Abstract
Myelomeningocele (MMC) is the most severe form of spina bifida and occurs in nearly 1 out of 1,000 births worldwide. MMC is a birth defect caused by incomplete closure of the neural tube. Incomplete closure causes the inappropriately exposed cells of the developing spinal cord to be progressively damaged by the surrounding amniotic fluid. This damage leaves children born with MMC to develop varying levels of paralysis, loss of bladder or bowel control, and musculoskeletal deformities. In addition, children diagnosed with MMC are more likely to develop hydrocephalus, a condition that leads to severe cognitive dysfunction due to an excessive buildup of cerebral spinal fluid around the brain. The most effective standard of care for MMC spina bifida patients is in utero surgical repair of the malformed neural tube. The recent Management of Myelomeningocele Study (MOMS) validated for the first time in humans that in utero surgical repair of the MMC neural tube defect improves the outcome of lower limb motor function compared to post-natal surgery. However, 58% of these children were still unable to walk at 30 months of age. Recently, the use of stem cell therapy in combination with in utero surgery has been shown to be a promising approach in improving motor function recovery in the large animal model of MMC. Since abnormally high levels of neuronal cell apoptosis are involved in the pathogenesis of MMC, effective treatment approaches to protect neurons from apoptosis plays an important role in mitigating the disease outcome. Using the fetal ovine model of MMC, our lab has shown that the use of early gestation human placental mesenchymal stromal/stem cells (PMSCs) during in utero surgery significantly improves lower limb motor function after birth. We have confirmed that PMSCs secrete higher levels of brain-derived neurotrophic factor (BDNF) and hepatocyte growth factor (HGF), both of which contribute to neuroprotection. Like other mesenchymal stromal/stem cells (MSCs) derived from other tissue sources, the regenerative capabilities of PMSCs are largely attributed to a paracrine mechanism. Among the secretome of PMSCs are extracellular vesicles (EVs), which are nano-sized particles that play an important role in cell-to-cell communication. As such, EVs have been found to participate in wound healing, neurodegeneration and neuroprotection through their delivery of beneficial cargos including nucleic acids, metabolites, cytokines, and other proteins to sites of injury.
While MSC-derived EVs have been used in several clinical applications, one critical limitation to their therapeutic potential is untargeted delivery. A recent biodistribution study revealed that, following systemic injection of MSC-derived EVs, a majority accumulated in the liver and spleen, while very few were delivered to the central nervous system (CNS). Therefore, how to effectively deliver PMSC-derived EVs to the CNS, represents a significant challenge and unmet need in treating CNS diseases such as MMC. Recent studies have demonstrated enhanced delivery to the CNS after conjugating the CNS-targeting ligand, rabies virus glycoprotein (RVG) peptide, onto the surface of MSC-derived EVs. We hypothesized that surface modification of PMSC-EVs with CNS targeting peptides would enable effective CNS targeting of PMSC-EVs and decrease neuron apoptosis in the MMC defect. In this current project, for proof of concept, our primary goal was to create RVG-conjugated PMSC-EVs and compare their neuroprotective capabilities to unconjugated PMSC-EVs in vitro using a neuroprotection assay established in our laboratory.
Following the culturing and isolating of PMSC-EVs, this study began by first characterizing isolated PMSC-EVs through nanoparticle tracking analysis (NTA) in order to determine their concentration and average size. Our results verified that the size of the PMSC-EVs that were isolated from PMSC cultures fall within the reported EV size profile. To further verify the identity of our PMSC-EV isolates, Western blotting was performed to check for the presence of specific EV-markers. Western blot analysis revealed the presence of all probed EV-markers and the absence of the endoplasmic reticulum protein, calnexin, further confirming proper PMSC-EV isolation. We then needed to confirm that our isolated EVs were innately neuroprotective. To confirm their neuroprotective ability, we performed a neuroprotection assay. The results from this experiment showed that compared to the phosphate-buffered saline (PBS) control group, the addition of our isolated PMSC-EVs were able to preserve neurons after staurosporine-induced apoptosis. Next, the CNS-targeting peptide RVG, was conjugated onto the EV surface through the established chemical strategy of copper-free click chemistry using dibenzylcyclootyne (DBCO) linkage. Following the creation of CNS-targeting EVs, we characterized RVG-EVs using NTA. These results showed that after surface modification, the size of our engineered EVs decreased. Despite their smaller size, they remained within the normal EV size range. Lastly, to evaluate the neuroprotective function of RVG-EVs, subsequent neuroprotection assays were performed. The results of the neuroprotection experiments were inconclusive since the unconjugated EVs that served as the control group showed no significant neuroprotective effect as previously shown. These results are likely due to an insufficient amount of EVs used; therefore, additional experiments with the proper ratio of neuronal cells to EVs (1:2000) are necessary to appropriately investigate the neuroprotective potential of RVG-EVs. These experimental results will advance the current knowledge of CNS-targeting peptide function on EV engineering and their potential to enhance the neuroprotective effects of PMSC-EVs. This study provides a preliminary step for exploring ways to enhance sufficient delivery of MSC-EVs to the CNS to offer neuronal protection from MMC-related damage.