Differentiation of HD-iPSC.

Induced differentiation of GABAergic neurons was performed as described previously (38). First, iPSCs were dissociated by 1× dispase (1 mg/mL), diluted by MEF growth medium (DMEM supplemented with 10% FBS, 1% NEAA, 2 mM L-glutamine), and seeded in ultra-low attachment dishes (Corning). Cells were incubated in hESC medium containing 100 nM LDN193189 and 10 µM SB431542, but without basic fibroblast growth factor (bFGF) for 3 d, followed by culturing in N2 medium (DMEM/F12, 1 × N2 supplement, 1% NEAA, 2 mM L-glutamine, 1 mM sodium pyruvate, 20 ng/mL bFGF, 100 nM LDN 193189, and 10 µM SB 431542) for another 2 d. Embryonic bodies (EBs) were then collected and placed on 6-cm dishes with MEF medium. 24 h post seeding, the growth medium was changed from MEF to N2 medium and replenished every other day for 2–3 consecutive weeks to produce neural progenitor cells (NPCs). For induced differentiation of GABAergic neurons from NPC, cells with rosette-like structures were picked by needles, transferred to Matrigel-coated dishes, and cultured for 3–4 wk in N2/B27 medium (mixture of DMEM/F12 and Neurobasal medium (1:1) supplemented with 0.5 × N2, 0.5 × B27, 1% NEAA, 0.5 mM sodium pyruvate, 2 mM L-glutamine, and 10 ng/mL bFGF). During this period, neurospheres were isolated/purified from cell population on a weekly basis. Mature GABAergic neurons were isolated and grown in B27 medium (Neurobasal medium supplemented with 1 × B27 supplement, 1% NEAA, and 2 mM L-glutamine) for 4–6 wk, with the medium being changed every 3 d. Differentiation of MSN GABAergic neurons was characterized by immunocytochemistry.