Additional Supporting Information may be found in the online version of this article.

STEM_1060_sm_SuppFig1.tif1195KFigure S1. The G-band analysis of human ESCs (A: KhES-1, B: KhES-2) showing no abnormalities.
STEM_1060_sm_SuppFig2.pdf455KFigure S2. The modified SDIA method can efficiently generate midbrain dopaminergic neurons comparable to the original SDIA method. (A) The results of the FACS analysis of the expression of undifferentiated (Oct4, SSEA-4) and neural (PSA-NCAM, NCAM) markers under each condition (means ± s.d.; *P<0.05) (B, C) The results of the quantitative PCR analysis of the expression of pluripotent (Oct4, Nanog) or neural (Sox1), midbrain (Pax2, Lmx1b, En1) markers on day 14. (D) The results of the quantitative PCR analysis of the expression of the midbrain DA progenitor (En1, Nurr1) or neuron (TH) markers on day 28. (E) The percentages of TH+ cells in the Tuj1+ cells on day 42. The data are presented as the means ± s.d. (**P < 0.01). (F) Immunofluorescence images of differentiated ESCs on O/L-coated slides for TH (red) and Tuj1 on day 42. Bars = 50 μm.
STEM_1060_sm_SuppFig3.tif2984KFigure S3. Behavioral changes and DA neuronal differentiation in the monkeys implanted with d14- or d28-spheres. (A) The neurological rating scale used for MPTP-treated monkeys [4, 5]. (B) The time course of changes in the neurological scores of the monkeys implanted with d14- (No. 52) or d28-spheres (No. 55, 56). (C) Immunofluorescence images of the d14- and d28-grafts nine months after transplantation, TH staining (left) or TH/DAPI dual staining (right). Bars=200 μm.
STEM_1060_sm_SuppFig4.tif1179KFigure S4. T2-weighted MR images of monkey No. 74 at 1 (A) and 3 (B) months after implantation.
STEM_1060_sm_SuppFig5.tif1800KFigure S5. The time course changes in the neurological score (A) and the amount of spontaneous movement (B) in each animal: control (54, 57, 66, 69), d35-spheres (62, 63), d45-spheres (65, 67, 68, 75). (C) Pixel changes for each animal during a 15-30 min period.
STEM_1060_sm_SuppFig6.pdf560KFigure S6. The growth of the hESCs, unsorted cells, and sorted PSA-NCAM+ cells in SCID mice. To investigate the growth of the human ESC (KhES-1)-derived NPCs that were generated using the protocol shown in Fig. 1A, we isolated neurally committed cells by flow cytometry using an anti-PSA-NCAM antibody [6]. (A) The FACS analysis of the expression of PSA-NCAM in undifferentiated ESCs, d7-, and d14-spheres was performed (***p<0.001). (B) Based on these results, and to see the difference between the sorted and unsorted cell populations more clearly, we compared the growth of the cells using d7- spheres. An RT-PCR analysis of the expression of pluripotent (Oct3/4), neural progenitor (Pax2, Pax6), and midbrain (Lmx1a) markers revealed that unsorted cells and the PSANCAM- population expressed low levels of Oct3/4, whereas the PSA-NCAM+ population did not. (C) Undifferentiated ESCs and unsorted d7-spheres (both after sham-FACS; n=8 and 4, respectively), and the sorted PSA-NCAM+ cells (n=9) were grafted (2x105 cells per animal) into the right putamen of SCID mice. The histological analyses revealed that the PSA-NCAM+ population tended to form smaller grafts than ESCs and unsorted d7-spheres, but the difference was not significant. The data are presented as the means ± s.d. (D, E) HE staining of the brains of transplanted SCID mice. Grafts derived from naïve ESCs showed clear margins (upper right in E) and teratoma-like findings, such as neural tissue (upper left), epithelium (lower left), and cartilage (lower right). In the case of the d7-grafts, those derived from both unsorted and PSA-NCAM+ cells showed neural structures with rosette formation. The bars in (D) = 1 mm, (E) = 100 μm. (F, G) Double-labeled immunofluorescence images for Nestin (green) / Ki67 (red), Oct3/4 (green) / Ki67 (red), or Nestin (green) / Pax6 (red). Bars = 1.0 mm (F), or 100 μm (G). In these SCID mice, the grafts contained no Oct3/4+ cells, even in those derived from undifferentiated ESCs, in which a large number of Nestin/Ki67+ cells were present, suggesting the proliferation of non-neural cells. In the grafts derived from d7-spheres, most of the Ki67+ cells also expressed Nestin and Pax6, suggesting that the NPCs were proliferating. (H) A comparison of the volume between grafts of hESCs (n=9), unsorted cells (n=7), and sorted PSA-NCAM+ cells (n=7) in testis (***P<0.001). The data are presented as the means±s.d. (I) Excised tumors from testis. (J) H-E staining of these tumors, showing teratomatous findings (ESCs) or neuroepithelial rosettes (unsorted, PSA-NCAM+). Bars in ESCs = 1.0 mm, in unsorted and PSA-NCAM+ cells = 100 μm.
STEM_1060_sm_SuppInfo.pdf88KSupplementary Method
STEM_1060_sm_SuppTab1.pdf7KSupplementary Table 1.
STEM_1060_sm_SuppTab2.pdf8KSupplementary Table 2.
STEM_1060_sm_SuppTab3.pdf8KSupplementary Table 3.
STEM_1060_sm_SuppTab4.pdf74KSupplementary Table 4.

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