Yong-Hee Rhee, Ji-Yun Ko, Mi-Yoon Chang, Sang-Hoon Yi, Dohoon Kim, Chun-Hyung Kim, Jae-Won Shim, A-Young Jo, Byung-Woo Kim, Hyunsu Lee, Suk-Ho Lee, Wonhee Suh, Chang-Hwan Park, Hyun-Chul Koh, Yong-Sung Lee, Robert Lanza, Kwang-Soo Kim, Sang-Hun Lee
Protein-based hiPSCs generate functional midbrain-like DA neurons in vitro.
(A–C) TuJ1+ cells differentiated from protein-based hiPSC-NPCs were positive for hNCAM (A), HuC/D (B), and MAP2 (C). (D) TuJ1+ cells differentiated from protein-based hiPSC-NPCs were electrophysiologically active neurons. Exemplary membrane potential response upon depolarizing current steps (30, 60, and 90 pA for 300 ms) was abolished by 500 nM tetrodotoxin. (E–M) Midbrain marker expression in protein-based hiPSC–derived DA neurons. TH+ cells from differentiated protein-based hiPSC-NPC cultures were immunoreactive for markers specific for neuronal (hNCAM, HuC/D, and MAP2; E–G) and for DA homeostasis (VMAT2 and DAT; H and I). Markers specific for midbrain (EN1 and Nurr1; J and K), A9 midbrain (GIRK2; L), and A10 midbrain (calbindin 1, 28 kDa; M) colocalized in subpopulations of TH+ cells. (N–P) Presynaptic DA neuronal functions, such as DA release (N and O) and DA uptake (P). (N) Typical HPLC chromatograms for DA released in the absence and presence of 56 mM KCl for 30 minutes compared with that of standard mixture (NE, norepinephrine; E, epinephrine). IS, internal standard (3,4-dihydroxybenzylamine) used for quantification of DA concentrations. (O and P) DA release and uptake values, respectively, of 4 independent cultures. In P, DAT-mediated specific DA uptake was calculated by subtracting nonspecific uptake (with nomifensine) from total uptake. *P < 0.01, **P < 0.05 vs. respective control, 2-tailed Student’s t test (O, left, and P) and 2-tailed paired t test (O, right).