PMC

Tumorigenesis and tumor volume after intracerebral transplantation of mice iPS cells. Transplanted-induced pluripotent stem (iPS) cells formed only a small tumor within the sham-operated brain at 14 and 28 days (A, C), whereas iPS cells formed much larger tumors in the postischemic brain at 14 and 28 days (B, D). ^**P<0.01, nonrepeated measures analysis of variance (ANOVA) and SNK test (E). Values are mean±s.d. SNK, Student-Newman-Keuls.

Undifferentiated induced pluripotent stem (iPS) cells formed tridermal teratoma in ischemic brain. Coronal brain sections obtained from the MCAO+iPS group (A to F) at 14 day after the transplantation, and stained with hematoxylin–eosin. The iPS cells expanded over the infacted area (A). The iPS-derived tumors consisted of ectodermal cells with neural tube-like cells (B), mesodermal cells with striated muscle fiber (C) and chondrocyte (D), and endodermal cells with immature (E) or cylinder-like (F) epithelium, scale bar=2 mm (A), 50 μm (B to F).

c-myc was prominently expressed in the tumors. c-myc was expressed in cells of the tumors at 14 day after the induced pluripotent stem (iPS) transplantation in the Sham+iPS (A) and the MCAO+PBS groups (D). c-myc expression was prominent at the interface of iPS tumor and ischemic host brain (D), as compared with sham brain (A), and c-myc stain was observed in the cylinder-like epithelial cells (B, C, E, F). (C) and (F) represent magnification of the boxed areas (c) and (f), respectively. Scale bar =2 mm (A, D), 500 μm (B, E), 100 μm (C, F). MCAO, middle cerebral artery occlusion; PBS, phosphate-buffered saline.

Behavioral analysis after the induced pluripotent stem (iPS) cell transplantation. Motor functional analysis performed every 7 days after the iPS cell transplantation. The clinical scores shown as Bederson's score (upper panel) and Rotarod time (lower panel) were not significantly different between the Sham+PBS (open circles) and the Sham+iPS (open squares) groups. After transient decrease in the MCAO grpups (filled circles and squares), the recovery of clinical scores delayed in the MCAO+iPS group (filled circles) as compared with the MCAO+PBS group (filled squares), but did not reach a statistically significant difference. MCAO, middle cerebral artery occlusion; PBS, phosphate-buffered saline.

Induced pluripotent stem (iPS) cells have a potential to produce both neuroblasts and mature neurons in normal and postischemic brains. iPS cells were originally labeled with Nanog-GFP before transplantation, and Dcx (a neuroblast marker) or NeuN (a mature neuronal marker) was stained in sections of the infracted area of the Sham+iPS (A to D) and the MCAO+iPS (E to H) groups. Small number of Nanog-GFP and Dcx double-positive cells were found in the tumors of the Sham+iPS group and the MCAO+iPS group at 14 day (A and E, arrowheads). A few NeuN-positive cells were found in the tumors of the MCAO+iPS group at 14 day (F, arrow). Scale bar =20 μm. GFP, green fluorescent protein; MCAO, middle cerebral artery occlusion; PBS, phosphate-buffered saline.

NAGO was abundantly expressed outside of tumors. NAGO, a protein marker of vascular endothelial cells, was observed at basal level at 28 day after the iPS cell transplantation in the Sham+PBS group (A), which was highly induced in the periphery of tumors in the Sham+iPS (B), and slightly induced in the MCAO+PBS groups (C). Such NAGO induction was more striking in the MCAO+iPS group (D); (a to d) represent magnification of the boxed areas in (A, B, C, D), respectively. Scale bar=2 mm (A), 100 μm (a). The intensity of NAGO staining was significantly higher in the MCAO+iPS group compared with Sham+PBS group (^**P<0.01). Statistical analysis: nonrepeated measures analysis of variance (ANOVA) and Bonferroni correction. Data represent mean±s.d. iPS, induced pluripotent stem; MCAO, middle cerebral artery occlusion; NAGO, N-acetylglucosamine oligomer; PBS, phosphate-buffered saline.