Figure 3. Transplanted human induce

Figure 3. Transplanted human induced pluripotent stem cell-derived long-term self-renewing neuroepithelial-like stem (hiPS-lt-NES) cells survive and differentiate in the injured spinal cord of nonobese diabetic-severe combined immunodeficient mice.(A): Survival of transplanted cells was checked every week using a bioluminescence imaging system; left, 2 hours after transplantation; middle, 4 weeks after SCI; and right, 8 weeks after SCI.(B): Time course of transplanted hiPS-lt-NES cell survival in SCI model mice. Optical signal intensity was measured using the bioluminescence imaging system. Quantification of the photon intensity revealed that approximately 20% of the transplanted cells survived 5 weeks after SCI; thereafter, the photon signals remained stable. Data are means6SEM (n¼6).(C):Sagittal sections of SCI model mice treated with hiPS-lt-NES cells at 8 weeks after transplantation. Sections were stained with anti-GFP antibody (green). The epicenter of the SCI is indicated (*). Higher-magnification images of the white dotted boxes (C-1 and C-2) show GFP-positive transplant-derived cells extending their processes into gray and white matter.(D): Immunostaining images, 8 weeks after injury, of hiPS-lt-NES cells grafted into spinal cord. Spinal cord sections were stained with anti-GFP (green), hGFAP (magenta) and Tuj1 (red) antibodies and with Hoechst (blue). (E): Confocal images, 8 weeks after injury, of hiPS-lt-NES cells transplanted into spinal cord, revealing transplanted cells which were double-positive for GFP and markers of neural lineages.(F): Quantitative analyses of Tuj1-positive neurons, hGFAP-positive astrocytes, and MBP-positive oligodendrocytes as in E. Data are means6SD (n¼3). Scale bars¼500lm in C, 100lm in C-1, C-2, D, and E. Abbreviations: GFP, green fluorescent protein; hGFAP, anti-human-specific glial fibrillary acidic protein; MBP, and SCI, spinal cord injury.
hiPS-Lt-NES Cell Transplantation Does Not Promote CST Axon Re-Extension After SCI
Since transplanted NSCs have been reported to play a supportive role in the re-extension of injured axons [29], we examined the effect of the transplanted hiPS-lt-NES cells on CST first-order axonal regeneration. We injected BDA into the bilateral motor cortices and labeled CST first-order neuron axons by anterograde tracing [21, 22]. Because BDA is not transported from first- to second-order neurons across the synapse, only the axons of first-order neurons in the CST are visualized by this method.
In the region caudal to the injury site, >50% of the labeled fibers observed at 5 mm anterior to the lesion site were detected in the intact mice, whereas almost no BDA-traced CST fibers could be seen in the SCI control or hiPS-lt-NES cell-transplanted mice (Fig. 4A). Quantitative analysis revealed that there was no significant difference in the number of BDA-labeled fibers between the SCI control and hiPS-lt-NES cell-treated groups at any position up to 5 mm on either side of the lesion site (Fig. 4B). Although we cannot exclude possibilities such as the re-extension of descending raphespinal axons, which are also important for the motor functional recovery of hind limbs [30, 31], these results suggest that the regeneration of CST axons, if it occurs, cannot be a major contributing factor in the recovery induced by hiPS-lt-NES cell transplantation in our SCI model.
Local Neurons Reconstruct Disrupted CST Neuronal Circuits in a Relay Manner
Recent studies have revealed that local endogenous and transplant-derived neurons can form new neuronal circuits and make synaptic connections after SCI [32–34]. To determine whether disrupted CSTs were reconstructed by forming a local neuronal relay, we injected WGA-expressing adenoviruses into the motor cortex of the hind limb area at 12 weeks after SCI. WGA, a plant lectin, has been widely used as a tracer of neuronal pathways [24, 25] because it is transported in axons and dendrites, and across synapses, to second- and even third-order neurons.
After injection, we could detect WGA-immunoreactive in-tracellular granule-like structures in MAP2ab-positive neurons (Fig. 5A). Furthermore, we found that hiPS-lt-NES cell-treated mice had more WGA/Map2ab double-positive cells than SCI control mice in the caudal region below the injured site (Fig. 5A, 5B). Taking these data and the results of the BDA experiment into consideration, it is suggested that WGA was transferred to the caudal area through the lesion site vianew synaptic connections, and that hiPS-lt-NES cell transplantation promoted the CST reconstruction without CST axonal re-extension. In support of this proposition, we could observe GFP-positive transplant-derived neurons adjacent to synaptophysin-positive patches (Fig. 5C-1 and 5C-1’). Furthermore, immunohistochemistry using species-specific antibodies for presynaptic markers revealed that transplant-derived neurons made synapses with endogenous neurons, suggesting that they reconstructed disrupted CST neuronal circuits in a relay fashion (Fig. 5C-2, 5C-2’, 5C-3, and 5C-3’).
Figure 4. hiPS-lt-NES cell transplantation does not promote corticospinal tract (CST) axon re-extension after SCI.(A): Representative pictures of BDA-labeled CST fibers (red) at 5 and 2 mm rostral and 2 mm caudal to the lesion site at 12 weeks after SCI. Hoechst (blue) shows nuclear staining. Scale bar¼50lm.(B): Quantification of the labeled CST fibers in the spinal cords of untreated, SCI control, and hiPS-lt-NES cell-transplanted mice. Thex-axis indicates specific locations along the rostro-caudal axis of the spinal cord, and the y-axis indicates the ratio of the mean number of BDA-labeled fibers at the indicated site to that at 5 mm rostral to the lesion site (thoracic vertebra [Th] 9). Intact mice were compared with SCI control mice. *,p

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Figure 3. Transplanted human induced pluripotent stem cell-derived long-term self-renewing neuroepithelial-like stem (hiPS-lt-NES) cells survive and differentiate in the injured spinal cord of nonobese diabetic-severe combined immunodeficient mice.(A): Survival of transplanted cells was checked every week using a bioluminescence imaging system; left, 2 hours after transplantation; middle, 4 weeks after SCI; and right, 8 weeks after SCI.(B): Time course of transplanted hiPS-lt-NES cell survival in SCI model mice. Optical signal intensity was measured using the bioluminescence imaging system. Quantification of the photon intensity revealed that approximately 20% of the transplanted cells survived 5 weeks after SCI; thereafter, the photon signals remained stable. Data are means6SEM (n¼6).(C):Sagittal sections of SCI model mice treated with hiPS-lt-NES cells at 8 weeks after transplantation. Sections were stained with anti-GFP antibody (green). The epicenter of the SCI is indicated (*). Higher-magnification images of the white dotted boxes (C-1 and C-2) show GFP-positive transplant-derived cells extending their processes into gray and white matter.(D): Immunostaining images, 8 weeks after injury, of hiPS-lt-NES cells grafted into spinal cord. Spinal cord sections were stained with anti-GFP (green), hGFAP (magenta) and Tuj1 (red) antibodies and with Hoechst (blue). (E): Confocal images, 8 weeks after injury, of hiPS-lt-NES cells transplanted into spinal cord, revealing transplanted cells which were double-positive for GFP and markers of neural lineages.(F): Quantitative analyses of Tuj1-positive neurons, hGFAP-positive astrocytes, and MBP-positive oligodendrocytes as in E. Data are means6SD (n¼3). Scale bars¼500lm in C, 100lm in C-1, C-2, D, and E. Abbreviations: GFP, green fluorescent protein; hGFAP, anti-human-specific glial fibrillary acidic protein; MBP, and SCI, spinal cord injury.hiPS-Lt-NES Cell Transplantation Does Not Promote CST Axon Re-Extension After SCISince transplanted NSCs have been reported to play a supportive role in the re-extension of injured axons [29], we examined the effect of the transplanted hiPS-lt-NES cells on CST first-order axonal regeneration. We injected BDA into the bilateral motor cortices and labeled CST first-order neuron axons by anterograde tracing [21, 22]. Because BDA is not transported from first- to second-order neurons across the synapse, only the axons of first-order neurons in the CST are visualized by this method.In the region caudal to the injury site, >50% of the labeled fibers observed at 5 mm anterior to the lesion site were detected in the intact mice, whereas almost no BDA-traced CST fibers could be seen in the SCI control or hiPS-lt-NES cell-transplanted mice (Fig. 4A). Quantitative analysis revealed that there was no significant difference in the number of BDA-labeled fibers between the SCI control and hiPS-lt-NES cell-treated groups at any position up to 5 mm on either side of the lesion site (Fig. 4B). Although we cannot exclude possibilities such as the re-extension of descending raphespinal axons, which are also important for the motor functional recovery of hind limbs [30, 31], these results suggest that the regeneration of CST axons, if it occurs, cannot be a major contributing factor in the recovery induced by hiPS-lt-NES cell transplantation in our SCI model.Local Neurons Reconstruct Disrupted CST Neuronal Circuits in a Relay Manner
Recent studies have revealed that local endogenous and transplant-derived neurons can form new neuronal circuits and make synaptic connections after SCI [32–34]. To determine whether disrupted CSTs were reconstructed by forming a local neuronal relay, we injected WGA-expressing adenoviruses into the motor cortex of the hind limb area at 12 weeks after SCI. WGA, a plant lectin, has been widely used as a tracer of neuronal pathways [24, 25] because it is transported in axons and dendrites, and across synapses, to second- and even third-order neurons.
After injection, we could detect WGA-immunoreactive in-tracellular granule-like structures in MAP2ab-positive neurons (Fig. 5A). Furthermore, we found that hiPS-lt-NES cell-treated mice had more WGA/Map2ab double-positive cells than SCI control mice in the caudal region below the injured site (Fig. 5A, 5B). Taking these data and the results of the BDA experiment into consideration, it is suggested that WGA was transferred to the caudal area through the lesion site vianew synaptic connections, and that hiPS-lt-NES cell transplantation promoted the CST reconstruction without CST axonal re-extension. In support of this proposition, we could observe GFP-positive transplant-derived neurons adjacent to synaptophysin-positive patches (Fig. 5C-1 and 5C-1’). Furthermore, immunohistochemistry using species-specific antibodies for presynaptic markers revealed that transplant-derived neurons made synapses with endogenous neurons, suggesting that they reconstructed disrupted CST neuronal circuits in a relay fashion (Fig. 5C-2, 5C-2’, 5C-3, and 5C-3’).
Figure 4. hiPS-lt-NES cell transplantation does not promote corticospinal tract (CST) axon re-extension after SCI.(A): Representative pictures of BDA-labeled CST fibers (red) at 5 and 2 mm rostral and 2 mm caudal to the lesion site at 12 weeks after SCI. Hoechst (blue) shows nuclear staining. Scale bar¼50lm.(B): Quantification of the labeled CST fibers in the spinal cords of untreated, SCI control, and hiPS-lt-NES cell-transplanted mice. Thex-axis indicates specific locations along the rostro-caudal axis of the spinal cord, and the y-axis indicates the ratio of the mean number of BDA-labeled fibers at the indicated site to that at 5 mm rostral to the lesion site (thoracic vertebra [Th] 9). Intact mice were compared with SCI control mice. *,p <.05; **, p<.01. There was no significant difference in the number of BDA-labeled fibers between hiPS-lt-NES cell-treated (blue line) and SCI control groups (red line). Data are means6SEM (n¼5). Abbreviations: BDA, biotinylated dextran amine; hiPS-lt-NES, human induced pluripotent stem cell-derived long-term self-renewing neuroepithelial-like stem; and SCI, spinal cord injury