| ObjectiveEcto-mesenchymal stem cells (EMSCs) are a special type of mesenchymal stem cells derived from embryonic neural crest, with self-renew ability and pluripotency. The purpose of this study was to investigate the sternness and pluripotency of the EMSCs derived from the nasal mucosa of rats, and explore its pluripotency on fibrin scaffolds to find new seed cells for stem cells/scaffolds graft in tissue engineering. Finally, EMSCs/Fibrin constructs were transplanted into the rat spinal cord injury (SCI) model, followed by evaluation of its effect on the recovery of spinal cord injury.Methods1. To explore the distribution of EMSCs in the rat nasal mucosa, frozen sections of rat nasal mucosa were prepared for determining the expression of marker proteins of EMSCs, including Nestin, CD 133 and CD44, by immunofluorescence staining.2. Purified with the differential adhesion method, the EMSCs were characterized by a variety of stem cell marker proteins and the ability to form stem cell spheres.3. Osteogenic differentiation of the EMSCs was induced in the osteogenic differentiation medium. The alkaline phosphatase activity of the cells was tested using NBT staining. Furthermore, the ossification efficiency of the differentiated EMSCs was assessed by detecting bone nodules on the surface of the induced osteoblasts using alizarin red S staining. The expression levels of Collagen-I, Osteocalcin, Osteopontin and Runx2 were detected using immunofluorescence staining and Western blotting.4. Neurogenic differentiation from the EMSCs was induced in neurogenic differentiation medium. The expression levels of Synapsin, Synaptotagmin, GAP43 and NF-200 were detected using immunofluorescence staining. Western blotting was performed to assess the efficiency of neurogenic differentiation.5. Schwann cells differentiation from the EMSCs was induced in Schwann cell differentiation medium. The expression levels of GFAP, p75, S100β and CNPase were detected using immunofluorescence staining. Western blotting was carried out to assess the efficiency of Schwann cell differentiation.6. The EMSCs were non-contact co-cultured with neural stem cells derived from rat spinal cord in Transwell to assess the EMSCs’ impact on neural stem cells’ differentiation into neurons in this system by immunofluorescence staining.7. The EMSCs were contact co-cultured with neural stem cells derived from rat spinal cord to assess the EMSCs’ impact on neural stem cells’differentiate into neurons in this system using dynamic imaging of living cells and immunofluorescence staining.8. RGD, antibodies against Integrinβ1 and BOC were, respectively, added into the contact co-culture system to evaluate the inhibition effects on the neural stem cells’ differentiation.9. Structural features of fibrin scaffolds were observed by scanning electron microscope (SEM) and transmission electron microscopy (TEM). Hematoxylin-eosin (Hematoxylin-eosin, HE) staining was employed to evaluate the biocompatibility of fibrin scaffolds with the EMSCs.10. Spontaneous differentiation of EMSCs on fibrin scaffolds was detected by immunofluorescence staining and Western blotting.11. Immunofluorescence staining, Western blotting, SEM and TEM were used to detect the EMSCs’differentiation into osteoblasts, neurons and Schwann cells on fibrin scaffolds using the same way as points 3-5 mentioned above.12. EMSCs/fibrin scaffold was grafted into the spinal cord injury model in rats to observe the migration of the EMSCs in the spinal cord, and the recovery of the hind limb functional status after injury was examined by Basso, Beattie, Bresnahan scoring (BBB scoring). Spinal cord neurons regeneration was detected by immunofluorescence staining, immunohistochemistry, TEM and Western blotting.Results1. Immunofluorescence staining of the frozen sections of rat nasal mucosa showed that CD133, Nestin and CD44 positive cells were widely distributed in both sides of the lamina propria of the nasal septum.2. EMSCs can form CD133, Nestin and Sox1 positive stem cell spheres when cultured in anti-adherent condition. Passage three EMSCs showed strong stemness after purified by differential adhesion method, which express neural crest stem cell and mesenchymal stem cell markers, including Nestin, Sox1, Sox10, S100, Snail, Vimentin, CD44, CD133, Sall4 and Integrin β1, a receptor of RGD-containing proteins of extracellular matrix (ECM).3. After two weeks of culture in osteogenic medium, the EMSCs showed significantly enhanced alkaline phosphatase activity and notably increased expression levels of osteoblast marker proteins Collagen-I, Osteocalcin, Osteopuntin and Runx2. Another week later, mineralized bone nodules could be observed via alizarin red S staining on the surface of the osteogenic-differentiated EMSCs.4. The expression of neuron marker proteins Synapsin, Synaptotagmin, GAP43 and NF-200 significantly increased after EMSCs were maintained neuronal differentiation medium for 14 days.5. The expression of Schwann cells marker proteins GFAP, p75, S100β and CNPase significantly enhanced after EMSCs were induced by Schwann cell differentiation medium for 14 days.6. When non-contact co-cultured with the neural stem cells derived from rat spinal cord in Transwell, EMSCs could form neuron networks with significantly increased synapses number and length.7. The EMSCs in contact co-culture with rat spinal cord origin neural stem cells showed positive expression of neuron marker GAP43 and negative of glia marker GFAP; however, when neural stem cell was cultured alone, both neuron marker GAP43 and glia marker GFAP were positively expressed.8. The growth of nerve fibers was inhibited by the addition of RGD, antibodies against Integrin β1 and BOC into contact co-culture system.9. SEM and TEM revealed the spongy morphology of the fibrin scaffolds with fibrinogen concentrations ranging from 12.5 to 50 mg/ml. As the fibrinogen concentration increased, the porosity decreased and density increased. When the fibrinogen concentration was 75 mg/ml, most parts of the scaffold agglomerated into large clots. HE staining showed that EMSCs grew well in low concentrations fibrin scaffold.10. When EMSCs were cultured on fibrin scaffold for 12 days, immunofluorescence staining and Western blotting results showed positive expression of Schwann cells marker proteins, including. CNPase, MBP and GalCer, and neurotrophic factors, such as BDNF, NGF, NT-3.11. Immunofluorescence staining, Western blotting, SEM and TEM results showed that EMSCs had a higher efficiency of induced differentiation into osteoblasts, neurons and Schwann cells in fibrin scaffolds than on poly-L-Lysine (PLL). Most importantly, EMSCs could reconstruct fibrin scaffold to make it more favorable for its growth.12. When EMSCs/Fibrin scaffold was grafted into the spinal cord injury in rats, EMSCs could migrate to both sides of the spinal cord. Twelve weeks after implantation, functional recovery of the hind limb was well as examined by BBB scoring, while negative results were observed in the control group. Furthermore, immunofluorescence staining, immunohistochemistry, TEM and Western blotting revealed significantly improved nerve regeneration with neurons coated in very thick myelin as well as the remarkably increased expression levels of the nerve regeneration-associated proteins GAP43, NF-200 and MBP in EMSCs/Fibrin transplantation group compared with those in the SCI group and fibrin group.Conclusions1. The EMSCs were widely distributed in Olfactory and respiratory lamina propria, which possessed a strong sternness that could be differentiated into osteoblasts, neurons and Schwann cells.2. Fibrin scaffolds possessed a good biocompatibility with the EMSCs, on which the EMSCs could spontaneously differentiate into Schwann cells.3. The EMSCs showed a higher efficiency of differentiation into osteoblasts, neurons and Schwann cells in fibrin scaffold than on PLL. Furthermore, EMSCs could reconstruct fibrin scaffold to make it more favorable for its growth.4. The hind limb functional status recovered well when EMSCs/Fibrin scaffold-was transplanted into the spinal cord injury model in rats. In summary, the EMSCs are a promising seed cell in tissue engineering, which can be used for autologous transplantation for spinal cord injury. |
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