Reduction of Inflammation and Enhancement of Motility after Pancreatic Islet Derived Stem Cell Transplantation Following Spinal Cord Injury

Abstract

Objective: Spinal cord injury (SCI) is a very serious health problem, usually caused by a trauma and accompanied by elevated levels of inflammation indicators. Stem cell-based therapy is promising some valuable strategies for its functional recovery. Nestinpositive progenitor and/or stem cells (SC) isolated from pancreatic islets (PI) show mesenchymal stem cell (MSC) characteristics. For this reason, we aimed to analyze the effects of rat pancreatic islet derived stem cell (rPI-SC) delivery on functional recovery, as well as the levels of inflammation factors following SCI.

Methods: rPI-SCs were isolated, cultured and their MSC characteristics were determined through flow cytometry and immunofluorescence analysis. The experimental rat population was divided into three groups : 1) laminectomy & trauma, 2) laminectomy & trauma & phosphate-buffered saline (PBS), and 3) laminectomy+trauma+SCs. Green fluorescent protein (GFP) labelled rPI-SCs were transplanted into the injured rat spinal cord. Their motilities were evaluated with Basso, Beattie and Bresnahan (BBB) Score. After 4-weeks, spinal cord sections were analyzed for GFP labeled SCs and stained for vimentin, S100β, brain derived neurotrophic factor (BDNF), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), vascular endothelial growth factor (VEGF) and proinflammatory (interleukin [IL]-6, transforming growth factor [TGF]-β, macrophage inflammatory protein [MIP]-2, myeloperoxidase [MPO]) and anti-inflammatory (IL-1 receptor antagonis) factors.

Results: rPI-SCs were revealed to display MSC characteristics and express neural and glial cell markers including BDNF, glial fibrillary acidic protein (GFAP), fibronectin, microtubule associated protein-2a,b (MAP2a,b), β3-tubulin and nestin as well as antiinflammatory prostaglandin E2 receptor, EP3. The BBB scores showed significant motor recovery in group 3. GFP-labelled cells were localized on the injury site. In addition, decreased proinflammatory factor levels and increased intensity of anti-inflammatory factors were determined.

Conclusion: Transplantation of PI-SCs might be an effective strategy to improve functional recovery following spinal cord trauma.

Keywords: Islets of langerhans; Regeneration; Spinal cord; Stem cells; Wounds and injuries.

Fig. 1.

Morphological characteristics of the rat pancreatic islets. Free floating rat pancreatic islets (A). Fibroblast-like cells are observed growing out and away from a pancreatic islets (B). rPI-SC morphologies (unstained) for late passage (C, P3–4th day). Flow cytometry analysis for P3 cells (C and D). Scale bars, 100 μm. FITC : fluorescein isothiocyanate, rPI-SC : rat pancreatic islet derived stem cell.

Fig. 2.

Representative panels of immunofluorescence stainings for phenotype identification of rPI-SC. The expression of cell markers of neurogenic (A-C and E-G) and mesenchymal markers (D) and inhibitors of pro-inflammatory cytokines (H, I). All markers were detected with FITC (green) labelled secondary antibodies. Nuclei were labeled with DAPI (blue) (Scale bars, 50 µm). BDNF : brain derived neurotrophic factor, GFAP : glial fibrillary acidic protein, MAP2a,b : microtubule associated protein-2a,b, IL-1ra : interleukin-1 receptor antagonis, FITC : fluorescein isothiocyanate, rPI-SC : rat pancreatic islet derived stem cell.

Fig. 3.

Immunofluorescence stainings in paraffin sections of rat spinal cord tissues. Four weeks after rPI-SC transplantation, GFP+ cells were migrated to the damaged site and survived in laminectomy+trauma+SC (group 3) animals’ sections. GFP+/vimentin+rPI-SCs were located in damaged area. Green : GFP, red : vimentin. Nuclei were labeled with DAPI (blue) (Scale bars, 50 µm). DAPI : 4',6-diamidino-2-phenylindole, dihydrochloride, GFP : green fluorescent protein, SC : stem cell, rPI-SC : rat pancreatic islet derived stem cell.

Fig. 4.

Immunofluorescence stainings in paraffin sections of rat spinal cord tissues. Four weeks after rPI-SC transplantation, GFP+ cells were migrated to the damaged site and survived in laminectomy+trauma+SC (group 3) animals’ sections. GFP+/CNPase+rPI-SCs were located in damaged area. Green : GFP, red : CNPase. Nuclei were labeled with DAPI (blue). Arrows indicate GFP+ cells. Scale bars is 50 µm. DAPI : 4',6-diamidino-2-phenylindole, dihydrochloride, GFP : green fluorescent protein, CNPase : 2’,3’-cyclic-nucleotide 3'-phosphodiesterase, SC : stem cell, rPI-SC : rat pancreatic islet derived stem cell.

Fig. 5.

Immunofluorescence stainings in paraffin sections of rat spinal cord tissues. Four weeks after rPI-SC transplantation, GFP+ cells were migrated to the damaged site and survived in laminectomy+trauma+SC (group 3) animals’ sections. GFP+/S100+rPI-SCs were located in damaged area. Green : GFP, red : S100β. Nuclei were labeled with DAPI (blue). Arrows indicate GFP+ cells. Scale bars is 50 µm. DAPI : 4',6-diamidino-2-phenylindole, dihydrochloride, GFP : green fluorescent protein, SC : stem cell, rPI-SC : rat pancreatic islet derived stem cell.

Fig. 6.

Immunofluorescence stainings in paraffin sections of rat spinal cord tissues. Four weeks after rPI-SC transplantation, GFP+ cells were migrated to the damaged site and survived in laminectomy+trauma+SC (group 3) animals’ sections. GFP+/BDNF+rPI-SCs were located in damaged area. Green: GFP, red : BDNF. Nuclei were labeled with DAPI (blue). Arrows indicate GFP+ cells. Scale bars is 50 µm. DAPI : 4',6-diamidino-2-phenylindole, dihydrochloride, GFP : green fluorescent protein, BDNF : brain derived neurotrophic factor, SC : stem cell, CNPase : 2’,3’-cyclic-nucleotide 3'-phosphodiesterase, rPI-SC : rat pancreatic islet derived stem cell.

Fig. 7.

A : Immunofluorescence stainings in paraffin sections of rat spinal cord tissues. Four weeks after rPI-SC transplantation, GFP+ cells were migrated to the damaged site and survived in laminectomy+trauma+SC (group 3) animals’ sections. GFP+/VEGF+rPI-SCs were located in damaged area. Green : GFP, red : VEGF. Nuclei were labeled with DAPI (blue). Arrows indicate GFP+ cells. Scale bars is 50 µm. B : Graphs of immunostaining intensities. The values were presented as mean±standard error. *p<0.05. DAPI : 4',6-diamidino-2-phenylindole, dihydrochloride, GFP : green fluorescent protein, VEGF : vascular endothelial growth factor, SC : stem cell, BDNF : brain derived neurotrophic factor, a.u. : arbitrary unit, rPI-SC : rat pancreatic islet derived stem cell.

Fig. 8.

A : Immunofluorescence stainings for anti-inflammatory (IL-1ra) and pro-inflammatory (IL-6, TGF-β1, MIP-2, and MPO) markers with and without rPI-SC injection in paraffin sections of rat spinal cord. All markers were detected with FITC (green) labelled secondary antibodies. Nuclei were labeled with DAPI (blue). Scale bars is 50 µm. B : Graphs of immunostaining intensities. The values were presented as mean±standard error. *p<0.05. rPI-SC : rat pancreatic islet derived stem cell, TGF-β1 : transforming growth factor-1, MPO : myeloperoxidase, MIP-2 : macrophage inflammatory protein-2, IL-6 : interleukin-6, IL-1ra : interleukin-1 receptor antagonis, a.u. : arbitrary unit.

Fig. 9.

The effect of T10–T11 SCI on general hind limb function over time after SCI. Deficits are expressed as a BBB locomotion score. The values were presented as mean±standard error. *p<0.05. BBB : Basso, Beattie and Bresnahan, PBS : phosphate-buffered saline, rPI-SC : rat pancreatic islet derived stem cell, SCI : spinal cord injury.