Research paper
Immunomodulatory and regenerative effects of the full and fractioned adipose tissue derived stem cells secretome in spinal cord injury

https://doi.org/10.1016/j.expneurol.2022.113989Get rights and content

Abstract

Adipose tissue derived stem cells (ASCs) are recognized to secret a myriad of molecules (secretome) know to modulate inflammatory response, promote axonal growth as well vascular remodeling and cellular survival. In previous works we have reported the benefit effects of ASCs transplanted to the injury site in a rat model of spinal cord injury (SCI). Emerging evidence have shown that the therapeutic actions of these cells are a consequence of their intense paracrine activity mediated by their secretome, which includes soluble bioactive molecules and vesicles. In this study, we intended to dissect the vesicular and protein individual function, comparing with whole secretome therapeutic effect. Therefore, we identified a beneficial effect of the whole secretome on neurite growth compared with protein or vesicular fraction alone and characterized their impact on microglia in vitro. Moreover, in a compression SCI mice model, from the motor tests performed, a statistical difference was found on beam balance test revealing differences in motor recovery between the use of the whole the secretome or their protein fraction. Finally, two different delivery methods, local or peripheral (IV), of ASC secretome were tested in vivo. Results indicate that when injected intravenously the secretome of ASCs has a beneficial effect on motor recovery of spinal cord injury animals compared with a single local injection and respective controls. Overall, our results showed that the whole secretome performed better than the fractions individually, raising ASC secretome mode of action as a synergy of proteic and vesicular fraction on SCI context. Also, when intravenously delivered, ASC secretome can promote SCI animal's motor recovery highlighting their therapeutic potential.

Introduction

Spinal Cord Injury (SCI) is a public health condition which affects more than 250,000 man, women and children every year. Studies point for higher incidences to be found in young individuals being the main causes of SCI traffic accidents, sport injuries and falls(Singh et al., 2014). Its consequence is devasting, as it leads to motor, sensory and autonomic complications and low perspectives of recovery.

SCI physical impact leads to immediate disruption of blood vessels, axons damage and neuronal death(Silva et al., 2014). Moreover, upon SCI an immune response is built, which often contributes to the lesion expansion related with the release of proteases, reactive oxygen species (ROS) and nitric oxide(Schnell et al., 1999; Stirling and Yong, 2008). Altogether, these events contribute to the exacerbation of the initial outcomes leading to the secondary injury which generate even more deleterious effect on the CNS(Silva et al., 2014). This secondary phase includes ischemia formation, release of ions (Na+, Ca2+) and glutamate excitotoxicity from senescent cells, altogether contributing to massive cellular death by activation of apoptotic pathways, worsening the pathology prognosis(Silva et al., 2014). In the chronic phase, it is formed a glial scar, which contain lesion debris, surrounding the lesion site. Ultimately, this extremely inhibitory environment prevents all possibilities of axonal regeneration(Silver and Miller, 2004).

To date there is no efficient therapy to SCI patients. A possible therapeutic solution may reside on the use of cell therapies, or their derivatives, in order to modulate that negative environment. Adipose Tissue derived Stem Cells (ASCs) have gained interest regarding their regenerative potential(Shammaa et al., 2020; Teixeira et al., 2013). Indeed, we have shown in rodent SCI models, that transplantation of ASCs could lead to benefit effects in both motor and histological parameters(Gomes et al., 2018a). Moreover, it was demonstrated in vivo and supported in vitro that the effects exerted by ASCs were mainly related with the intense paracrine activity of their secretome(Gomes et al., 2018a). However, cell transplantation for a condition like SCI still poses challenges. For instance, a relatively high numbers of o cells are required and cellular death often occurs 24 h after transplantation. The use of cell secretome as a therapeutic method can be presented as an alternative to this as it brings advantages when compared with cellular transplantation since it is easier to storage, have better shelf life and does not disclose some of the complications associated to cellular transplantation.

Under this scope the secretome of stem cells of different sources has shown to be able to participate in regenerative processes in different models of injury and degeneration. For instance it was previously reported that ASC secretome is a potent modulator of neural and glial cells survival and differentiation(Ribeiro et al., 2012a), as well as promoter of neurite growth(Assunção-Silva et al., 2018) and inductor of de novo vascularization(Hsiao et al., 2012). Indeed, ASC secretome mediates monocytes anti-inflammatory actions(Guillén et al., 2018a; Manferdini et al., 2017) (by reducing monocytes recruitment, phagocytosis and oxidative stress), which could be beneficial in a lesion microenvironment, where a uncontrolled inflammatory response may lead to deleterious effects. This data suggests that, ASC secretome has an intrinsic capability to modulate the lesion site in multiple parameters, providing neuroprotection, induce neurite growth, re-vascularization and immunomodulation, which may preserve the remaining cells besides regeneration(Hsiao et al., 2012; Kilroy et al., 2007; Niada et al., 2018; Pires et al., 2016). Moreover, some studies already show the effect of secretome from other mesenchymal stem cells in vivo(Cantinieaux et al., 2013a; Kanekiyo et al., 2018) and other authors show the effect of adipose stem cells secretome in vitro(Lopatina et al., 2011; Martins et al., 2017; Serra et al., 2018).

Although it is now widely accepted that the secretome of ASCs mediates most of their regenerative actions(Pinho et al., 2020), little is known on the influence of its respective proteic and vesicular components(Marote et al., 2016). In fact, to our knowledge is yet to be demonstrated whether any of these fraction disclose similar, improved or worst outcomes when used in SCI context. Indeed, each secretome fraction may have a different mechanism of action being protein fraction mainly through protein-receptor interaction wheres vesicular fractions needs internalization pathways (Baglio et al., 2012). Therefore, each protein and/or vesicular fraction can mediate different effect on injury repair.

Therefore, here we assess the therapeutic value not only of the ASC secretome but also of their individual proteic and vesicular fractions, in different in vitro and in vivo models. Having this in mind, we compare their neurite growth capacity and their immunomodulatory potential using dorsal root ganglia and microglia-enriched primary cultures, respectively. Also, we compare the therapeutic value of each ASC secretome individual fraction with the total ASC secretome on a SCI mice model. These studies revealed a predominant effect using ASC secretome as a whole and therefore we tested this approach in a compression mice model of SCI using two distinct administration methods (acute local injection and multiple intravenously injections). This final experiment revealed significant improvements at motor level in SCI mice which received IV injections, demonstrating the secretome therapeutic potential on SCI context.

Section snippets

Cell culture and secretome collection

Adipose Stem Cells (ASCs), were collected from human donors, according to the procedure described by Dubois et al.(Dubois et al., 2008) and kindle provided by Professor Jeffrey Gimble (LaCell LLC, New Orleans, Lousiana, USA). ASCs in passage 6 (P6) were seeded in complete α-MEM medium [Invitrogen, USA; supplemented with 10% Fetal Bovine Serum exosome-free (FBS, Biochrom AG, Germany) and 1% Penicilin-Streptomycin (P/S, Invitrogen USA)]. FBS Bovine Serum exosome-free was obtained from the

ASC secretome present vesicles with a large diameter range that are isolated by an ultracentrifugation protocol composing the vesicular fraction

Dynamic light scattering allowed to analyse the size distribution of vesicles present on the total ASC secretome but also on the different fractions (Fig. 1A).

The size distribution profile of ASCs secretome vesicles (in graphs represented as conditioned media – CM) ranged between 50 and 1200 nm (Fig. 1B), even after the 100× concentration protocol needed for the in vivo studies. After the ultracentrifugation protocol, the vesicular fraction was enriched on vesicles with 300 nm (Fig. 1C). The

Discussion

During the current century, several cellular therapies have been used to tackle the devastating conditions caused by SCI. The use of mesenchymal stem cells was particular intense during this time. However several data suggests that the benefits achieved after transplantation are rather obtained by the role of the paracrine factors than cellular differentiation or replacement(Gomes et al., 2018b, Gomes et al., 2020).

Along these lines, stem cells secretome has been recently proposed to be a cell

Conclusions

With this work using both in vitro and in vivo models, we present data that strongly suggests ASC secretome as an interesting therapeutic opportunity for SCI. We observed that total ASC secretome promoted a significantly higher value for neurite outgrowth and revealed a positive effect on activated microglial cells. Importantly, on spinal cord injury mouse models the administration of total ASC secretome reveals positive effect on motor recovery, namely on fine coordination tests, such as the

Funding statement

The authors want to acknowledge the financial support from Prémios Santa Casa Neurociências – Prize Melo e Castro for Spinal Cord Injury Research (MC-04/17). This work was funded by FEDER, through the Foundation for Science and Technology (FCT) and national funds, under the scope of the POCI-01-0145-FEDER-029206; PTDC/MED-NEU/31417/2017; POCI-01-0145-FEDER-016739; UIDB/50026/2020 and UIDP/50026/2020. CR thanks the FCT for the contract under the Stimulus of Scientific Employment (DL57/2016

Declaration of Competing Interest

There are no conflicts of interest.

Acknowledgments

Dr. Jeff Gimble and LaCell, Inc. for kindly providing the adipose tissue derived stem cells used in this study.

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    These authors contributed equally to this work.

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