E-cadherin regulates biological behaviors of neural stem cells and promotes motor function recovery following spinal cord injury

Abstract

Stem cell-based repair strategies for spinal cord injury (SCI) are a highly studied area of research. Multiple gene-modified stem cells have been transplanted into SCI models, in the hope of generating more neurons to repair a damaged nervous system. However, the results are not always successful, as the grafted cells may be unable to survive in the injured spinal cord. E-cadherin, a transmembrane adhesion protein, has been identified as an epithelial-to-mesenchymal transition marker and is vital for morphological structure maintenance and the functional integrity of epithelial cells. At present, few studies have examined the association between E-cadherin and neural stem cells (NSCs). The present study investigated the expression of E-cadherin in subcultured NSCs and differentiated NSCs. Furthermore, the effect of E-cadherin on NSC viability, migration, differentiation and neurosphere formation was assessed. An in vivo study was used to assess the long-term survival of grafted NSCs. Additionally, the protective effect of E-cadherin on SCI was assessed by analyzing tissue repair, Basso Mouse Scale scores and the expression of inflammatory cytokines. The results of the present study suggested that E-cadherin was able to promote NSC viability and neurosphere formation; however, it had no significant effect on NSC differentiation. To conclude, grafted NSCs with highly expressed E-cadherin facilitated motor function recovery following SCI by reducing the release of inflammatory cytokines.

Keywords: E-cadherin; inflammatory cytokines; neural stem cells; spinal cord injury.

Figure 1.

Isolated NSCs are able to proliferate and differentiate. (A) The undifferentiated NSCs were observed under microscopes. (B) NSCs could express Nestin and form neurospheres. (C) DAPI staining of NSCs. (D) NSCs were able to differentiate into neurons. (E) NSCs were able to differentiate into astrocytes. NSC, neural stem cells; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; Tuji1, neuron-specific class III β-tubulin.

Figure 2.

E-cadherin increases in subcultured NSCs but decreases subsequent to induced differentiation. (A) Reverse transcription-quantitative polymerase chain reaction revealed that E-cadherin was significantly increased in P5 NSCs compared with P0 cells, and decreased in differentiated NSCs. (B) The change was further confirmed by western blotting. (C) Expression of β-catenin, the core component of E-cadherin/β-catenin complex, was increased in subcultured NSCs, as confirmed by flow cytometry. **P<0.01 as indicated or vs. P0-NSCs. NSC, neural stem cells; D-NSCs, differentiated NSCs; P0, primary cells; P5, passage 5.

Figure 3.

The role of E-cadherin in regulating the biological behaviors of NSCs. (A) Western blotting revealed that the lentivirus was able to significantly increase E-cadherin protein expression following transfection into NSCs. (B) MTT results indicated that E-cadherin appeared to facilitate NSCs viability. (C) Data obtained from a Transwell experiment demonstrated that E-cadherin inhibited cell migration. (D) The immunostaining revealed that E-cadherin did not affect the fate determination of NSCs, as it did not alter the percentage of differentiated neurons (Tuj1). (E) E-cadherin increased the number of neural spheres and maintained the stemness of NSCs. *P<0.05 and **P<0.01 vs. NSC. NSC, neural stem cells; Tuj1, neuron-specific class III β-tubulin; NC, negative control; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 4.

E-cadherin improved the survival of grafted NSCs in the short term. (A) T9 laminectomy was performed to exposure spinal cord. (B) Subsequent to clamping the spinal cord at T9 for 30 sec, two red lines were observed at either sides of the midline (black arrow). (C) The disruption of the tissue was observed in the injured spinal cord. (D) A total of 3 months post surgery, the injured spinal cord was able to form a large cavity surrounded by GFAP positive glial cells. (E) Transplanted NSCs were able to migrate to the lesion site, and E-cadherin improved the survival of NSCs around the epicenter in the short term. *P<0.05 vs. NSC. NSC, neural stem cells; GFAP, glial fibrillary acidic protein. DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; SCI, spinal cord injury.

Figure 5.

Grafted NSCs with highly expressed E-cadherin are able to promote motor function recovery and create a better microenvironment for neural regeneration. (A) Immunostaining of GFAP in spinal cord tissues. (B) NSCs with E-cadherin group increased tissue sparing the most compared with PBS treatment and NSCs transplantation. (C) NSCs with E-cadherin transplantation obtained the highest BMS scores. *P<0.05 vs. NSCs; ^#P<0.05 vs. PBS. NSCs, neural stem cells; BMS, Basso Mouse Scale; GFAP, glial fibrillary acidic protein; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 6.

E-cadherin alleviated secondary damage by reducing the release of inflammatory cytokines. (A) The release of inflammatory cytokines (IL-1β, IL-6, IL-8, MCP-1, iNOS and TNF-α) was increased in the PBS group 3 days after transplantation. (B) INF-γ was able to induce the ectopic expression of IL-1β, iNOS and TNF-α released by activated macrophages, and NSCs alone were able to reduce the expression of these cytokines, while NSCs with E-cadherin exhibited the most marked inhibitory effect. (C) Cytokines were detected in NSCs alone in response to INF-γ treatment. No obvious difference was observed before and after INF-γ treatment. *P<0.05, **P<0.01 as indicated; ^##P<0.01 vs. NSCs. NSCs, neural stem cells; IL, interleukin; MCP-1, monocyte chemoattractant protein 1; iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor α; IFN-γ, type II interferon.