Combined NSC transplantation and VPA administration improves functional recovery of hind limbs without CST axon reextension. As VPA has been shown to have effects that are likely to be beneficial to treatment of the injured CNS, such as neuroprotection (2731), induction of neuronal differentiation (26), and promotion of neurite outgrowth (32), we examined the response of SCI model mice to different combinations of VPA administration and NSC transplantation. We prepared NSCs from embryonic forebrains of 3 different Tg mouse lines ubiquitously expressing either GFP (GFP-Tg) (33), GFP and LUC (GFP.LUC-Tg), or GFP, LUC, and the diphtheria toxin (DT) receptor human heparin-binding EGF-like growth factor (TR6) (TR6.GFP.LUC-Tg) (see Methods). The expression of GFP, LUC, and TR6 in NSCs enabled us to distinguish transplanted cells from host cells, to trace the survival of transplanted cells based on LUC activity in a noninvasive fashion, and to specifically ablate transplanted cells (see below), respectively. To obtain a homogeneous population of NSCs, we used adherent monolayer culture (3436). The embryonic forebrains were dissociated and cultured with EGF and basic FGF (bFGF) (36) (Supplemental Figure 1, A and B; supplemental material available online with this article; doi:10.1172/JCI42957DS1). These cells uniformly expressed the stem cell markers Sox2 and nestin but did not express differentiation markers (Supplemental Figure 1, C and D). Under the appropriate conditions for each lineage, these NSCs differentiated into neurons, astrocytes, or oligodendrocytes (Supplemental Figure 1, E and F). NSCs from different Tg mice behaved similarly in these culture conditions (data not shown). NSCs that had been cultured and passaged 510 times in the presence of both EGF and bFGF to maintain the undifferentiated state were used for transplantation studies.

Undifferentiated NSCs were transplanted into the SCI epicenter 7 days after injury. Nontransplanted control and transplanted mice were then intraperitoneally administered VPA or saline daily for 7 days (Figure 1A), whereafter we monitored their hind limb motor function using the open field locomotor scale (BBB score) (79, 37) for 6 weeks. Remarkably, we found that the simultaneous treatment of SCI model mice with NSCs and VPA resulted in a dramatic recovery of hind limb function compared with either treatment alone (Figure 1B and Supplemental Videos 14). There were no significant differences among the data obtained from each SCI model mouse group transplanted with the 3 distinct NSCs. Functional recovery of each treated SCI model mouse reached a plateau at around 6 weeks, the level of which was sustained for more than 3 months. Since mice treated with VPA alone showed no further improvement compared with untreated mice, it is most likely that VPA affected the function of transplanted cells.

A combination of NSC transplantation and VPA administration improves functional recovery of hind limbs without CST axon reextension. (A) Schematic of the NSC transplantation and VPA injection protocol. (B) Time course of functional recovery of hind limbs after SCI. GFP-NSCs, GFP.LUC-NSCs, and TR6.GFP.LUC-NSCs were transplanted into the SCI epicenter 7 days after injury as indicated. Combined treatment with NSC transplantation and VPA administration resulted in the greatest functional recovery. Data represent mean SEM. **P < 0.001 compared with SCI models with no treatment; *P < 0.01 compared with SCI models with no treatment (repeated measures ANOVA). NSC+VPA, total n = 21. (C) Representative pictures of BDA-labeled CST fibers at 5 mm rostral and 5 mm caudal to the lesion site. BDA was injected into the motor cortices 12 weeks after SCI. 2 weeks after the injection, mice were fixed and spinal cord sections were stained. Representative results for a GFP-NSCtransplanted spinal cord are shown. Blue, Hoechst nuclear staining. Scale bar: 20 m. (D) Quantification of the labeled CST fibers in the spinal cords of intact mice, SCI mice receiving no treatment, and SCI mice undergoing combined NSC/VPA treatment. Eight 30-mthick serial parasagittal sections from individual spinal cords were evaluated. The x axis indicates specific locations along the rostrocaudal axis of the spinal cord, and the y axis indicates the ratio of the number of BDA-labeled fibers at the indicated site to that at 6 mm rostral to the lesion site (Th9). **P < 0.001 compared with SCI models without treatment; *P = 0.188 There is no significant difference in the number of BDA-labeled fibers between NSC+VPA-treated mice (blue line) and SCI model mice with no treatment (yellow line) (repeated measures ANOVA). All data shown are from at least 3 experiments in parallel conditions, with error bars representing SEM.

We next sought to determine the basis for this improvement in locomotor function. Since transplanted NSCs have been reported to play a supportive role in the reextension of injured axons (14), we analyzed whether CST axons were regenerated by anterograde labeling using biotinylated dextran amine (BDA) (6, 16, 17). Because BDA was injected into the motor cortex, only the axons of first-order neurons in the CST could be visualized (Figure 1C). In our SCI model mice, the caudal part of the injured site was completely devoid of CST axons (Figure 1, C and D), and the same was true in mice that had undergone combined NSC transplantation and VPA administration (Figure 1, C and D). These data indicated that CST axons did not reextend in mice treated with both NSCs and VPA and therefore that some other mechanism was responsible for the animals dramatic functional locomotor improvement.

Transplanted NSCs encompass the lesion site and extend their processes. Given that host CST axon reextension was not involved in the observed hind limb recovery, we decided to focus on the transplanted cells. We analyzed the migration, morphology, neuronal marker expression, and viability of these cells after coadministration with VPA. Transplant-derived cells migrated to both rostral and caudal areas and displayed processes that extended into the gray matter and dorsal funiculus within 5 weeks of transplantation (Figure 2). Between 20% and 40% of the transplanted cells were found to be surviving in the injured spinal cord after 8 weeks, and 17% still remained viable more than 1 year after transplantation (data not shown). About 20% of the surviving cells had differentiated into microtubule-associated protein 2positive (MAP2-positive) neurons with elongated processes within 5 weeks after transplantation (Figure 2, B and C, and Figure 3, E and F). Survival of the transplanted NSCs was not significantly influenced by VPA administration (Supplemental Figure 8).

Transplanted NSCs migrate from the injection site and encompass the lesion site. Representative results of GFP-NSCtransplanted SCI model mice are shown. (A) A series of immunostaining images of injured spinal cord at 6 weeks after injury. SCI mice received combination treatment with NSC transplantation and VPA administration. Specimens were picked up every 150 m and stained with anti-GFP (green) and MAP2 (not shown) antibodies and Hoechst (blue). The epicenter of the SCI is indicated (*). Scale bar: 1 mm. (B and C) Higher-magnification images of the white boxes in A. GFP-positive transplanted NSCs differentiated into MAP2-positive neurons and extended their processes. Scale bar: 50 m.

VPA promotes neuronal differentiation of transplanted NSCs. Representative results of GFP-NSCtransplanted SCI model mice are shown. (A) Confocal images of NSCs 1 week after transplantation into the injured spinal cords. Spinal cord sections from VPA-treated (+) and untreated () mice were stained with anti-GFP (green), anti-doublecortin (DCX) (immature neuronal marker, red) and anti-GFAP (magenta) antibodies, and Hoechst (blue). VPA administration resulted in an increase in the number of DCX-positive neuronal precursors among transplanted cells (lower panel). Scale bar: 20 m. (BD) The percentages of DCX-, GFAP-, and MBP-positive cells in GFP-positive transplanted cells were quantified. **P < 0.01; *P < 0.05 compared with controls (Students t test). (E) Confocal images of NSCs 5 weeks after transplantation into injured spinal cords. Spinal cord sections from VPA-treated (+) and untreated () mice were stained with anti-GFP (green), anti-MAP2 (neuronal marker, red) and anti-GFAP (magenta) antibodies, and Hoechst (blue). VPA administration increased the numbers of MAP2-positive neurons (lower panel). Scale bar: 20 m. (F and G) The percentages of cells positive for MAP2 or GFAP in GFP-positive transplanted cells in E were quantified. **P < 0.01; *P < 0.05 compared with control (Students t test). All data shown in BD, F, and G are from at least 15 confocal images of 3 individuals in parallel experiments, with error bars representing the SD.

HDAC inhibition promotes neuronal differentiation of NSCs and is critical for transplantation-induced hind limb recovery. In contrast to previous studies, which have indicated that very few transplanted NSCs differentiate into neurons in the injured CNS environment (8, 10, 11, 20), many neurons were observed in the spinal cord after coadministration with VPA. We next examined in more detail the contribution of VPA to differentiation of cultured and transplanted NSCs. To analyze differentiation in vitro, NSCs were treated with either VPA or valpromide (VPM), an amide analog of VPA that is also an antiepileptic but is not an HDAC inhibitor (24), under differentiation culture conditions. VPA enhanced histone acetylation (Supplemental Figure 2A) and promoted neuronal differentiation and neurite outgrowth of the NSCs (Supplemental Figure 3, AC); it also inhibited astrocytic and oligodendrocytic differentiation of NSCs (Supplemental Figure 3, DG). A different HDAC inhibitor, trichostatin A (TSA), also enhanced histone acetylation (Supplemental Figure 2A) and neuronal differentiation of NSCs (not shown) (26). In contrast, VPM neither enhanced histone acetylation nor induced neuronal differentiation, suggesting that HDAC inhibition has an important role in regulating fate determination in NSCs.

We then assessed the histone acetylation status and differentiation profiles of transplanted NSCs. VPA administration enhanced histone acetylation in transplanted cells in the spinal cord (Supplemental Figure 2, B and C). When we examined the differentiation status of transplanted cells 1 week after transplantation, neuronal but not glial differentiation was greatly enhanced by VPA administration (Figure 3, AD, and Supplemental Figure 4A). A similar differentiation tendency of transplanted NSCs to that at 1 week was observed at 5 weeks after transplantation: there was a dramatic increase in the number of cells positive for MAP2 (a relatively late differentiation marker of neurons in comparison with DCX) in VPA-administered mice (Figure 3, EG, and Supplemental Figure 4B). Furthermore, VPM administration to the SCI mice neither promoted neuronal differentiation nor enhanced hind limb motor function, suggesting that HDAC inhibition has an essential role in regulating fate determination of transplanted NSCs and improvement of motor function in vivo (Supplemental Figure 5, AC). In light of the above findings that the percentage of neurons generated from transplanted NSCs increased dramatically with VPA administration, whereas those of astrocytes and oligodendrocytes declined, we anticipated that these neurons would be likely to play a major role in regenerating the disrupted neuronal circuitry of the injured spinal cord.

Transplant-derived neurons reconstruct disrupted neuronal circuits in a relay manner. We next asked how the disrupted neuronal circuits were regenerated following the combined treatment with NSC transplantation and VPA administration. Wheat germ agglutinin (WGA), which can be transsynaptically transported, is one of the best known tracers of neural pathways (38). WGA protein can be transferred across synapses to second- and third-order neurons, permitting functional neuronal circuits to be tracked in the CNS. We injected WGA-expressing adenoviruses into the motor cortex of mouse brain 12 weeks after SCI. In uninjured mice, WGA was detected as intracellular granule-like structures in neurons localized in the ventral horn throughout the spinal cord (Figure 4, A and B). In untreated SCI model mice, WGA granules were almost completely absent from the caudal region below the injured site (Figure 4, A and C). Surprisingly, although we could not observe CST axonal reextension through the lesion site (Figure 1, C and D), WGA granules were clearly present in caudal large neurons located in the spinal cords of mice treated with both NSC and VPA (Figure 4, A and D). Intriguingly, moreover, transplant-derived neurons in or close to the lesion site contained WGA granules (Figure 4E), which were received from more rostral neurons. These data imply that WGA was conveyed through the lesion site to the caudal area via transplant-derived neurons. Considering this finding, together with the fact that WGA could be detected in caudal neurons without CST axonal reextension in mice that had undergone the combined treatment, it seemed conceivable that the transplant-derived neurons reconstructed the disrupted neuronal circuits, thereby acting as relays for transmitting signals between endogenous neurons whose interconnection had been abolished by the injury. In mice that received NSC transplantation alone after SCI, the percentage of WGA-positive cells among MAP2ab-positive cells in the caudal region was higher than that in untreated mice (Figure 4C) but lower than that in mice receiving combined NSC transplantation and VPA administration (Supplemental Figure 6), reflecting the degree of hind limb functional improvement (Figure 1C).

Transplant-derived neurons reconstruct disrupted neuronal circuits in a relay manner. (A) Representative pictures of WGA-labeled neuronal cell bodies located in the ventral horn at 14 weeks after SCI. Spinal cord sections were stained with anti-WGA (red) and -MAP2ab (magenta) antibodies and Hoechst (blue). Scale bar: 20 m. Intense WGA immunoreactivity was observed as intracellular granule-like structures. Left panels show the rostral area (Th4Th7), and right panels show the caudal area (Th11 to lumbar vertebra [L] 1). In uninjured mice, WGA injected into the bilateral motor cortices was transsynaptically transported to neurons in areas rostral and caudal to the injured site (top panels). In the SCI model mice that did not receive treatment, very little WGA was observed in caudal areas (middle panels). However, in spinal cords of animals that underwent dual treatment with NSC and VPA, WGA was clearly observed in neurons in the caudal areas (bottom panels). Representative results of GFP-NSCtransplanted SCI model mice are shown. (BD) The percentages of WGA-positive cells in the neurons localized in the ventral horn were quantified. **P < 0.05 (Students t test). All data shown are from at least 30 images, containing more than 600 cells, from 3 individuals (5 images per area) in parallel experiments, with error bars representing SD. (E) Representative confocal images of WGA-labeled transplant-derived MAP2-positive neurons. Sections were stained with anti-WGA (red), anti-MAP2ab (magenta) and anti-GFP (green) antibodies, and Hoechst (blue). Granule-like WGA structures (yellow arrowheads) could be seen in the GFP and MAP2abdouble-positive transplant-derived neurons. Scale bar: 10 m.

In support of the notion of a relay function for transplant-derived neurons, immunoelectron microscopy revealed that GFP-positive transplant-derived neurons received projections from endogenous neurons (Figure 5, A and B) and that the axon terminals of transplant-derived neurons made synapses with endogenous neurons localized in the ventral horn (Figure 5, CE).

Transplant-derived neurons make synapses with endogenous neurons. (A) Immunoelectron microscopy image of a sagittal section of dual-treated (GFP-NSC and VPA) injured spinal cord (rostral area). A GFP-positive dendrite (Den) made synapses with GFP-negative endogenous axon termini (At) (yellow arrowheads). Scale bar: 1 m. (B) In other rostral regions, a dendrite of a GFP-positive transplant-derived neuron made a synapse (yellow arrowheads) with the axon terminus of a GFP-negative endogenous neuron. Scale bar: 1 m. (C) Sagittal section of dual-treated (NSC and VPA) injured spinal cord (caudal area) stained with anti-GFP antibody (dark brown). The epicenter of the SCI is indicated (*). Scale bar: 500 m. (D) High-magnification image of a large neuron localized in the ventral horn in the white rectangle in C. GFP-positive transplanted neurons extended their processes toward an endogenous neuron (yellow arrowheads). Scale bar: 100 m. (E) Immunoelectron microscopy image of the boxed area in D. GFP-positive axon termini made synapses with the dendrite of a GFP-negative endogenous large neuron (yellow arrowheads). Scale bar: 1 m.

Transplanted cells contribute directly to functional recovery of hind limb movement in SCI mice. To determine whether the transplanted cells made a direct contribution to the functional recovery of hind limbs after SCI, we performed specific ablation of transplanted cells using the toxin receptormediated cell knockout (TRECK) method (Figure 6A and refs. 39, 40). For this purpose, we prepared NSCs from the embryonic forebrains of GFP.LUC Tg and TR6.GFP.LUC Tg mice (Figure 6A and Supplemental Figure 7, A and B). Almost all of the transplanted TR6.GFP.LUC-NSCs were specifically ablated following DT administration (Figure 6, B and C). Furthermore, after ablation of the transplanted cells, the BBB scores of SCI model mice that had undergone combined TR6.GFP.LUC-NSC transplantation and VPA administration declined rapidly to levels similar to those observed in untreated and VPA onlytreated mice. These results were superimposed on the graph in Figure 1B, with the observation period extended to 12 weeks after SCI, as shown in Figure 6D (for clarity, the data for GFP-NSC.VPA and GFP.LUC-NS in Figure 1B were removed). These data indicate that the transplanted cells, in the presence of VPA, made a direct and major contribution to the functional recovery of hind limb movement in SCI model mice.

Ablation of transplanted cells abolishes hind limb motor function recovery. (A) Schematic of the protocols for NSC transplantation and for detection and ablation of transplanted cells. NSCs derived from GFP.LUC- or TR6.GFP.LUC-Tg mice were transplanted into SCI model mice 1 week after injury. VPA was intraperitoneally administered every day for 1 week. Survival of transplanted cells and locomotor function of the mice were monitored weekly for 14 weeks. (B) Survival of transplanted cells was checked every week using a bioluminescence imaging system. 6 weeks after injury (5 weeks after transplantation), each mouse received 2 DT administrations. By the following week, LUC activity had completely disappeared in mice transplanted with TR6.GFP.LUC-NSCs (lower panel). (C) Sagittal sections from SCI model mice transplanted with GFP.LUC- and TR6.GFP.LUC-NSCs 2 weeks after DT injection. All transplanted cells were ablated with DT (lower panel). Scale bar: 1 mm. (D) Time course of the changes in BBB scores in SCI model mice. The hind limb function of mice that had undergone dual treatment with TR6.GFP.LUC-NSCs and VPA dropped drastically after DT administration (black line). *P < 0.0001 compared with GFP.LUC-NSCtransplanted, VPA-administered, and DT-injected SCI model mice (blue line) (repeated measures ANOVA). Data are mean SEM. VPA, n = 8; no treatment, n = 8. (E) Twelve weeks after injury, groups of SCI model mice received NMDA injections, as indicated, into the injury epicenter, to ablate local neurons in the gray matter (blue, black, and yellow lines with triangles). *P < 0.0001 compared with non-NMDAinjected mice in each group (blue, black, and yellow lines with circles) (repeated measures ANOVA). Data represent mean SEM.

Both endogenous and transplant-derived local neurons play an important role in improving hind limb motor function. It has been shown recently that local neurons in the spinal cord play an important role in spontaneous functional recovery after SCI (41, 42). In our SCI model, we also observed slight but significant spontaneous recovery of hind limb function in untreated mice, and similar levels of recovery were sustained after ablation of transplanted cells (Figure 6D). We thus hypothesized that these recoveries were attributable to endogenous local neurons in the spinal cord. Furthermore, it seemed likely that the much higher recovery observed in mice with the combined treatment but without cell ablation (Figure 6D) was effected by transplant-derived local neurons in addition to the endogenous ones. To evaluate the involvement of these local neurons in our treatment regime, we divided each treated mouse group analyzed in Figure 6D into 2 subgroups (except for the TR6.GFP.LUC-NCStransplanted only and VPA-administered only groups). The axon-sparing excitotoxin NMDA was injected at 12 weeks after SCI into the injury epicenter in the injured spinal cords of the mice in 1 subgroup for each treatment to ablate local neurons in the gray matter (4345). In uninjured mice, NMDA injections had no significant effect on hind limb function (data not shown). However, as shown in Figure 6E, NMDA injections completely reversed both spontaneous and treatment-provoked functional recovery of hind limb movement in SCI model mice, indicating that both endogenous and transplant-derived local neurons indeed play an important role in the restoration of hind limb motor function.

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JCI - Neurons derived from transplanted neural stem cells ...