Schwann cells (SCs) and olfactory ensheathing glia (OEG) have shown promise for spinal cord injury repair. We sought their in vivo identification following transplantation into the contused adult rat spinal cord at 1 week post-injury by: (i) DNA in situ hybridization (ISH) with a Y-chromosome specific probe to identify male transplants in female rats and (ii) lentiviral vector-mediated expression of EGFP. Survival, migration, and axon-glia association were quantified from 3 days to 9 weeks post-transplantation. At 3 weeks after transplantation into the lesion, a 60-90% loss of grafted cells was observed. OEG-only grafts survived very poorly within the lesion (<5%); injection outside the lesion led to a 60% survival rate, implying that the injury milieu was hostile to transplanted cells and or prevented their proliferation. At later times post-grafting, p75(+)/EGFP(-) cells in the lesion outnumbered EGFP(+) cells in all paradigms, evidence of significant host SC infiltration. SCs and OEG injected into the injury failed to migrate from the lesion. Injection of OEG outside of the injury resulted in their migration into the SC-injected injury site, not via normal-appearing host tissue but along the pia or via the central canal. In all paradigms, host axons were seen in association with or ensheathed by transplanted glia. Numerous myelinated axons were found within regions of grafted SCs but not OEG. The current study details the temporal survival, migration, axon association of SCs and OEG, and functional recovery after grafting into the contused spinal cord, research previously complicated due to a lack of quality, long-term markers for cell tracking in vivo.

Due to an ever-growing population of individuals with chronic spinal cord injury, there is a need for experimental models to translate efficacious regenerative and reparative acute therapies to chronic injury application. The present study assessed the ability of fluid grafts of either Schwann cells (SCs) or olfactory ensheathing glia (OEG) to facilitate the growth of supraspinal and afferent axons and promote restitution of hind limb function after transplantation into a 2-month-old, moderate, thoracic (T8) contusion in the rat. The use of cultured glial cells, transduced with lentiviral vectors encoding enhanced green fluorescent protein (EGFP), permitted long-term tracking of the cells following spinal cord transplantation to examine their survival, migration, and axonal association. At 3 months following grafting of 2 million SCs or OEG in 6 µl of DMEM/F12 medium into the injury site, stereological quantification of the three-dimensional reconstructed spinal cords revealed that an average of 17.1 ± 6.8% of the SCs and 2.3 ± 1.4% of the OEG survived from the number transplanted. In the OEG grafted spinal cord, a limited number of glia were unable to prevent central cavitation and were found in patches around the cavity rim. The transplanted SCs, however, formed a substantive graft within the injury site capable of supporting the ingrowth of numerous, densely packed neurofilament-positive axons. The SC grafts were able to support growth of both ascending calcitonin gene-related peptide (CGRP)-positive and supraspinal serotonergic axons and, although no biotinylated dextran amine (BDA)-traced corticospinal axons were present within the center of the grafts, the SC transplants significantly increased corticospinal axon numbers immediately rostral to the injury-graft site compared with injury-only controls. Moreover, SC grafted animals demonstrated modest, though significant, improvements in open field locomotion and exhibited less foot position errors (base of support and foot rotation). Whereas these results demonstrate that SC grafts survive, support axon growth, and can improve functional outcome after chronic contusive spinal cord injury, further development of OEG grafting procedures in this model and putative combination strategies with SC grafts need to be further explored to produce substantial improvements in axon growth and function.

Transgenic mice carrying the bacterial lacZ reporter gene under the control of the regulatory elements of the human myoD gene have been produced. The developmental expression of the myoD reporter transgene in somites, limb buds, visceral arches, and cephalocervical regions was studied in transgenic embryos by beta-gal staining. In somites, the spatiotemporal pattern of transgene expression was different from other muscle-specific regulatory and structural genes and revealed that myoD-expressing cells arise in distinct patterns in somites that are dependent on position along the anterior-posterior (AP) body axis (occipital and cervical vs thoracic and more posterior myotomes). Transgene expression did not follow a strict anterior to posterior sequence of activation and therefore was not strictly correlated with somite developmental age. Moreover, the pattern of transgene expression along the dorsal-ventral myotomal axis was dependent on somite position along the anterior-posterior axis. While myoD expression is first detected after the myotome is well-formed, transgene expression in the dorsal and ventral medial lips of the dermatome suggests a function for myoD in the expansion of the myotome. Whole-mount in situ hybridization confirmed that these unique patterns of transgene expression in somites, as well as expression in limb buds, visceral arches, and other myogenic centers, are concordant with the distribution of endogenous myoD transcripts. These results shed new light on the developmental differences between myotomes at different positions along the AP and DV axis and demonstrate a unique axial pattern of somitic myoD expression, suggesting a specific role of myoD in myotome lineage determination and differentiation.