(a) Amphetamine-induced rotations in mice receiving matched dopamine neuron grafts. (b) Spontaneous rotation behavior in the same mice. (c, d) Cylinder test (c) and adhesive tape–removal test (d) were additional spontaneous behavior measures used to assess graft function and restoration of behavioral asymmetry. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to pretransplantation values (Dunnett test). (e) Representative graft showing large numbers of eGFP⁺ (left) and tyrosine hydroxylase–positive cells (right). (f) Girk2 immunohistochemistry confirmed the presence of substantia nigra (A9) type midbrain dopamine neurons located primarily at the graft periphery. Right panel shows a higher power image of cells in left panel. Scale bar in e, 100 μm; scale bars in f, 50 μm in left panel and 10 μm in right panel. All mouse experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Sloan Kettering Institute for Cancer Research.

(a, b) Percentage tyrosine hydroxylase–positive cells out of total cells as shown by a representative sample of six ntES lines derived from different individual mice (a) or among lines derived from a single mouse (b). Whereas all ntES lines were capable of generating tyrosine hydroxylase–positive neurons, they did show significant variability. Data were derived from three independent sets of differentiations. TH/Tuj1, tyrosine hydroxylase–positive cells over β-tubulin–positive cells. (c) Representative spectral karyotype analysis of an ntES line, showing euploidy. The majority of the lines maintained a high proportion of karyotypically normal cells, with loss of a sex chromosome representing the most common abnormality. (d–g) ntES cell line performance was also tested in vivo by grafting dopamine neuron progeny from 2 different lines into a group of allogenic (BALB/c) and congenic (129/Sv) mice. Behavioral analysis shows statistically significant improvement in amphetamine rotation scores in the congenic hosts compared to the allogenic hosts (d). A similar pattern was noted in a battery of spontaneous (non–drug-induced) behavioral assays, the cylinder test (e), tape removal test (f) and beam traversal test (g). *P < 0.05, **P < 0.01 and ***P < 0.001 in a and b (Scheffe test, compared against each other) and in (d–g) (Dunnett test, compared to pre-transplantation scores).