Neurotrophin-3 gene-modified Schwann cells promote TrkC gene-modified mesenchymal stem cells to differentiate into neuron-like cells in poly(lactic-acid-co-glycolic acid) multiple-channel conduit.

Rapid progress in the field of nerve tissue engineering has opened up the way for new therapeutic strategies for spinal cord injury (SCI). Bone marrow-derived mesenchymal stem cells (MSCs) could be differentiated into neural lineages, which can be used as a potential cell source for nerve repair. Schwann cells (SCs) have been reported to support structural and functional recovery of SCI. In this study, we co-cultured neurotrophin-3 (NT-3) gene-modified SCs and NT-3 receptor tyrosine protein kinase C (TrkC) gene-modified MSCs in a three-dimensional porous poly(lactic-acid-co-glycolic acid) (PLGA) conduit with multiple channels in vitro for 14 days. Our results showed that more than 50% of the grafted MSCs were MAP2- and ß-III-tubulin-positive cells, and the MSCs expressed a high level of ß-III-tubulin detected by Western blotting, indicating a high rate of neuronal differentiation. Furthermore, immunostaining of PSD95 revealed the formation of a synapse-like structure, which was confirmed under electron microscopy. In conclusion, co-culture of NT-3 gene-modified SCs and TrkC gene-modified MSCs in the PLGA multiple-channeled conduit can promote MSCs' differentiation into neuron-like cells with synaptogenesis potential. Our study provides a biological basis for future application of this artificial MSCs/SCs/PLGA complex in the SCI treatment.

Transplantation of artificial neural construct partly improved spinal tissue repair and functional recovery in rats with spinal cord transection.

Delivery of cellular and/or trophic factors to the site of injury may promote neural repair or axonal regeneration and return of function after spinal cord injury. Engineered scaffolds provide a platform to deliver therapeutic cells and neurotrophic molecules. To explore therapeutic potential of engineered neural tissue, we generated an artificial neural construct in vitro, and transplanted this construct into a completely transected spinal cord of adult rats. Two months later, behavioral analysis showed that the locomotion recovery was significantly improved compared with control animals. Immunoreactivity against microtubule associated protein 2 (Map2) and postsynaptic density 95 (PSD95) demonstrated that grafted cells had a higher survival rate and were able to differentiate toward neuronal phenotype with ability to form synapse-like structure at the injury site; this was also observed under the electron microscope. Immunostaining of neurofilament-200 (NF-200) showed that the number of nerve fibers regrowing into the injury site in full treatment group was much higher than that seen in other groups. Furthermore, Nissl staining revealed that host neuron survival rate was significantly increased in rats with full treatments. However, there were no biotin dextran amine (BDA) anterograde tracing fibers crossing through the injury site, suggesting the limited ability of corticospinal tract axonal regeneration. Taken together, although our artificial neural construct permits grafted cells to differentiate into neuronal phenotype, synaptogenesis, axonal regeneration and partial locomotor function recovery, the limited capacity for corticospinal tract axonal regeneration may affect its potential therapy in spinal cord injury.

NT-3 gene modified Schwann cells promote TrkC gene modified mesenchymal stem cells to differentiate into neuron-like cells in vitro.

Reports of neuronal differentiation of bone marrow derived mesenchymal stem cells (MSCs) suggested the possibility that these cells could serve as a source of treatment for spinal cord injury. However, the percentages of neuron-like cells differentiated from the MSCs were relatively low both in vitro and in vivo. Here, we investigated whether co-culture of human neurotrophin-3 (NT-3) gene modified Schwann cells (SCs) and human NT-3 receptor tyrosine protein kinase C (TrkC) gene modified MSCs could increase differentiation of neuron-like cells from MSCs. It was shown that MSCs were significantly promoted to differentiate into neuron-like cells, as evidenced immunocytochemically by the expression of neuronal markers, including nestin, beta-III-tubulin, MAP2 and PSD95, 7 days after co-culture. However, the expression of glial fibrillary acidic protein (GFAP)--an astrocyte marker in these cells--was not so obvious. These results demonstrate that the binding of overexpressed NT-3 in SCs and its receptor TrkC in MSCs can be considered to stimulate the increased rate of neuronal differentiation.

Synaptic transmission of neural stem cells seeded in 3-dimensional PLGA scaffolds.

To explore therapeutic potential of engineered neural tissue, we combined genetically modified neural stem cells (NSCs) and poly(lactic acid-co-glycolic acid) (PLGA) polymers to generate an artificial neural network in vitro. NSCs transfected with either NT-3 or its receptor TrkC gene were seeded into PLGA scaffold. The NSCs were widely distributed and viable in the scaffold after culturing for 14 days. Immunoreactivity against Map2 was detected in >70% of these grafted cells, suggesting a high rate of differentiation toward neurons. Immunostaining of synapsin-I and PSD95 revealed formation of synaptic structures, which was also observed under electron microscope. Furthermore, using FM1-43 dynamic imaging, synapses in these differentiated neurons were found to be excitable and capable of releasing synaptic vesicles. Taken together, our artificial PLGA construct permits NSCs to differentiate toward neurons, establish connections and exhibit synaptic activities. These findings provide a biological basis for future application or transplantation of this artificial construct in neural repair.