Fig. 1. Differentiation of cynomolgus monkey ES cells treated with RA in vitro. (A) A colony of undifferentiated cynomolgus monkey ES cells showed flat configuration as compared with that of undifferentiated mouse ES cells. (B) Cynomolgus monkey ES cells became to form floating cell aggregates, EBs, at day 5. (C) RA-treated cells cultured on a dish coated with gelatin at day 17 showed axon like processes at the periphery of EB. (D) The axon like processes looked interacting each other and came to overlay to those of another EB at day 17. The scale bar represents 20 μm (A–C) and 40 μm (D).

Fig. 2. Expression of neural markers in RA-treated cynomolgus monkey cells by RT-PCR. (A) Schedule for induction of neural cells from cynomolgus monkey ES cells and their transplantation to mice with cryogenic brain injury. Undifferentiated ES cells were cultured in a floating fashion for 4 days. 1 μM RA was added twice to the cell culture at day 4 and day 6. 1 × 10⁵ RA-treated cells at day 8 were transplanted to underneath the motor cortex having cryogenic injury. Immunosuppressants were given to the recipients from day of the transplantation to that of sacrifice. RT-PCR was conducted using mRNAs from the RA-treated cells at days 8, 12 and 22. (B) mRNA expression of several neural cell markers in the RA-treated cells was determined by RT-PCR. The RA-treated cells derived from cynomolgus monkey ES cells at day 8, day 12 and day 22 were used. Diethylpyrocarbonate (DEPC)-treated water served as negative controls (NC). The RA-treated cells at day 8 (cultured for 4 days without RA, followed by 4 days culture with RA) that expressed nestin, was used for transplantation. ND, not done.

Fig. 3. Expression of neural cell antigens in RA-treated cynomolgus monkey ES derived neural cells in vitro by immunocytochemical staining. The cells were cultured on the 8-well chamber slides. After fixation, the cells were stained with the indicated antibody. Brown color indicates immunopositive cells and blue color represents counter staining for nuclei with hematoxylin. (A) Some of the RA-treated cells derived from cynomolgus monkey ES cells at day 8 were βIII tubulin-positive with axon like processes. (B) The RA-treated cells at day 14 had axons expressing NFM. (C) Approximately 50% of the cells expressed Islet1 at day 14, suggesting their differentiation into motoneurons. Islet1-positive cells were typically observed here. (D) A few of the cells locating peripheral site of EB expressed GFAP, suggesting that some of them differentiated to glial cells. (E) GalC-positive cells were hardly found in the culture period throughout, even though we have detected weak expression of GalCase mRNA in the cells (Fig. 2B). The scale bar represents 10 μm (A, B, C, D, E).

Fig. 4. Recovery of motor function in mice with experimental brain injury transplanted with the cynomolgus monkey ES cell derived neural cells. The motor cortex of normal mice was damaged by cryogenic injury at day 1. Mice were transplanted with either the graft cells or vehicle at day 8. Motor function was evaluated by the beam walking test and the rotarod test. In the experiments, RA-treated cells or vehicle (PBS) were injected. Data shown were mean ± SD at each point after injection. The numbers of footfaults in beam walking test were significantly decreased in mice transplanted with the neural cells (n = 18) as compared with vehicle (PBS)-treated mice (n = 18) after day 22 (* in beam walking test; day 22–day 29, P < 0.01). The scores of the rotarod test were significantly improved in the neural cell transplanted mice (n = 18) as compared with vehicle-treated mice (n = 18) (* in the rotarod test; day 22–day 29, P < 0.05). However, it is evident that neural cell transplantation did not restore their motor function completely when compared with control mice without brain injury (n = 9). The SD of the control mice without brain injury were less than 5% of the mean, thus were omitted. We have conducted the experiments three times and the representative result was presented. V, vehicle; N, neural cell transplantation; C, uninjured control.

Fig. 5. Adaptation of the neural cells derived from cynomolgus monkey ES cells to mouse brain tissue. (A) Schematic representation of panel B. The brain was cryoinjured (a closed arrow) for use as a model of brain damage. The graft cells were injected to the periventricular area of left hemisphere (underneath the lesion). The dotted parallel lines indicate the position of needle insertion. The abbreviations used in the panels were as follows; VL, lateral ventricle; CP, caudate putamen; CC, corpus callosum; MO, motor cortex. (B) The RA-treated neural cells were injected underneath the motor cortex with cryogenic injury. A representative result (day 14) with HE staining was shown. (C) A microscopic image of the mouse brain with cryogenic injury. The motor cortex of left hemisphere was widely damaged, and the depth of the cryogenic injury was restricted to cortex and exactly reached to the upper surface of corpus callosum. A representative of five independent experiments was shown. (D) Higher magnification of the damaged motor cortex in panel C. (E) Schematic representation of panel F. Red dots indicate the location of the transplanted cells. (F) Two color immunostaining of mouse brain with the neural cell graft at day 10, 2 days after transplantation. Single cell suspension of the monkey ES cell derived neural cells at day 8 was used as a tissue graft. Green color was anti-NFM antibody and red color was anti-human nuclei antibody. A cluster of the grafted cells located near the injection site (a white box). (G) Higher magnification of the boxed area in panel F. (H) Schematic representation of panel I. Red dots indicate the location of the transplanted cells. (I) Two-color immunostaining of mouse brain with the neural cell graft at day 36, 28 days after transplantation. Green color was anti-NFM antibody. Red color was anti-human nuclei antibody, detecting the grafted cells in the cortical region of the damaged brain (white arrows), suggesting their migration to the lesion from the injection site. The grafted cells dispersed over the motor cortex. (J) Higher magnification of panel I. White arrowheads indicate the anti-human nuclei antibody-positive transplanted cells in the injured cortex. Three individual mice both at day 10 and at day 36 were analyzed and representative results obtained from each group of mice were shown. The scale bars represent 100 μm (BCD), 50 μm (FI) and 10 μm (G, J). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Expression of neural markers in transplanted RA-treated cynomolgus monkey cells in host brain tissue. After transplantation of the RA-treated neural cells whose nuclei were anti-human nuclei antibody positive, the brains were recovered at days 10 and 28 for analyses. Panel A was higher magnification of the injection site (a boxed area in Fig. 5F). Panels B, C, D, E, F, G and H, I, J were higher magnifications of the injured cortical region (arrows in Fig. 5I). (A) Neural cells derived from cynomolgus monkey ES cells were positive for anti-human nestin antibody, which did not react with mouse nestin, on day 10, 2 days after transplantation. (B, C, D) Two color staining with anti-human nuclei (red) and anti-β IIItubulin (green). The anti-human nuclei antibody-positive neural cells derived from cynomolgus monkey ES cells were positive for the neural marker on day 28. (E, F, G) Two-color staining with anti-human nuclei (red) and anti-synaptophysin (green). The anti-human nuclei antibody-positive nucleus was surrounded by synaptophysin immunostaining (an arrow). Circular synaptophysin immunostaining lacking anti-human nuclei antibody staining (an arrowhead) located near the anti-human nuclei antibody-positive nucleus (an arrow), suggesting possible synaptic connection between the host and the grafted neural cells. (H, I, J) Two-color staining with anti-human nuclei (red) and anti-Islet1 (green). The anti-human nuclei antibody-positive nucleus was simultaneously positive for Islet1 immunostaining (an arrow). Islet1 immunostaining lacking anti-human nuclei antibody staining (an arrowhead) located near the anti-human nuclei antibody-positive nucleus (an arrow). Three individual mice both at day 10 and at day 36 were analyzed and representative results were shown. The scale bars represent 5 μm (A, B, C, D) and 10 μm (E, F, G, H, I, J). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. Perfusion MRI studies revealed recovery of the local perfusion pattern by the transplantation of cynomolgus monkey ES cell-derived neural cells. The blood flow patterns in the brain of normal mice (n = 3) (A, B), the mice with cryogenic injury and PBS injection (n = 6) (C, D), and the mice transplanted with neural precursor cells (n = 8) (E, F) were analyzed at day 14 after the injection/transplantation by functional MRI study as described in Materials and methods. Representative results of proton-density images (A, C, E) and perfusion images (B, D, F) of each group of mice were shown.