Combined treatment with enteric neural stem cells and chondroitinase ABC reduces spinal cord lesion pathology

Abstract

Background: Spinal cord injury (SCI) presents a significant challenge for the field of neurotherapeutics. Stem cells have shown promise in replenishing the cells lost to the injury process, but the release of axon growth-inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs) by activated cells within the injury site hinders the integration of transplanted cells. We hypothesised that simultaneous application of enteric neural stem cells (ENSCs) isolated from the gastrointestinal tract, with a lentivirus (LV) containing the enzyme chondroitinase ABC (ChABC), would enhance the regenerative potential of ENSCs after transplantation into the injured spinal cord.

Methods: ENSCs were harvested from the GI tract of p7 rats, expanded in vitro and characterised. Adult rats bearing a contusion injury were randomly assigned to one of four groups: no treatment, LV-ChABC injection only, ENSC transplantation only or ENSC transplantation+LV-ChABC injection. After 16 weeks, rats were sacrificed and the harvested spinal cords examined for evidence of repair.

Results: ENSC cultures contained a variety of neuronal subtypes suitable for replenishing cells lost through SCI. Following injury, transplanted ENSC-derived cells survived and ChABC successfully degraded CSPGs. We observed significant reductions in the injured tissue and cavity area, with the greatest improvements seen in the combined treatment group. ENSC-derived cells extended projections across the injury site into both the rostral and caudal host spinal cord, and ENSC transplantation significantly increased the number of cells extending axons across the injury site. Furthermore, the combined treatment resulted in a modest, but significant functional improvement by week 16, and we found no evidence of the spread of transplanted cells to ectopic locations or formation of tumours.

Conclusions: Regenerative effects of a combined treatment with ENSCs and ChABC surpassed either treatment alone, highlighting the importance of further research into combinatorial therapies for SCI. Our work provides evidence that stem cells taken from the adult gastrointestinal tract, an easily accessible source for autologous transplantation, could be strongly considered for the repair of central nervous system disorders.

Keywords: ChABC; Enteric neural stem cells; Spinal cord injury; Stem cells.

Fig. 1

Spinal cord contusion injuries resulted in difficulty traversing the horizontal ladder, which improved over the course of the study. Spinal cord injuries were induced with an Infinite Horizons impactor. Applied force (a) and spinal cord displacement (b) were consistent between animals. Following injury, the ability of each rat to traverse the horizontal ladder was assessed at regular time points (c). All rats improved over the course of the study. At the 16 week time point, there was a significant difference between the ENSCs+ChABC and the non-treated group (* indicates significance (p = < 0.05) and between the ENSCs+ChABC and the ENSC-only group (§ indicates significance (p = < 0.005). Data are represented as mean ± s.e.m

Fig. 2

In vitro cultures of rat-derived ENSCs contain a heterogenous population of dividing progenitor cells and various neuronal subtypes. a Representative FACS plot showing isolation of p75 FITC-labelled ENSCs. Following FACS, 1 week-old p75+ cultures were analysed by qRT-PCR, revealing expression of Sox10 (neural crest cell progenitor cell marker), Tuj1 (pan-neuronal marker), Gls1 (glutamine), nNos (neuronal nitric oxide), Tph1 (serotonin), ChAT (acetylcholine), Gad (GABA) and S100b (glia) (b). c–h In vitro characterisation of ENSCs prior to transplantation. p75-sorted cell cultures displayed a characteristic neuronal morphology by 1 week in culture, including extension of fine interneuronal processes (c), and formed dense neurospheres by around 2 weeks (d). A small number of dividing cells were detected by Ki67+ staining (e). However, the vast majority of cells stained positive for the pan-neuronal marker TuJ1 (f), indicating neuronal differentiation. A subpopulation of cells stained positive for specific neuronal subtype markers, including nNOS (g) and 5HT (h). Data are represented as mean ± SEM. Scale bar—c, d 200 μm, e 50 μm, and f, g, h 100 μm

Fig. 3

Immunoflourescent staining reveals the presence of CSPG breakdown products and ENSCs 16 weeks post-transplantation. Longitudinal cryosections of the spinal cords harvested at 16 weeks post-injury were analysed for markers indicative of successful treatment. a–d Representative images of the spinal cords in the untreated (a), ChABC-treated (b), ENSC-treated (c) and ENSC+ChABC-treated (d) groups. Asterisks indicate lesion cavity. Chondroitin-4-sulfate disaccharides (C4S), a breakdown product of CSPGs, were detected by immunoflourescent staining following treatment with ChABC (b, d). In rats receiving ENSC transplantation, ENSCs could be seen within the spinal cord injury zone (c, d). ENSCs extended substantial processes from the transplantation site (e, arrowheads), often for several millimetres (f, arrows indicate maximum detected length of a single GFP+ fibre). The boxed area in f is shown at a higher magnification in (f’). Arrowheads indicate the path of the GFP+ fibre. Scale bar—d 1 mm, e 200 μm, and f 500 μm

Fig. 4

Application of ChABC has no effect on ENSC spread/behaviour. a, b Representative images of transplanted ENSCs within the ENSC-treated (a) and ENSC+ChABC-treated (b) spinal cords. At the time of tissue harvest (16 weeks post-injury), numerous GFP+ cells were found within sagittal spinal cord sections of both the ENSC-only group (a) and the ENSC+ChABC groups (b). c–f Serial spinal cord sections spanning the extent of GFP+ cell detection were analysed for cell spread across the anterior/posterior (c), dorsal/ventral (d) and left/right planes (e), as well as for cell survival (f). No significant differences were detected in any parameter between animals treated with ENSCs+ChABC or with ENSC-only. Data are represented as mean ± s.e.m. Scale bars—a, b 500 μm

Fig. 5

Combined treatment of SCI with ENSCs and ChABC resulted in significant reductions in tissue pathology, as assessed by eriochrome cyanine staining. a–d Representative sagittal sections of the spinal cords from each treatment group harvested from animals sacrificed 16 weeks post-injury and stained for eriochrome C. e–h Summary data of cavity area (e, f) or injured tissue area (g, h) analysis. ENSC transplantation or ChABC application applied as single treatments did not significantly affect the cavity size or the area of injured tissue. However, in the combined treatment group, there was a significant decrease in both cavity area and the area of injured tissue compared to the non-treated group. Data are represented as mean ± s.e.m. *Indicates significance (p ≤ 0.05). Scale bar—a 500 μm

Fig. 6

Transplanted ENSCs project axons through and past the injury site to reach caudal spinal cord regions. Fluorogold, a retrograde tracer, was injected caudal to the injury site (T12) at 15 weeks post-injury (1 week prior to sacrifice of animals at week 16). a Representative tile scan of the injury epicentre and rostral spinal cord of a rat treated with ENSCs only. Asterisk indicates injury cavity. a’ High magnification of boxed area in (a), indicating FG-labelled neurons (arrows). Frequent instances of transplanted, GFP+ ENSCs (b) co-labelled with FG (b’) were found (b” shows merged image of b and b’). Following incubation with a fluorogold antibody and development with diaminobenzidine (c), the number of FG+ cells rostral to the lesion was quantified using unbiased stereology (d). Data are represented as mean ± s.e.m. *Indicates significance (p ≤ 0.05). Scale bars: a 500 μm, b 50 μm, and c 25 μm