Investigação do potencial terapêutico e análise patentária de linhagens de células-tronco mesenquimais e suas vesículas extracelulares para a dor neuropática

Abstract

Neuropathic pain is a chronic pain syndrome that affects a significant proportion of the population and has devastating effects on patients' quality of life and work activity. Although there are therapeutic options, most patients benefit little or do not respond to treatment. Therefore, regenerative therapies with mesenchymal stem cells and their derivatives may help to fill this therapeutic gap. AIM: This study aimed to evaluate the therapeutic potential of a human mesenchymal stem cell line (MSC), an MSC-derived line overexpressing the leukemia inhibitory factor (MSC-LIF) and their extracellular vesicles (VE-MSC and VE-LIF) in a model of neuropathic pain. It also aimed to explore the mechanisms underlying MSC’s and VE-MSC’s antinociceptive effects. Additionally, we aimed to perform a patent mapping focusing on MSC’s derivatives. METHODS: MSCs obtained from a biobank were genetically modified to overexpress LIF and characterized by flow cytometry and in vitro cell differentiation assays. The extracellular vesicles were isolated by ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, and Dotblot. Cytokines released by macrophages stimulated in vitro and treated with MSC, EV-MSC, MSC-LIF, or EV-LIF were quantified by ELISA. Using the model of neuropathic pain induced by partial ligation of the sciatic nerve in C57Bl/6 mice, the antinociceptive effect of all treatments was evaluated by Hargreaves and von Frey filaments tests. Gait function was assessed on a treadmill. Knockout mice for interleukin-10 (IL-10 KO) and pharmacological antagonism assays with a selective CXCR2 antagonist (SB225002; 1 mg/kg, i.p.) were used to investigate the mechanisms of action of MSC and EV-MSC. Sections of the spinal cord (L4-L5) and serum from mice were collected for gene expression analysis by RT-qPCR, cytokine and chemokine analysis by ELISA. Additionally, the DWPI database was used to retrieve the patents. RESULTS: Macrophages treated with MSC, EV-MSC, MSC-LIF, or EV-LIF showed reduced levels of TNFα, while IL-10 production was increased, indicating the possible application in sensory neuropathy. A single injection of MSC (1x106), EV-MSC (7.45x109 ± 2.25 x108 particles/mL), MSC-LIF (1x106) and EV-LIF (2.47 x109 ± 7.13 x107 particles/mL) provided complete and long-lasting relief of thermal hypernociception and improvement of gait functional parameters. Treatment with MSC and VE-MSC also completely relieved the mechanical hypernociception associated with neuropathy. Conversely, treatment with MSC-LIF and EV-LIF transiently and partially reduced the mechanical nociceptive behaviors of neuropathic mice. The participation of IL-10 in the therapeutical effect of MSC and EV-MSC was demonstrated since their antinociceptive effect was only partial in IL-10 KO mice compared to wild animals. In addition, IL-10 levels were elevated in the spinal cords of mice 14 days after treatment with MSC and EV-MSC but not at the end of the experimental period, or after treatment with MSC-LIF or VE-LIF. Preproenkephalin gene expression was regulated by MSC and VE-MSC. In addition, acute treatment with SB225002 partially reversed the thermal antinociceptive effect of MSC and EV-MSC, while this antagonist completely reversed mechanical antinociception. However, serum levels of CXCL1, a CXCR2 ligand, did not differ between the experimental groups 14 days after treatment with MSC or EV-MSC and were elevated only in neuropathic animals at the end of treatment. Regarding the patent search, 150 families of patents protecting mesenchymal stem cell’s derivatives were found, most of which were dedicated to the protection of extracellular vesicles and exosomes. There was an exponential increase in these patents from 2015 onwards. China was the country with the highest number of approved patents, followed by the United States. CONCLUSION: The effect profile of stem cells and extracellular vesicles was quite similar, showing that extracellular vesicles can mediate the effect of mesenchymal stem cells, even those from genetically modified cells. In addition, the improvement of both nociceptive and functional parameters by treatment with MSC and EV-MSC reinforces the proposal for future application of these therapies for the treatment of neuropathic pain. Although the genetic modification of MSC via overexpression of LIF has been deleterious to the antinociceptive effect of MSC, this work has advanced the understanding of MSC’s mechanism of action in neuropathic pain, which seems to involve increased production of the anti-inflammatory cytokine IL-10 and to be maintained by the activation of CXCR2 receptors. Finally, the patent analysis also indicated a growing interest in the research and development of cell-free therapy products. Thus, this work corroborates the potential of cell-free therapy with MSC extracellular vesicles and its application in neuropathic pain.