"Introduction: Spinal cord injury (SCI) is a condition that hampers the communication between the brain and the body, resulting in several comorbidities that decrease the patient’s life quality. The limited regeneration after SCI is mainly attributed to the injury complexity composed of several interconnected mechanisms. Although reestablishment of lost nerve tracts is essential for functional recovery, it is also important to restore the destroyed blood vessels. Adipose-derived stem cells (ASCs) have been addressed as therapeutic agents do their ability to modulate several repair processes, among neuronal and vascular growth, both at a paracrine and non-paracrine level. However, until now, ASCs did not elicit a total satisfactory repair. To enhance the regenerative features of ASCs, several approaches are being explored. Inflammatory pre-conditioning has shown to augment the anti-inflammatory properties of these cells, but with few reports showing the effects at a neuronal and vascular level.
Therefore, we aimed to understand the impact of pro-inflammatory (LPS+IFN-γ) and pro-regenerative molecules (IL-10) on the neuroregenerative and angiogenic potential of ASCs at three levels: indirect contact, secretome and direct contact.
Methodologies: The neuroregenerative potential was evaluated by analysis of the neurites produced by rat dorsal root ganglia (DRG) explants. The angiogenic potential was assessed by plating human umbilical vein endothelial cells (HUVECs) on a matrigel matrix, with further morphological analysis of the formed vessels. Each assay was adapted to the type of cell communication studied. Gene analysis for several vascularization and axonal growth-related molecules was performed after ASCs stimulation.
Results: ASCs induced neurite growth in all DRG assays. In the secretome and direct contact assay, the control did not induce any growth. No differences between inflammatory stimulations were found regarding neurite area, length and arborization pattern, in any DRG assay. LPS+IFN-γ enhanced the vascular potential of ASCs secretome, forming more vessel-like structures with higher average length and interconnectivity. This effect was lost in the indirect co-culture, with no differences between stimulations at any level. IL-10 did not alter ASCs behavior. Inflammatory stimulation led to alterations in ASCs gene expression, but without a clear shifted phenotype.
Conclusion: Overall, this work showed that ASCs and their secretome can modulate neurite and vascular processes essential for successful CNS regenerative applications. Furthermore, pro-inflammatory stimulation enhanced secretome angiogenic properties, holding great potential to enhance ASCs therapeutic properties.
Funding: This work has been funded by grants from Prémios Santa Casa Neurociências ‐ Prize Melo e Castro for Spinal Cord Injury Research (MC-04/17); PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) FEDER, through the Competitiveness Internationalization Operational Programme (POCI), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the projects TUBITAK/0007/2014; PTDC/DTP-FTO/5109/2014; POCI-01-0145-FEDER-029206; POCI-01-0145-FEDER-031392; PTDC/MED-NEU/31417/2017; NORTE-01-0145-FEDER-029968; project UIDB/50026/2020 and UIDP/50026/2020 ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122); and by the projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the ERDF."