Long-Distance Migration and Colonization of Transplanted Neural Stem Cells

A recent study reported remarkable survival and exuberant axon outgrowth from transplants of neural stem cells (NSCs) in a fibrin matrix with growth factors that were grafted into a complete spinal cord transection site in rats (Lu et al., 2012). As part of the NIH-supported replication project (Facilities of Research Excellence-Spinal Cord Injury), we repeated key parts of that study. Because this is a surgical intervention that may depend on skills that require extensive experience, we felt that the goals of the replication would be best served if the same surgeon performed the lesion surgeries and transplants. Accordingly, Dr. P. Lu traveled to UCI to perform the surgical procedures with aid of our staff. Also, because the cells could be considered to be a ‘‘product’’ that was obtained for use in much the same way as stem cells from a company, the cells were prepared at UCSD and were delivered to UCI on the day of the grafting procedure. Rats (n = 20) received complete spinal cord transections at thoracic level 3 (T3) and 2 weeks later received transplants of neural stem cells (NSCs) isolated from E14 rat embryos that express GFP. Spinal cord injury and transplant surgeries were done on 4 separate days over a 2 week period. By 9–10 weeks, NSC grafts had grown and differentiated to completely fill in the space left at the site of the spinal cord injury (Figure 1A). There was exuberant axon outgrowth from the grafts into the host spinal cord as reported by Lu et al. (2012) (Figure 1B). We were surprised, however, to find ectopic colonies of graft-derived cells at long distances from the transplant site, including in the central canal of the spinal cord, adhered to the surface of the spinal cord, and in the fourth ventricle of the brainstem. An example of ectopic colonies in the central canal is illustrated in Figure 1A in a horizontal section through the center of the spinal cord that had been immunostained for GFP to reveal graft-derived cells. Ectopic colonies of graft-derived cells were evident in the central canal approximately 4–7 mm rostral to the transplant. The central canal was expanded around the ectopic cell masses. Having discovered ectopic colonies of NSC-derived cells at long distances from grafts of NSC’s, we screened for ectopic colonies in other locations. Examination of cross-sections taken from cervical through high lumbar levels of the spinal cord revealed ectopic colonies of graftderived cells in the central canal at cervical levels in four rats (Figures 1C and 1J), and on the pial surface of the spinal cord in nine rats. Figure 1D illustrates a colony capping the dorsal horn near the dorsal root entry zone. Examination of sections through the brain revealed ectopic colonies adhered to the brainstem and in the fourth ventricle in four rats (Figure 1F). Overall, ectopic cell masses were seen in rats that received transplants on all four of the separate days. The ectopic cell masses extendedGFPpositive axons into the surrounding host parenchyma. The masses in the central canal extended axons into the spinal cordparenchyma formingahaloof labeled axons around the central canal (Figure 1E). Ectopic colonies capping the dorsal horn extended GFP-labeled axons into the dorsal horn and dorsal root (Figure 1D). The ectopic colony in the floor of the fourth ventricle extended axons into the dorsal part of the tegmentum near the nucleus of the solitary tract (Figures 1G and 1H). Immunostaining of colonies with celltype-specific markers revealed GFAPpositive cells with astrocyte morphology (Figure 1I) and MAP2-positive processes resembling dendrites (Figure 1J). Immunostaining with Ki67, a marker for proliferating cells, revealed abundant Ki67-positive cells in the ectopic mass in the brainstem (Figure 1K), indicating ongoing proliferation.