Animals

All procedures involving animals were approved by the guidelines for animal experiments of Kyoto University and the guide for the care and use of laboratory animals of the Institute of Laboratory Animal Resources (ILAR; Washington, DC, USA). All attempts were made to minimize the suffering and the number of animals used in this study. Male Crlj:LE rats were group-housed under a 12 h light and dark cycle, with food and water available. A total of 76 rats were used in this study as follows: 57 rats were lesioned, transplanted and used for the neuroanatomical and behavioral study (37 two-week-old rats, 10 seven-week-old rats, 10 twelve-week-old rats), 12 two-week-old rats were lesioned and used for the behavioral study, and 7 two-week-old rats were used as control in the behavioral study. A total of 80 C57BL/6-Tg[CAG-EGFP] mouse fetuses of either sex32 were used to acquire graft tissues. All rats were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan), and all mice were from Japan SLC (Shizuoka, Japan).

Animal model with lesion in motor cortex area

Rats were anesthetized with isoflurane (1.5%) in a mixture of O 2 and N 2 O (50%: 50%) and clamped in a stereotactic apparatus to keep their head at the horizontal position (Narishige, Tokyo, Japan). After checking for the absence of the pain reflex by pinching the cranial skin, a small midline incision in the skin and a small window of the skull over the motor cortex was made using a drill (Minitor Co., Tokyo, Japan). The motor cortex was aspirated from −1.0 to 1.0 mm rostral to the bregma and from 0.5 to 2.5 mm lateral to the midline in 2-week-old rats with the corpus callosum left intact, and −1.5 to 1.5 mm rostral to the bregma and from 0.5 to 3.5 mm lateral to the midline in 7- and 12-week-old rats (Fig. 1a). Body temperature was maintained in the normothermic range (37–38 °C) with a feedback-controlled heating pad and incubator (Biomachinery Co., Chiba, Japan).

Cortical tissue harvesting and transplantation

The cortical tissues of E14.5 mice were harvested and transferred to HBSS (Gibco) and kept at 4 °C until the transplantation. Then, the tissues were transplanted into the anterior part of the lesioned motor cortex of 3-, 8-, and 13-week-old rats 1 week after the lesion.15,25,26 Rats were anesthetized and clamped with the same methods, and a small craniectomy in the anterior part of the lesion was made using the drill. The mouse tissues were sucked up with a sterile 22 gauge needle and transplanted into four sites (1.0 µl/site) targeting the frontal cortex in the anterior part of the lesion (from the bregma: anterior 1.5 mm, lateral 1.0 mm and 2.0 mm, vertical 0.5 mm and 1.0 mm in 3-week-old rats; anterior 2.0 mm, lateral 1.0 mm and 3.0 mm, vertical 1.0 and 1.5 mm in 8- and 13-week-old rats). The injection was held for 1 min or longer (Fig. 1b).33 The rats received intraperitoneal injections of the immunosuppressant Cyclosporin A (10 mg/kg) (Wako) every day starting 5 days before the transplantation until the day before euthanization. As older rats showed poorer graft survival, we decided to use 3-week-old rat as the host for the remaining experiments (Suppl. Fig. 4).

Behavioral analysis

We examined the motor functional recovery after treatment using the foot fault test. The apparatus consisted of a grid floor (1.5 cm × 1.5 cm per grid). Each rat was placed at one end of the grid and monitored by video recording for 2 min as they traversed the grid. The number of affected forelimb and hindlimb placement errors was scored, and the percentage of foot faults out of total steps was calculated.34,35 The test was performed the day before the lesion, before the cell transplantation, and 1 week and 2 weeks after the transplantation. Baseline scores of intact 2-week-old rats (n = 7) were recorded. The other rats were randomly divided into the following four experimental groups: lesion and vehicle injection group (LV; n = 6), lesion and vehicle injection with TMT group (LVT; n = 6), lesion and transplantation group (LTx; n = 11) and lesion and transplantation with TMT group (LTxT; n = 10). Seven rats out of the LTx group and two rats out of the LTxT group were excluded from the analysis because the transplanted tissues did not engraft sufficiently according to immunohistochemical analyses. An observer blinded of the therapeutic interventions performed the behavioral analysis by video recordings.

Treadmill training

TMT was used as the rehabilitation therapy (Harvard Apparatus, MA, US). We set the time period of TMT to 2 weeks after the transplantation, because cortical neuroblasts transplanted into damaged adult motor cortex developed appropriate projections to cortical and subcortical targets 2 weeks after grafting.36 Next, we set the speed of the TMT to 15 cm/s, because 3-week-old rats could not run above this velocity over a long-time. Finally, we determined the effective TMT time as follows. We divided 16 rats into four equally sized groups as lesion only group, lesion + 20 min TMT (TMT 20 min group), lesion + 40 min TMT (TMT 40 min group), and lesion + 60 min TMT (TMT 60 min group) (n = 4, respectively). Two days after the lesion, rats began treadmill running 10 min/day at a speed 10 cm/s and slope of 0 degrees. For successive days, the running speed was increased 1 cm/s every day until 1 week after the lesion. Then, TMT 20 min, TMT 40 min, and TMT 60 min groups started TMT at 15 cm/s for 1 week. The foot fault test was performed at the day before the lesion, and 1 week and 2 weeks after the lesion. At 2 weeks after the lesion, the success rate was significantly higher in only the TMT 40 min group compared with the lesion only group (Suppl. Fig. 5). Therefore, the TMT group in our experiments is represented by the TMT 40 min group. In our experiment (Fig. 2a), 2 days after the lesion, rats began treadmill running 10 min for 5 days at a speed 10 cm/s and slope of 0 degrees. The running speed was increased 1 cm/s every day until the transplantation. One day after the transplantation, the rats started TMT at 15 cm/s for 40 min.

Immunostaining

Fourteen days after the transplantation, rats were transcardially perfusion-fixed with 4% paraformaldehyde (PFA) (Wako), and brains and spinal cords were fixed with 4% PFA for 24 h, transferred to 30% sucrose in phosphate-buffered saline (PBS), and preserved at 4 °C. The brains of four mouse fetuses were fixed with 4% PFA for 4 h and preserved using the same method. They were then embedded with O.C.T. compound (Sakura finetek, Torrence, CA, USA) and cut with a cryostat (coronal section of the brain with 50 µm, sagittal section of the brain with 30 µm and longitudinal section of the spinal posterior column lower than C1 level with 30 µm) (CM-1850, Leica Biosystems) and preserved in antifreeze (30% glyceol (Nacalai tesque), 30% ethylene glycol (Wako) and 40% PBS) at −30 °C before use. The cryosections were attached to MAS-coated glass slides (Matsunami, Osaka, Japan). Immunohistochemical analysis of the cryosections was carried out after permeabilization with 0.3% Triton X-100 (Sigma-Aldrich) and blocking in 2% skim milk (BD). The primary antibodies used were anti-GFP (rabbit and rat, 1:1000, MBL International), anti-CTIP2 (rat, 1:500, Abcam), anti-SATB2 (mouse, 1:200, Abcam), anti-PAX6 (rabbit, 1:500, Covance), anti-TBR1 (rabbit, 1:500, Abcam), anti-TBR2 (rabbit, 1:500, Abcam), anti-NRP1 (rabbit, 1:250, ECM Biosciences), anti-FOXG1 (rabbit, 1:1000, Takara Bio Inc.), anti-CUX1 (rabbit, 1:100, Santa Cruz), anti-C-FOS (rabbit, 1:1000, Santa Cruz) and anti-Synaptophysin (guinea pig, 1:1000, Synaptic Systems). For visualization, the secondary antibodies used were Alexa Fluor 488 goat anti-rabbit (1:400, Invitrogen), Alexa Fluor 488 goat anti-rat (1:400, Invitrogen), Alexa Fluor 488 goat anti-guinea pig (1:400, Invitrogen), Alexa Fluor 594 goat anti-rat (1:400, Invitrogen), Alexa Fluor 594 goat anti-mouse (1:400, Invitrogen), Alexa Fluor 594 goat anti-rabbit (1:400, Invitrogen), and Alexa Fluor 647 goat anti-rabbit (1:400, Invitrogen). The tissue samples were mounted on glass slides with fluorescent mounting medium containing 4,6-dimamidino-2-phenylindole (DAPI). We used 20 brain sections per animal for the histological analysis. The analysis was performed using the coronal sections with maximum graft area in all specimens, and we defined GFP/DAPI double-positive cells as engrafted cells.

The immunoreactive cells and fibers were analyzed using a fluorescence microscope (BZ-9000, Keyence) or confocal laser microscope (LSM700, ZEISS; CQ1, YOKOGAWA). The number of positive cells labeled by each primary antibody in the graft and the host brain were counted in the coronal sections. We performed cell counting using Image J (NIH). The original figures in Fig. 1d and Supplementary Fig. 1 were taken by a confocal laser microscope (CQ1, YOKOGAWA). The fluorescence emission was viewed through a dry emersion objective (UPLSAPO20X, Olympus), and Z-stack images were taken in 5 µm intervals over 30 µm. Maximum intensity projection (MIP) images of GFP/DAPI were made using CellPathfinder software (YOKOGAWA) and converted to a tiled figure using Image J.

Retrograde tracing of transplanted grafts

Fourteen days after the transplantation and TMT, four rats were anesthetized and clamped as described above. Following the incision of the skin overlying a cervical region, laminectomy of C1 was performed, and 0.3 µl of 4% Fast blue (Polysciences, Warrington, PA, USA), and 4% Dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) in artificial cerebrospinal fluid (Harvard Apparatus, Holliston, MA, USA) were injected into four sites (0.3 µl/site, vertical 0.5 mm) targeting a posterior column at C1–2 level over 1 min using a sterile 26 gauge needle. Then, 21 days after the transplantation, the rats were transcardially perfusion-fixed and preserved by the same methods.

Statistical analysis

Statistical analyses were performed using PRISM (GraphPad Software). For the comparison of two groups, the significance of differences was determined by Mann–Whitney test; for the comparison of more than three groups, one-way ANOVA with Tukey’s multiple comparisons test was done. Behavioral data were analyzed with two-way ANOVA with Tukey’s multiple comparisons test. Differences were considered statistically significant when probability values were <0.05. The data are presented as the mean ± standard error of the mean (SEM).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.