Fig. 1. Characterization of MASH1-transfected cells in vitro. (A) The MASH1-transfected cells were cultured on 8-well chamber slides, and hematoxylin–eosin (HE) staining was conducted. Some of the cells were spindle shape and had axon like processes. (B–D) Higher magnifications of the MASH1-transfected cells. HE staining. (E) Inverted microscopic view of the MASH1-transfected cells. (F) Confocal microscopic analysis disclosed that the MASH1-transfected cells were positive for MASH1. (G) Some of the MASH1-transfected cells were positive for NFM. (H) They were Islet1-positive. (I) They were weakly Nestin-positive. (J) They were βIIItubulin-positive. (K) They were Lim1/2-positive. The scale bar represents 100 μm (A), 25 μm (B–E), and 10 μm (F–K).

Fig. 2. Calcium mobilization of MASH1-transfected cells upon KCl induced depolarization. (A) Confocal image of MASH1-transfected cells loaded with the Ca²⁺ indicator dye fluo-3-AM, before depolarization. The basal fluorescence was relatively low and almost homogenous. (B) Stimulation with 100 mM K+ solution depolarized the neurons and elicited increased fluorescence signals. (C) Stimulation with 1 μM ionomycin as a positive control for Ca²⁺ entry. The lower panel indicates the fluorescence intensity of individual cells shown in panels A–C. Each line represents the fluorescence intensity of each cell. Data shown were representative of 5 separate experiments.

Fig. 3. The loss of Nogo receptor (NgR) expression in MASH1-transfected cells. (A) RT-PCR analyses of MASH1-transfected cells for NgR. Total RNA was extracted from undifferentiated ES cells and MASH1-transfected cells, and RT-PCR was performed. Note that the downregulation of NgR was observed in MASH1-transfected cells compared with undifferentiated ES cells. Embryonic mouse brain and adult mouse liver were used as a positive control (PC) and a negative control (NC), respectively. (B) Western blotting for NgR protein expression in undifferentiated ES cells and MASH1-transfected cells. Note the downregulation of NgR in MASH1-transfected cells. Actin was used as a control of intracellular protein expression. Cell lysates from COS-7 cells transfected with mouse NgR expression plasmid were used as a positive control (PC) and those from nontransfected COS-7 cells as a negative control (NC) for NgR protein expression.

Fig. 4. Recovery of motor functions by the transplantation of MASH1-transfected neural progenitor cells after complete spinal cord transection. The spinal cords of C57BL/6 mice were completely transected. The mice were transplanted with MASH1-transfected neural cells (MASH) on day 0. RA-treated cells were similarly transplanted. Mice with spinal cord injury were injected with vehicle (PBS) alone (SCI). Sham operation in other mice without SCI was simultaneously performed (Sham). Motor function was evaluated using the BBB open field test (left panel) and vertical grid test (right panel) at each time point. Neural cell transplantation did not restore their motor function completely, as compared with the sham-operated mice (Sham; n = 10). Significant improvements of both scores were observed in mice transplanted with the MASH1 cells (MASH; n = 11) and RA-treated cells (RA-treated; n = 12) as compared with the control mice injected with vehicle alone (SCI; n = 12) after day 14 (both, day 14–day 56, P < 0.01).

Fig. 5. Electrophysiological evaluation of the transected spinal cord transplanted with MASH1-transfected neural cells. MEP conducted in the spinal cords was measured. The spinal cord was promptly removed from mice with sham operation (Normal), SCI mice with vehicle injection, and SCI mice with MASH1-transfected neural cell transplantation. A square wave pulse of 5 mV intensity and 5-ms duration repeated at 5 Hz was delivered via an electrode placed at C7/8, and recordings were made at L4/5 with an amplification (gain 10,000×; bandpass 10–500 Hz). Averaged MEP over 500 pulses was obtained. An open arrow indicates the MEP of sham operated spinal cord. Closed arrows indicate the MEP of transplanted spinal cord.

Fig. 6. Histology of spinal cords after transplantation of MASH1-transfected cells. Spinal cords of mice were completely transected at a level of T7/8, and a vacant cavity was made in the spinal cord. A single cell suspension of the MASH1-transfected neural cells was used for transplantation. The spinal cord was removed from mice at the indicated time after transplantation. Representative of 5 separate experiments was shown. (A) HE staining of the normal spinal cord. (B) HE staining of the injured spinal cord after SCI. (C) HE staining of the injured spinal cord with vehicle injection at day 7. (D) HE staining of the spinal cords 7 days after cell transplantation. (E) HE staining of the injured spinal cord with vehicle injection at day 28. (F) HE staining of the spinal cords 28 days after MASH1-transfected cell transplantation. (G) Elastica van Gieson staining of the injured spinal cord with vehicle injection on day 28 revealed extensive scarring of the injury site. (H) Elastica van Gieson staining of the spinal cords 28 days after MASH1-transfected cell transplantation (the center of the injured site of spinal cord). A large number of cells accumulated in the lesion, and mild scarring was observed. The scale bar represents 320 μm (A–F) and 16 μm (G and H).

Fig. 7. In vivo characterization of ES derived neural cells transplanted in SCI mice. Left upper panel, RT-PCR to characterize the cells in vivo. Left lower panel, Nogos/NgR expression in SCI site with transplantation of the MASH1-transfected cells. From both mice with or without transplantation of MASH1 cells at 28 days after transplantation, small pieces of tissue in the center of the injured site of spinal cord were collected to avoid contamination of the host spinal cord, total RNA was extracted and RT-PCR performed. Mouse brain was used as a positive control (PC) and liver as a negative control (NC). Center panels. Two color immunofluorescence (merged figures) to detect NgR in the SCI site (T7/8) with MASH1 cell transplantation, with RA-treated cell transplantation and with vehicle injection alone at day 7. MASH1 cells were labeled with GFP and were transplanted to the SCI site (T7/8). Cells in normal spinal cord (red fluorescence; (A) a lower magnification; (B) a higher magnification), those in injured spinal cord with vehicle injection (red fluorescence; (C) a lower magnification; (D) a higher magnification) and those in the injured spinal cord with RA-treated cell transplantation (red fluorescence; (G) a lower magnification; (H) a higher magnification) expressed cell surface NgR. However, spinal cord transplanted with the MASH1-transfected cells (green fluorescence) scarcely expressed NgR (red fluorescence in panel E, a lower magnification; F a higher magnification) at the site of transplantation. Right panels. The sites below injury (T12/L1) were similarly analyzed for their NgR expression. NgR was similarly expressed on the T12/L1 of normal spinal cord (I), SCI with vehicle injection (J), MASH1 cell transplantation (K), and RA-treated cell transplantation (L). MASH1-transfected cells (GFP-positive) located the site below injury (M). Each scale bar indicates 10 μm.

Fig. 8. Confocal microscopic analyses of the mouse spinal cord transplanted with the MASH1-transfected cells. 28 days after transplantation of the MASH1-transfected cells whose nuclei were GFP-positive. Upper panels. Spinal cord injury (SCI NFM). A merged figure of NFM immunostaining and GFP of the SCI site. RA-treated cells NFM. A merged figure of NFM immunostaining and GFP of the SCI site transplanted with the RA-treated cells. MASH NFM. A merged figure of NFM immunostaining and GFP of the SCI site transplanted with the MASH1-transfected cells. A scale bar indicates 10 μm. Lower panels. (A) GFP. (B) Anti-NFM staining. (C) Merge. GFP-positive nuclei of the transplanted neural cells were surrounded by NFM-positive axons. (D) GFP. (E) Anti-Lim1/2 staining. (F) Merge. A GFP-positive nucleus was overlaid Lim1/2 immunostaining at perimeter of the lesion. It was seen that transplanted cells were migrated to the permissive portion to avoid an impertinent environment for growth and regeneration. (G) GFP. (H) Anti-synaptophysin staining. (I) Merge. Circular synaptophysin immunostaining around the GFP-positive nucleus at the perimeter of the lesion. It suggested a possible synaptic connection between the host and the grafted neural cells. (J) GFP. (K) Anti-Islet1 staining. (L) Merge. A GFP-positive nucleus overlaid Islet1 immunostaining, suggesting that motoneuron precursors migrated to a better place for growth and regeneration. The cartoon on the right side indicates the position of each panel.