Genetically modified human umbilical cord blood cells as a promising strategy for treatment of spinal cord injury.

Spinal cord injury (SCI) continues to be a pressing health and social problem. The injury leads to neuronal and glial cell death accompanied by degeneration of nerve fibers. There are currently no particularly effective treatments. SCI causes profound disability of people affected and has attracted increased attention in the international field of neuroregeneration. For the past two decades, much hope has been placed in cell therapies for the restoration of both structure and function of the injured spinal cord. Embryonic and neural stem cells, olfactory ensheathing cells, microglia-like cells, Schwann cells, mesenchymal stem cells, human umbilical cord blood cells (UCBCs) and many other cell types have been investigated (Xu and Onifer, 2009; Ronaghi et al., 2010; Sabapathy et al., 2015). It has been shown that graft cells support tissue sparing, exert a trophic effect on neurons and glia, promote axonal growth, and participate in their remyelination.

The current literature suggests that after SCI, cell therapy is effective at promoting neuroregeneration. However, the optimal cell type for transplantation after neurotrauma has not been determined due to differences in experimental conditions, such as localization (cervical, thoracic, lumbar), injury type (contusion, compression, hemisection, complete transection, selective shutdown tracts, etc.), the severity of the injury, methods of cultivation and preparation of cells for transplantation, time of administration after SCI, site of cell injection (site of injury, intrathecal or intravenous), the presence or absence of immunosuppression, and many other factors. It appears that researchers are guided by the general criteria when choosing a cell intended for transplantation, such as oncogenic and infectious safety, low invasiveness of the fence material, which is important for clinical use. The main limitations of stem and progenitor cell transplants in SCI are their poor survival rates and uncontrolled differentiation. Globally clinical trials suggest that the use of autologous cells, especially cells of mesenchymal origin, have a high likelihood of being effective in treatment of neurotrauma. In addition to fitting the criteria for safety and invasiveness, transplantation studies in model systems and the results of clinical trials collectively suggest that hUCBCs are very suitable for cell therapy and warrant further research into their use for stimulation of regeneration of SCI.

Previously, our group, and others, have demonstrated that hUCBCs possess many of the properties that allow them to overcome many of the major hurdles of SCI regeneration (). It has been demonstrated that transplantation of hUCBCs in SCI depresses the inflammatory response, provides a neurotrophic influence and stimulates neovascularization (Chen et al., 2008). Injection of hUCBCs into the bloodstream after SCI reduces the expression of pro-apoptotic genes and promotes neuronal survival (Dasari et al., 2009). At 6 weeks after local transplantation of hUCBCs into the area of SCI, Chua et al reported a significant improvement in behavioral recovery (BBB rating scale) with increased levels of cytokines and growth factors with neuroprotective, angiogenic and anti-inflammatory actions (Chua et al., 2010). Using a model of SCI in dogs, Lee at al. (2011) demonstrated the ability of hUCBCs to enhance remyelination by formation of peripheral type myelin sheaths. In this study, axons were remyelinated by P0-positive Schwann cells with functional recovery being maintained for three years after the cell transplantation. Recently, our group demonstrated that transplantation of human umbilical cord blood mononuclear cells (hUCB-MCs) reduced cavity volume and tissue retention at the lesion site (both white and gray matter) (Mukhamedshina et al., 2016).

Human umbilical cord blood cells (hUCBCs) possess many properties which allow them to overcome the hurdles of regeneration in spinal cord injury.

It should be noted that clinical trials are being conducted with hUCBCs. Kang et al. have shown that transplantation of MSCs from hUCBCs (UCMSCs) into a 37-year-old female with SCI improved both functional recovery and sensory perception (Kang et al., 2005). Transplantation UCMSCs in combination with CD34+ hUCBCs into a 29-year-old male with an L1 SCI resulted in recovery of muscle, bowel, and sexual function along with a decrease in ASIA score from “A” to “D” (Ichim et al., 2010).

For cell therapy to be more effective in stimulation of neuroregeneration and restoration of lost functions of the spinal cord, there is a need for both the continued presence of high levels of transplanted cells in the damaged tissue and the ability of cells to readily home to the injured area. It has been shown that hUCBCs have high survival rates and strong migration potential both by intravenous and intraspinal administration (Garbuzova-Davis et al., 2003; Mukhamedshina et al., 2015).

Despite many important advances in cell transplantation research for the treatment of SCI, it has become clear that standalone cell therapy leads to only a modest improvement in structural parameters and to insufficient restoration of function. Therefore, genetically modified cells with enhanced expression of therapeutic genes (e.g. genes for neurotrophic factors genes) have attracted the attention of researchers as potential stimulators of posttraumatic spinal cord regeneration. Simultaneous delivery into the damaged tissues of several neurotrophic factors appears to be the most effective, with co-delivery of neurotrophic and angiogenic factors being the most efficient at stimulating neuroregeneration. As example is the combination of vascular endothelial growth factor (VEGF) and glial-derived neurotrophic factor (GDNF) genes. Until recently the use of such gene combinations had not been investigated.

Gene delivery into cells using viral vectors is currently the leading method of therapy directed to CNS disorders. As a gene delivery system we used adenoviral vectors since they provide high titers of the recombinant virus, efficiently transfer genes into dividing and non-dividing cells, do not integrate into the genome, and have high expression of introduced genes. However, adenoviruses have the potential to induce nonspecific inflammation and cellular immune responses, which reduces the duration of transgene expression to weeks or months. However, other viral vectors also have disadvantages such as transient expression of transgenes (herpes virus), transduction of dividing cells only (retrovirus), small insert size for transgene constructs (adeno-associated virus) and doubts about safety (lentivirus). Therefore, the search for the most efficient and safe gene delivery system is still ongoing.

We investigated the possibility of strengthening posttraumatic spinal cord regeneration by transduction of hUCB-MCs with adenoviral vectors encoding genes for neurotrophic and angiogenic factors such as VEGF and GDNF (hUCB-MCs + Ad5-VEGF + Ad5-GDNF) (Mukhamedshina et al., 2016). Our results indicate that hUCB-MCs administered directly into the lesion site (immediately following SCI) survived 30 days after injection providing the possibility of expressing products of recombinant therapeutic genes for this therapeutic period, which completely overlaps the window during which secondary damage responses rapidly unfold. Examples of such responses include increased levels of proinflammatory cytokines, oxidative stress and excitotoxicity, all of which lead to neural cell death and axonal degeneration. The duration of grafted cell survival in the area of SCI in our experiments is consistent with similar work using transplanted mesenchymal precursors. It should be noted that these positive findings were obtained in the absence of immunosuppression. It is anticipated that further studies will demonstrate longer persistence in the SCI area of grafted hUCB-MCs that are capable of continual expression of therapeutic genes. Further, auto- or paracrine signaling could support the viability of the transplanted cells and/or their proliferation extending the survival of transduced hUCB-MCs in rat spinal cord.

Transplantation of hUCB-MCs transduced with adenoviral vectors expressing VEGF and GDNF at the site of SCI induced behavioral recovery that correlated with tissue sparing. The efficiency of hUCB-MCs + Ad5-VEGF + Ad5-GDNF transplantation into the area of SCI was higher than transplanted hUCB-MCs transduced with adenoviral vectors expressing a non-therapeutic gene (enhanced green fluorescent protein, EGFP) for both pathological cavitation and gray/white matter sparing. These results can be explained by the action of therapeutic gene products in targeting cells in the damaged tissue. An increase in the number of regenerating fibers in the white matter of the spinal cord as a result of gene therapy (unpublished data) could result from the direct effect of neurotrophic factors produced by the grafted cells on oligodendrocytes and remyelination. Amplification of myelinated fiber regeneration may also be a consequence of reducing the volume of cavities and increased white matter sparing.

We have demonstrated that transplantation of hUCB-MCs transduced with adenoviral vectors expressing VEGF and GDNF genes into the site of SCI reduce glial scar formation and induce prominent axonal sparing/regeneration in comparison to the other constructs tested. Thirty days after SCI and transplantation of genetically modified VEGF and GDNF genes in hUCB-MCs, it was observed that growth associated protein 43 positive (GAP43+) fibers in the lesion zone had no detectable glial fibrillary acidic protein (GFAP) and grew through small and medium-sized cystic cavities (Figure , ’) While in the group with transplanted hUCB-MCs transduced with adenoviral vectors expressing a non-therapeutic gene (hUCB-MCs + Ad5-EGFP), GAP43 expression was located in the islet of lesion zone and also distal to the lesion zone (Figure , ”). Astroglial scars are known to inhibit the regeneration of nerve fibers. We hypothesize that decreased synthesis of chondroitin sulphate proteoglycans due to reductions in glial scar tissue contributed to sprouting nerve fibers after transplantation of the genetically modified hUCB-MCs.

Figure 2

Expression of GFAP and GAP43 in the lesion site of spinal cord in experimental groups.

Panoramic view of glial scar formation at the lesion site using GFAP (red) in experimental groups with transplantation of hUCB-MCs transduced with adenoviral vectors expressing VEGF and GDNF genes after SCI (A, A’) and transplantation of hUCB-MCs transduced with adenoviral vector expressing EGFP (B, B’’). The dashed boxes indicate enlarged areas of A’ and B’’. (A’) GAP43+ fibers (green) grew through small and medium-sized cystic cavities. (B’’) GAP43 expression was located in the islet of lesion zone, surrounded by glial scar (GFAP). Nuclei are stained with DAPI (blue). Scale bars: 750 µm (A), 500 µm (B) and 20 µm (A’, B’’). Figure 2A, B from Mukhamedshina et al., 2016. hUCB-MCs: Human umbilical cord blood mononuclear cells; GFAP: glial fibrillary acidic protein; GAP43: growth associated protein 43; SCI: spinal cord injury; VEGF: vascular endothelial growth factor; GDNF: glial-derived neurotrophic factor.

Thus, the transplantation of hUCB-MCs transduced with adenoviral vectors expressing VEGF and GDNF genes after SCI supports functional tissue plasticity and its ability to regenerate. This approach permits the delivery into the damaged area of neurotrophic and angiogenic factors, which reduce the severity of retrograde axonal degeneration, support tissue reconstruction including remyelination, and elongation and collateral branching of axons to form synapses.

The study was supported by grants 15-04-07527 (AAR) and 16-34-60101 (YOM) from Russian Foundation for Basic Research. YOM was supported by a Presidential Grant for government support of young scientists (PhD) from the Russian Federation (MK-4020.2015.7). This work was performed in accordance with Program of Competitive Growth of Kazan Federal University and a subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities.