Experimental Neurology 354 (2022) 114069 Contents lists available at ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr New insights into peripheral nerve regeneration: The role of secretomes Estefania Contreras, Sara Bolívar, Xavier Navarro, Esther Udina * Institute of Neurosciences, Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Bellaterra, Spain A R T I C L E I N F O A B S T R A C T Keywords: Neurons of the peripheral nervous system retain the intrinsic capability of regenerate their axons after injury, by Nerve regeneration triggering a complex activation response. This genetic switch is dependent of signals from the injured axon. Secretome Schwann cells (SCs) in the distal stump of an injured nerve also play an active role in the local regulation of Exosome axonal programs, by using cell-to-cell contacts but also secreted signals, the so-called secretome. Secretome Schwann cell Macrophage contains all the proteins (cytokines, growth factors and others) secreted by the cell and includes extracellular Mesenchymal stromal cell vesicles. The released vesicles can transport signaling proteins and both coding and regulatory RNAs, thus facilitating multilevel communication. It is nowadays clear that secretome of SCs is fundamental to both orchestrate Wallerian degeneration and to sustain axonal regeneration. Therefore, the use of secretome has emerged as an alternative to cell therapy in the field of tissue regeneration. In fact, separate components of SC secretome have been extensively used in experimental models to enhance peripheral nerve regeneration after injury. However, the most used secretome in neural therapies has been the one derived from mesenchymal (MSC) or other derived stem cells. In fact, the effects of cell therapy with MSCs have been mainly associated with the secretion of bioactive molecules and extracellular vesicles, which constitute their secretome. In this review, we first describe the role of SC and macrophage secretomes on Wallerian degeneration and axonal regeneration after peripheral nerve injury. Then, we review the different works reported in the literature that have used secretomes of SCs or MSCs in the treatment of peripheral nerve injuries in experimental models, to highlight the use of secretomes as a promising cell-free therapeutic approach, that reduces some of the risks associated with the use of cells, such as tumor formation or rejection. In contrast to most neurons in the central nervous system, mature related with neural activity and neurotransmission, whereas up- neurons of the peripheral nervous system are able to regenerate after regulating some transcriptional factors related with growth and cyto­ axotomy. Peripheral neurons retain intrinsic growth capability after skeleton elements (He and Jin, 2016). Therefore, the neuron switches injury, triggering a complex activation response in both the soma and from a transmission state to a pro-regenerative state. Different tran­ the axon of the injured neuron. At the neuronal body, injury regulates a scription factors have been implicated in this switch, like c-Jun, Jun D, gene expression program, that mainly consist in downregulating genes ATF3, sox11 and STAT 3 (Allodi et al., 2012; Raivich and Makwana, Abbreviations: ATF3, Activating Transcription Factor 3; ASC, Adipose-derived MSCs; bFGF, Basic fibroblast growth factor; BMSC, Bone marrow derived MSC; BDNF, Brain derived neurotrophic factor; CNTF, Ciliary neurotrophic factor; CM, Conditioned media; DPSC, Dental pulp stem cells; DNA, Deoxyribonucleic acid; DRG, Dorsal root ganglia; EGF, Epidermal growth factor; ECM, Extracellular matrix; EV, Extracellular vesicles; GDNF, Glia derived neurotrophic factor; GAP43,, Growth associated protein 43; HGF, Hepatocite growht factor; SHED, Human exfoliated deciduous teeth; IGF-1, Insulin growth factor 1; IL-10, Interleukin-10; IL-1α, Interleukin-1α; IL-1β, Interleukin-1β; IL-6, Interleukin-6; JAKs, Janus kinases; LIF, Leukemia inhibitory factor; mTOR, Mammalian target of Rapamycin; MSCs, Mesenchymal stromal/stem cells; miRNAs, MicroRNAs; EV, Microvesicles; MCP-1, or chemokine C–C motif ligand 2, CCL2, Macrophage inflammatory protein 1αMIP-1α, Monocyte chemoattractant protein-1; NGF, Nerve growth factor; GTPase, Nucleotide guanosine triphosphate hydrolase; OECs, Olfactory ensheathing cells; PAP-III, Pancreatitis-associated protein III; PTEN, Phosphatase and tensin homolog; PLA2, Phospholipase 2; PDGF, Platelet-derived growth factor; PDGFα, Platelet- derived growth factor alpha; PTN, Pleiotrophin; qRT-PCR, Real-time quantitative reverse transcription- Polymerase chain reaction; RNAs, Ribonucleic acids; SCs, Schwann cells; sSiglec-9, Secreted ectodomain of sialic acid-binding Ig-like lectin-9; STATs, Signal transducer and activator of transcription proteins; SLN, Superior laryngeal nerve; TNF-α, Tumor necrosis factor α; TNFβ, Tumor necrosis factor β; VEGF, Vascular endothelial growth factor; VEGFα, VEGF alpha. * Corresponding author. E-mail address: esther.udina@uab.cat (E. Udina). https://doi.org/10.1016/j.expneurol.2022.114069 Received 1 September 2021; Received in revised form 5 February 2022; Accepted 3 April 2022 Available online 6 April 2022 0014-4886/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). E. Contreras et al. Experimental Neurology 354 (2022) 114069 2007). Activation of the JACK/STAT pathway through mammalian activating an injury-specific program that partially involves non-coding target of Rapamycin (mTOR) is also a key element to promote the ribonucleic acids (RNAs) and deoxyribonucleic acid (DNA) methylation intrinsic growth ability of neurons. In fact, central neurons do not (Arthur-Farraj et al., 2017). Activation of c-jun allows SC to become a activate mTOR after injury, leading to failure of regeneration, whereas cell specialized to support regeneration by increasing their proliferative forced activation of mTOR (Liu et al., 2010; Park et al., 2008) promotes capability and the expression of neurotrophic and chemotactic factors growth of corticospinal and retinal ganglion axons. (Arthur-Farraj et al., 2012). These factors recruit hematogenous mac­ The genetic switch at the neuronal body is dependent of signals from rophages that together with SCs (Catenaccio et al., 2017), contribute to the injured axon. At the lesion site, plasma membrane of the affected the phagocytose of myelin and axonal debris (Brück et al., 1996). axons is disrupted, leading to a massive calcium entrance and disorga­ Moreover, repair SCs have the ability to elongate (Gomez-Sanchez et al., nization of the cytoskeleton. This calcium input triggers signaling cas­ 2017) and align inside the endoneurial tubes, forming the bands of cades that spread to the cell body. If the injured neuron has the intrinsic Büngner, that will create a regenerative pathway for the growing axons. ability to regenerate, the tip of the injured axon will be rearranged to become a growth cone (He and Jin, 2016). Moreover, part of the 1. SC secretome intrinsic ability of neurons to grow is localized at the same axon, since local mRNA translation and protein synthesis at the axonal level is SCs in the distal stump of an injured nerve also play an active role in fundamental for a successful regeneration (Gumy et al., 2010; Terenzio the local regulation of axonal programs, including axonal extension and et al., 2018; Verma, 2005). In fact, mTOR, a key protein to enhance local proteins synthesis (Court et al., 2008; Court and Álvarez, 2005). mRNA translation and protein synthesis (Saxton and Sabatini, 2017), is The axon-glia interaction is mediated by cell-to-cell contacts but also by activated in injured axons. Activated mTOR would regulate both its own secreted signals, the so called secretome. Secretome contains all the translation as well as local translation of other factors, such as importin proteins (cytokines, growth factors, and other proteins) secreted by the β1 and STAT3 (Terenzio et al., 2018). These molecules retrogradely cell and includes extracellular vesicles (EV) (Fig. 1). It is nowadays clear signal injury and thus, contribute to the activation of the intrinsic that the secretome of SCs is fundamental to both orchestrate Wallerian growth program of neurons (Abe and Cavalli, 2008). degeneration and to sustain axonal regeneration. In fact, the decreased However, regeneration is not just a neuron-key event. At the distal ability of SCs to maintain the secretion of trophic factors during long level, a permissive milieu for axonal regeneration has to be created. periods of time can explain the failure of regeneration through chroni­ Therefore, the distal nerve stump undergoes Wallerian degeneration, a cally denervated nerves (Höke et al., 2002; Sulaiman and Gordon, process that leads to the lysis and elimination of axon and myelin debris. 2000). Schwann cells (SCs) are the glia of peripheral nerves and in the mature state can be myelinating (the ones that ensheath and myelinate a single 1.1. Cytokines and chemokines axon) or Remak SCs (the ones that ensheath a group of unmyelinated axons). During Wallerian degeneration, SCs switch to a repair pheno­ Denervated SCs overexpress several trophic factors and cytokines, type. The activation of the transcription factor c-jun triggers this con­ with a specific time course of expression for each factor (Boyd and version from a mature to a repair state (Arthur-Farraj et al., 2012), Gordon, 2003). These secreted factors have been implicated in neuronal Fig. 1. Effects of glial and macrophage secretomes in nerve regeneration. Schwann cells (SCs) and macrophages secrete growth factors, cytokines and extracellular vesicles that enhance axonal regeneration either directly or indirectly. Regenerating axons receive several neurotrophic factors (such as GDNF, IGF-1, or PTN), microvesicles and EV (containing several proteins, miRNA and mRNA) from SCs and macrophages. These signals, particularly those from the EV, induce an upre­ gulation of mTOR and a downregulation of regeneration inhibiting factors (PTEN, Strouty2, Rho GTPase), thus promoting axon regeneration and neuronal survival. Some factors from SCs and macrophages also contribute to regeneration indirectly by recruiting more hematogenous monocytes and activating SCs, which create a permissive environment to axonal regrowth. 2 E. Contreras et al. Experimental Neurology 354 (2022) 114069 survival, axonal growth, cell differentiation and axon remyelination injured nerve analyzed. Thus, GDNF is markedly increased in nerve (Chen et al., 2007) and also in the immune response of the distal nerve roots whereas insulin growth factor 1 (IGF-1) and vascular endothelial stump, contributing to recruitment of immune cells (Chen et al., 2015). growth factor (VEGF) expression is limited to peripheral nerves. Cytokines and chemokines secreted by SCs are fundamental for Denervated nerves containing motor axons expressed higher levels of macrophage chemoattraction and regulation that lead to myelin clear­ pleiotrophin (PTN) than the ones containing sensory axons, whereas ance in Wallerian degeneration. The most important ones are inter­ hepatocyte growth factor (HGF), BDNF and NGF were markedly upre­ leukin-1α (IL-1α), interleukin-1β (IL-1β), monocyte chemoattractant gulated in cutaneous nerves and dorsal roots (Brushart et al., 2013; Höke protein-1 (MCP-1, or chemokine C–C motif ligand 2, CCL2), macro­ et al., 2006). Although it is clear that SC derived trophic factors are key phage inflammatory protein 1α (MIP-1α), pancreatitis-associated pro­ molecules to promote axonal regeneration after nerve injury (see re­ tein III (PAP-III), leukemia inhibitory factor (LIF) and interleukin-6 (IL- views by (Allodi et al., 2012; Boyd and Gordon, 2003; Raivich and 6) (Perrin, 2005). The chemokines MCP-1 and MIP-1a probably Makwana, 2007; Terenghi, 1999), there are not consistent evidences in contribute to higher extent to recruitment of macrophages compared to the literature that related the different neurotrophic profiles of motor IL-1β, since blocking antibodies against the two chemokines have and sensory SCs with specificity of motor and sensory axon regeneration stronger impact on macrophage recruitment (Perrin, 2005). Denervated respectively (Bolívar et al., 2020). As an example, BDNF has been SCs upregulate pro-inflammatory molecules such as tumor necrosis extensively used to promote regeneration of motor axons (Boyd and factor α (TNF-α), Il-1α and β (Shamash et al., 2002) within hours. Besides Gordon, 2001; Vögelin et al., 2006). On the other hand, although initial its role in macrophage recruitment, Il-1 β contributes to SC proliferation studies pointed that PTN could enhance motor axons regeneration (Chu (Conti et al., 2002; Perrin, 2005). Some of these cytokines, such as IL-1β, et al., 2009) a recent study showed that this factor attracts sensory axons TNFα and MCP-1 also modulate the phagocytic ability of the macro­ when confronted to a combination of GDNF and BDNF, a mixture that phages in injured nerves (Perrin, 2005; Shamash et al., 2002), probably promotes motor regeneration in a Y tube repair model (Anand et al., by stimulating phospholipase 2 (PLA2). Breakdown of compact myelin is 2017). an early event in the degenerating nerve, and it is mediated by early PLA2 secretion by SCs prior to the entry of hematogenous macrophages 1.2.1. Other proteins to the nerve (Martini et al., 2008). SCs also secrete several molecules of the extracellular matrix (ECM), The cytokines LIF and IL-6 are also secreted by SCs and upregulated such as laminin, a major component of the basal lamina and an abundant within hours after nerve injury (Banner and Patterson, 1994; Kurek component of the ECM of peripheral nerves (Gonzalez-Perez et al., et al., 1996), suggesting that they are early factors of injury (Ito et al., 2013). Laminin is considered one of most important ECM molecules to 1998). LIF acts on macrophages but is also retrogradely transported by sustain axonal regeneration and its expression increases after nerve axons to promote the neuronal regenerative response (Curtis et al., injury (Wallquist et al., 2002). Laminin gives the regenerative promot­ 1994). In contrast, IL-6 secreted at the injury is not retrogradely trans­ ing capability to basal lamina scaffolds after nerve injury, and the use of ported (Kurek et al., 1996) and has a role in the local inflammatory anti-laminin antibodies dramatically reduces the ability of axons to response. Interestingly, mRNA of anti-inflammatory cytokines grow through basal lamina scaffolds (Wang et al., 1992). interleukin-10 (IL-10) and tumor necrosis factor β (TNFβ) are parallelly upregulated to those of pro-inflammatory cytokines, and probably 1.3. Extracellular vesicles contribute to modulation of pro-inflammatory changes in the injured nerve to guarantee a safe immune response (Perrin, 2005). EV are vesicles secreted by cells that contain cytosol sealed by a lipid bilayer membrane and allow cells to exert paracrine effects by inter­ 1.2. Neurotrophic factors changing information through their selected cargos (proteins, lipids, RNAs and DNAs). Some authors divide these EV, that are quite hetero­ Besides these cytokines and chemokines, denervated SCs secrete a geneous, into exosomes or microvesicles (MV) (Bruno et al., 2019). plethora of neurotrophic factors, that are key to promote axonal growth However, these terms have not a deep consensus in the literature, and (Boyd and Gordon, 2003). This ability is dependent on the creation of a some authors classify exosomes and MV on the basis of differential dynamic gradient. SCs secrete these factors while denervated, but when centrifugation (EV that sediment when centrifugation at 7000–10,000 they regain contact with the regenerating axons, the expression of ×g are exosomes, whereas when sedimenting at 10,000 g are micro­ neurotrophic factors and their receptors is suppressed, and thus, trophic vesicles). Others prefer a biogenetic definition, being MV larger and the factor secretion is limited to SCs located distally to the regenerative front ones originate from the plasma membrane itself, whereas exosomes are in the distal nerve. Expression of these factors follows a specific time smaller and originating from multi-vesicular bodies, that can fuse to course but for a limited period of time (Höke et al., 2002). Failure of lysosomes to be degraded. When fused with the plasma membrane, chronically denervated SCs to maintain sufficient levels of trophic fac­ exosomes can be extracellularly released. Finally, some authors use MV tors accounts for poor axonal regeneration from 2 months after dener­ or exosomes indistinctly, to refer to EV (Gould and Raposo, 2013). In vation (Sulaiman and Gordon, 2000). Interestingly, chronically fact, the exosome term is quite extended in the literature as a synonym of denervated SCs can be reactivated by administering the cytokine TGFβ EV. Therefore, in this work, we will use the most general term EV, (Sulaiman and Gordon, 2002), secreted by proliferating SCs and mac­ following the International Society for Extracellular Vesicles reco­ rophages during Wallerian degeneration. mendations (Witwer and Théry, 2019), independently of the term used The mRNA of the neurotrophin nerve growth factor (NGF) shows a by the authors of the cited studies. biphasic upregulation in the injured nerve, with peaks first at 6 h and In fact, both MV and exosomes mediate cell-to-cell communication later at 3 days, that last for 14 days (Heumann et al., 1987). The second (Raposo and Stoorvogel, 2013; Simons and Raposo, 2009). The released peak correlates with macrophage invasion in the degenerating nerve and vesicles can transport signaling proteins and both coding (mRNAs, that is dependent on IL-1β secreted by macrophages (Lindholm et al., 1987). can translate proteins) and regulatory (microRNAs -miRNAs- that can Upregulation of mRNA of brain derived neurotrophic factor (BDNF) and suppress protein production) RNAs, thus facilitating multilevel glia derived neurotrophic factor (GDNF) follow a slower course. There is communication (Rajendran et al., 2014) and modulating the phenotype a continuous slow increase of BDNF mRNA starting 3 days after injury of the target cells. that reaches a plateau at 21–28 days (Meyer et al., 1992). GDNF mRNA In the nerves, transfer of molecular cargos between glial cells and also shows a slow increase that peaks at 7 days and last for at least one neurons through EV has been described (Lopez-Verrilli et al., 2013; more week (Naveilhan et al., 1997). A distinctive profile of secreted Lopez-Verrilli and Court, 2012). In fact, some works have pointed that neurotropic factors by SCs has been proposed dependent on the type of EV from different sources can mediate regeneration by targeting the 3 E. Contreras et al. Experimental Neurology 354 (2022) 114069 mTOR pathway (Tang, 2018). As pointed above, the ability of this et al., 2001). At 4 days, an important infiltration of circulating mono­ pathway to locally enhance messenger RNA (mRNA) translation and cytes can be observed in the injured nerve (Mueller et al., 2003). These protein synthesis is a key event for axonal regeneration (Terenzio et al., monocytes, which will be differentiated into macrophages in the tissue, 2018). EV of SCs can be internalized by axons, and transfer poly­ are recruited from the blood by cytokines and chemokines secreted by ribosomes and genetic material to the axon after axonal damage and SCs (Perrin, 2005; Tofaris et al., 2002); see above). Some of these fac­ during regeneration (Court et al., 2008). These EV can act at a local tors, such as CCL2, TNF-α, IL-1α and IL-1β, can also be produced by level, by enhancing neurite growth in vitro and increasing axon growth macrophages (Kiguchi et al., 2013; Shamash et al., 2002), thus rein­ in vivo. The effects seem mediated by the ability of glial EV to suppress forcing the recruitment of more monocytes. Hematogenous macro­ the inhibitory activity of Rho GTPase on the growth cone (Lopez-Leal phages are the main cells contributing to remove myelin and axonal and Court, 2016; Lopez-Verrilli and Court, 2013). debris during Wallerian degeneration. The phagocytic ability of mac­ MicroRNAs are non-coding RNAs that impact on protein expression rophages is regulated by MCP-1 and IL-1β; neutralization of these mol­ at a post-transcriptional level and can regulate about 60% of mamma­ ecules leads to an important reduction of the number of phagocytic lian genes (Friedman et al., 2008). An important amount of these miR­ macrophages in the injured nerve (Perrin, 2005). Macrophages can also NAs has been detected in axons. Although miRNA can be transported secrete the anti-inflammatory cytokine IL-10 during Wallerian degen­ from the neuronal soma (Kosik, 2006), recent findings of the ability of eration (George et al., 2004) and thus it is proposed that they could EV to transfer non-coding RNA to other cells (Valadi et al., 2007) sup­ regulate themselves to switch off their pro-inflammatory responses port a direct role of SCs on local transfer of miRNA. In this sense, SC (Martini et al., 2008). modify their EV cargo when switching to a pro-regenerative phenotype On the other hand, macrophages produce a wide range of factors, after nerve injury, allowing these EV to promote neurite growth. This such as proteases and growth-promoting factors/cytokines, and stimu­ growth ability is dependent on the increased expression of miRNA-21 late ECM remodeling to promote peripheral nerve regeneration (Gaudet (López-Leal et al., 2020). Together with miRNA-21, miRNA-222 has et al., 2011). In fact, macrophage-conditioned medium improves neurite also been found in SC EV (Ching et al., 2018; López-Leal et al., 2020) outgrowth in vitro (Luk et al., 2003). One of the proposed candidates for and, by downregulating different growth inhibitors in axons, as PTEN the pro-regenerative effects of macrophage secretome is oncomodulin (López-Leal et al., 2020; Zhou et al., 2012) and Sprouty2 expression (Zigmond and Echevarria, 2019). In addition, macrophages also secrete (Strickland et al., 2011), both miRNAs enhance neurite growth of dorsal factors that affect SCs, as for example IL-1 (Gaudet et al., 2011). The root ganglia (DRG) neurons (Strickland et al., 2011; Zhou et al., 2012). hypoxic environment induced in the bridge of a cut injured nerve at­ Therefore, the interchange of miRNA through EV at the injury site can tracts macrophages, that secrete VEGF-A, polarize endothelial cells and mediate multidirectional transfer of information from the main cells mediate the migration of SCs through the bridge that unites the two implicated in the regenerative response after nerve injury. nerve stumps, facilitating axonal regeneration (Cattin et al., 2015). Interestingly, miRNAs are more abundant in the axons compared to Finally, macrophages can also contribute to axonal regeneration the neuronal soma (Natera-Naranjo et al., 2010). By locally regulating through release of EV (Qing et al., 2018). miR-223 contained in gene expression in the axon, miRNAs can affect axonal protein synthesis, macrophage derived EVs promotes SC migration and proliferation both local energy metabolism, and the modulation of axonal outgrowth and in vitro and in vivo, and their secretion of NGF and laminin in vitro branching (Kaplan et al., 2013). These miRNAs may have a compart­ (Zhan et al., 2015). A recent study has described that macrophage mentalized and differentiated action at the soma, axons and growth derived EV contain enzymes that produce reactive oxygen species. When cones (Iyer et al., 2014). It has been reported that nerve injury induces a endocytosed by axons, these enzymes are retrogradely transported to set of 22 miRNAs that coordinate SC differentiation and dedifferentia­ the soma, where they induce the inactivation of PTEN and the conse­ tion through combinatorial modulation of positive and negative gene quent enhancement of the intrinsic neuronal growth (Hervera et al., regulators in the acute phase injury (Adilakshmi et al., 2012). 2018). EV from SCs have also mRNA of proteins related with regeneration (Ching et al., 2018), such as Growth associated protein 43 (GAP43), a 3. Secretome: an emerging alternative to cell therapy to neural growth-associated protein related with growth cone guidance, promote axonal regeneration and Tau protein, that stabilizes microtubules. EV also contain two members of the Rho GTPase family, Rac1 and RhoA (Ching et al., 2018). Cell-free treatment is an emerging alternative to cell therapy in the Rac1 is a regulator of cytoskeletal dynamics and plays a critical role in field of tissue regeneration, and therefore, the use of the secretome axon growth and guidance (Hua et al., 2015), whereas RhoA is an in­ instead of the cell itself has been postulated as a promising therapeutical hibitor of regeneration, and local translation of this molecule mediates tool. Moreover, pre-conditioning the cells of interest can alter the growth cone collapse (Wu et al., 2005). composition of the secretome (Ferreira et al., 2018), thus becoming an interesting strategy to increase their pro-regenerative potential. Finally, 2. Secretome of macrophages EV can also be used as a therapeutic delivery vehicle (Ha et al., 2016), with the advantage of its lack of immunogenicity. Studies using secre­ During Wallerian degeneration, activation of SCs is accompanied by tomes to promote nerve regeneration are sumarized in Table 1 (in vivo) recruitment of immune cells into the lesion site, including neutrophils, and Table 2 (in vitro). macrophages and lymphocytes. Macrophages play a crucial role removing myelin debris and promoting SC activation, and also 3.1. Use of SC secretome in peripheral nerve injury contribute to the creation of a permissive environment for axonal regeneration in the distal stump, by releasing different pro-regenerative Separate components of the SC secretome have been widely used in factors, including cytokines and chemokines, growth factors and ECM experimental models to enhance peripheral nerve regeneration after molecules (Chen et al., 2015; Gaudet et al., 2011). Recent work also injury. In fact, addition of trophic factors, normally secreted by repair strengthens the important role of macrophage derived endosomes on SCs, to the injured nerve promotes neuronal growth. Pioneer work from Wallerian degeneration and axonal regeneration (Hervera et al., 2018). Lindsay demonstrated that NGF and BDNF applied on DRG cultures Peripheral nerves have resident macrophages, that can induce in­ promoted neurite growth, but were not needed for neuronal survival flammatory response in the acute phase by secreting different pro- (Lindsay, 1988). Plenty of works have posteriorly evaluated the effects inflammatory cytokines, like IL-13 and IL-1β (De Francesco-Lisowitz of trophic factors secreted by repair Schwann cells on neuronal growth et al., 2015). Resident macrophages proliferate and contribute to in vitro and in vivo (see reviews by (Allodi et al., 2012; Boyd and Gor­ phagocytosis of myelin debris already at 2 days post-injury (Mueller don, 2003; Raivich and Makwana, 2007; Terenghi, 1999). Initial works 4 E. Contreras et al. Experimental Neurology 354 (2022) 114069 Table 1 Table 1 (continued ) In vivo experimental studies evaluating therapeutical role of MSC and SC Reference Nerve model Therapeutical Outcome secretomes in nerve injury models. approach Reference Nerve model Therapeutical Outcome Kano et al., Facial nerve section Graft soaked with Increased number approach 2017 repaired with a SHED-CM of regenerating Rich et al., Sciatic nerve Silicone chamber Increased number collagen graft axons 1989 section fiiled with NGF of regenerating Improved myelinated axons functional recovery Hollowell Sciatic nerve Silicone chamber No increased Tsuruta Supra-laringeal Intravenous SHED- Improved recovery et al., 1990 transection (8 mm filled with NGF number of et al., 2018 nerve injury CM of swallow function gap) regenerating motor Increased density of and sensory axons regenerating axons through the chamber Derby et al., Sciatic nerve Silastic or Enhanced presence focused on the role of NGF on nerve regeneration. Addition of NGF in a 1993 transection (10–15 semipermeable of axons and non- silicone chamber used to repair a sciatic nerve section improved number mm gap) chambers filled neuronal cells in the of regenerating axons (Rich et al., 1989). The same approach used to with NGF chamber at early periods repair a nerve gap was (Derby et al., 1993) or was not (Hollowell et al., Increased 1990) effective in enhancing axon growth into the tubes. On the other regenerated hand, application of NGF after axotomy delays the onset of regeneration unmyelinated and (Gold, 1996), probably by attenuating the response of neurons to injury myelinated axons in (Mohiuddin et al., 1999). Local infusion of BDNF improved nerve the chamber at early time points regeneration in neural conduits (Vögelin et al., 2006), but not when Vögelin et al. Sciatic nerve Fascia tubes with Faster growth of applied acutely after cut and suture of the sciatic nerve (Boyd and 2006 transection (20 mm capsules to axons into the tube Gordon, 2001)). In contrast, the same authors described pro- gap) continuously Decreased regenerative effects of BDNF when applied in a chronically denervated release BDNF neuropathic pain Boyd and Sciatic nerve Continuous Increased number nerve, that usually has a poorer ability to sustain regeneration (Boyd and Gordon, section and delayed administration of of motor neurons Gordon, 2003). 2001 repair (chronically BDNF regenerating into Neural guides that delivery GDFN used to repair a nerve gap denervated) the distal stump enhanced regeneration (Fine et al., 2002). Similar to BDNF, GDNF No effect in acute applied to the proximal stump of chronically denervated nerves repair (fresh distal nerve) increased regeneration of motor neurons (Boyd and Gordon, 2003). On Fine et al., Sciatic nerve Artificial guide Increased the other hand, application of IGF-1 into a crush site improved func­ 2002 transection (15 mm delivering GDNF myelinated axons tional recovery (Emel et al., 2011), whereas slow release of LIF after a gap) into the conduit nerve section increased muscle mass and function (Tham et al., 1997). Increased number of motor and Trophic factors secreted by SC, either alone or combined, have also been sensory neurons used in experimental studies to selectively potentiate regeneration of regenerating motor and sensory axons in vitro (Allodi et al., 2011) and in vivo (Anand Boyd and Sciatic nerve Continuous Increased number et al., 2017; Lotfi et al., 2011). Gordon, section and delayed administration of of motor neurons Besides the amount of evidence of the potential role of trophic factors 2003 repair (chronically GDNF regenerating into denervated) the distal stump to enhance axonal regeneration, it is nowadays accepted that any Leong et al. Section and repair Slow release of LIF Improved muscle effective strategy to significantly improve functional recovery after 1999 of gastrocnemius mass and function nerve injury has to contemplate multiple neurotrophic and neurotropic nerve factors acting synergistically and in a defined sequence. Moreover, the Emel et al., Sciatic nerve crush IGF-1 on the crush Improved 2011 injury site functional recovery administration of these factors has to be limited in time and create a Lopez- Sciatic nerve crush Intraneural Increased axonal correct gradient to promote long distance regeneration. In fact, long Verrilli in rats injection of SC regeneration lasting expression of GDNF by transfecting SCs with viral vectors trap­ and Court, derived MV Improved ped regenerating axons in the site of application in a nerve transection 2013 functional recovery model (Tannemaat et al., 2008), thus limiting the extent of regeneration. Marconi Sciatic nerve crush Intravenous MSC Increased axon et al. 2012 in mice (ASC) sprouting In recent years, the discovery that SCs also secrete EV has pointed to Reduced new therapies to mimic the positive effects of SCs on axonal regenera­ inflammatory tion avoiding the limitations of cell therapy. In this line, it was shown response in the that SC EV enhanced neurite growth in vitro. The effect was specific, nerve since fibroblast EV did not have any effect on that study (Lopez-Verrilli Sun et al., Sciatic nerve Enclosed MSC in Improved 2019 transection (10 mm the conduit to favor functional recovery et al., 2013). In an in vivo model, daily injections of EV into the nerve gap) in rat, repaired release of their Increased distal to a crush injury produced two times longer regenerating axons with an engineered secretome myelination of and a positive response to the pinch test at a longer distance from the conduit regenerating axons crush site, indicating that EV greatly enhance the rate of regeneration Zhang et al., Sciatic nerve OEC plus Marginal 2020 transection (10 suspension of MV synergistic effect, after nerve injury (Lopez-Verrilli et al., 2013). This work demonstrated mm) in rat, repaired from MSC with improved the valuable cargo of SC EV for axonal regeneration and also pointed to with a chitosan tube motor and sensory its potential therapeutical role. recovery Nevertheless, cell therapy with SCs has some limitations in humans, Sugimura- Sciatic nerve SHED-CM Improved mainly related with the sacrifice of a healthy nerve to obtain autologous Wakayama transection (10 reinnervation of et al., 2015 mm) in rat, repaired target organs cells. Therefore, use of mesenchymal stromal cells (MSCs) has been an with a silicone tube alternative widely explored in the field to improve axonal regeneration and functional recovery (see below). Probably this is the reason that the use of MSC derived secretome has also received more attention (Fig. 2). 5 E. Contreras et al. Experimental Neurology 354 (2022) 114069 Table 2 In vitro studies evaluating the pro-regenerative effects of MSC and SC secretome on neurons. References In vitro model Therapeutical approach Outcome Proposed mechanisme Lindsay, 1988 DRG primary NGF and BDNF Enhanced neurite Factors not needed for neuronal survival cultures growth Lopez-Verrilli et al., 2013 DRG primary SC MV Enhanced neurite Intact proteins on the MV surface, as well as explants growth MVcontent Ching et al., 2018 NG108–15 neuron CM or concentrated of EV from ASC Enhanced neurite Proregenerative mRNA and miRNA from MV culture differenciated towards SC-like growth Sugimura-Wakayama DRG primary SHED-CM Neuritogenesis NGF, BDNF, NT3, CNTF, GDNF, VEGF, HGF et al., 2015 cultures contained in the CM Fig. 2. Therapies based on mesenchymal stem cells (MSCs) to improve nerve regeneration. MSCs are relatively easy to isolate from bone marrow, adipose tissue, Wharton's jelly or dental pulp. These cells have been widely used as regenerative therapy for the damaged nervous system. A promising option to enhance axonal regeneration after severe nerve lesions that need surgical repair consists of using artificial nerve grafts filled with different factors to bridge the nerve stumps. The use of MSCs in the nerve conduits as well as the secretome of these cells (conditioned medium) has been proved to have beneficial effects in axonal regeneration after nerve injuries. In fact, a recent paper used conditioned medium from primary cultures after spinal root injury (Torres-Espín et al., 2013) and spinal cord injury of SCs, that contained EV, to promote neurite growth in vitro, and found (Ruzicka et al., 2017; Sykova et al., 2021; Torres-Espín et al., 2014). similar effect when using a conditioned medium from cultures of adi­ However, transplanted MSCs did not sustain these positive effects for pose stem cells that had been differentiated into a SC-like phenotype long periods of time due to their poor survival when grafted within the (Ching et al., 2018). neural tissue (Torres-Espín et al., 2014). Although in these studies cell transplants moderately improved neuronal survival and axonal regen­ 3.2. Use of MSC secretome in peripheral nerve injury eration, they presented disadvantages, such as the high number of cells needed for clinical application and the need of autologous source or MSCs are adult multipotent progenitor cells found in many organs alternatively immunosuppressive therapy (Torres-Espín et al., 2015). and tissue types. Due to their relative ease of isolation and expansion in Considering the reduced time that the cells survive within the host culture, MSCs have been used as a multi-purpose cell-based therapy, in tissue after transplantation, it is mostly considered that the therapeutic which transplanted MSCs would reach damaged tissues and help to effects of MSCs, as well as other derived stem cells, are mainly associated reduce neuroinflammation and even differentiate in specific cell types with the secretion of bioactive molecules and extracellular vesicles, (Wu et al., 2020). Over the last decades, substantial work has assessed which constitute their secretome (Wu et al., 2020). Thus, cells secretome the impact of MSCs as regenerative therapy on nervous system damage might be used as a cell-free therapeutic approach, avoiding risks, such as (Volkman and Offen, 2017). Thus, the intraspinal injection of a sus­ tumor formation or rejection, associated with the use of cells. The pro­ pension of MSCs promoted axonal regeneration and neuronal survival teomic analysis of MSC secretomes derived from different sources has 6 E. Contreras et al. Experimental Neurology 354 (2022) 114069 revealed the existence of several trophic factors, such as VEGF, IGF-1, biocompatible nerve conduit filled with a neurotropic matrix and seeded platelet-derived growth factor (PDGF) and HGF, as well as anti- with competent pro-regenerative cells has been pursued for decades, as a inflammatory cytokines (Shin et al., 2021). However, the secretome promising option to enhance nerve regeneration and become an alter­ composition is not definitively characterized and varies significantly native to the classical autologous nerve graft for repairing long gap in­ between cultures dependent on subject donor, original tissue source and juries. Mostly SCs but also MSCs have been used embedded in a hydrogel culture conditions used (Lv et al., 2020; Rizk et al., 2016). Moreover, filling a variety of conduits with significant success in enhancing nerve studies revealed the existence of MSC subpopulations that co-express regeneration and allowing regeneration over critical long gaps (for re­ neurotrophins and other neuro-regulatory molecules, which may views see (Deumens et al., 2010; Sarker et al., 2018). However, the need differentially contribute to MSC effects on neuronal survival and axonal of an autologous cell source (Dezawa et al., 2004; Rodrı́guez et al., 2000) regeneration (Crigler et al., 2006). Nevertheless, it has to be noted that or the administration of immunosuppression (Udina et al., 2004) have the composition of stem cells secretome is dependent on the microen­ been reported as needed for optimizing the effect of the cell graft to vironment in which the cells are placed, making it possible to precon­ reach the functional outcome of autologous graft repair. dition them to improve specific profiles most adequate to the injured Using the conduit-supported approach Sun et al. (2019) enclosed tissue (Ferreira et al., 2018). MSCs in the wall of conduits made of co-spun polycaprolactone and Marconi et al. (2012) found that an intravenous administration of gelatin methacrylate. This increased cell local preservation, for up to 4 adipose-derived MSCs (ASC) 1 week after sciatic nerve crush injury in weeks, while when injected within a matrix into the conduit lumen the mice, produced presence of a restricted number of undifferentiated ASC cell abundance started to decline after just 1 week. By spatially together with an increase of axonal sprouting and reduction of inflam­ restricting cell enclosure, the released secretome allowed a directional matory infiltrates in the injured nerves up to 3 weeks. They also showed chemokine gradient of endogenous SCs in nerve conduits. This resulted that ASCs produced in culture neuroprotective factors such IGF-1, BDNF in significantly enhanced functional recovery and axonal myelination or basic fibroblast growth factor (bFGF), that may contribute to pe­ compared with non-cell or lumen-cell conditions after repair of a 10 mm ripheral nerve regeneration. Moreover, ASCs escape immune system gap in the rat sciatic nerve. This study offers proof that controlled cell surveillance, because they possess cell surface antigens that are poorly seeding to produce neurotrophic gradients within an engineered nerve recognized by T cells; therefore, MSC can be transplanted as an auto­ conduit is a potential strategy for secretome application in peripheral graft, allograft, and even xenograft (Bai et al., 2009; Marconi et al., nerve regeneration. 2012). Zhang et al. (2020) added human umbilical cord MSCs-derived EV to Interestingly, application of conditioned media (CM) or concentrated olfactory ensheathing cells (OECs) in culture and observed that EV extracellular vesicles derived from adipose-derived stem cells differen­ promoted survival and migration of OECs in hypoxic conditions, and tiated towards a SC-like phenotype significantly enhanced in vitro effectively increased BDNF secretion. Using a 10 mm sciatic nerve defect neurite growth of NG108–15 neurons, similarly to media from primary rat model repaired with a chitosan-collagen conduit, they reported that SCs. qRT-PCR demonstrated that the obtained EV contained mRNAs and prefilling the conduit with an OECs plus EV suspension synergistically miRNAs known to play a role in nerve regeneration and these molecules promoted motor and sensory recovery of the injured sciatic nerve, were upregulated by the SC differentiation protocol (Ching et al., 2018). although differences between groups were marginal. However, when exploring the cargo of these EV, the authors found dif­ Sugimura-Wakayama et al. (2015) investigated the effects of CM ferences depending on their origin. Thus, EV from differentiated adipose derived from stem cells obtained from human exfoliated deciduous teeth cells expressed higher amount of GAP43 and Tau mRNA than SC EV, but (SHED). In vitro, SHED-CM stimulated neuritogenesis of DRG. In vivo lower amounts of Rac and RhoA mRNA. Moreover, EV of adipose stem they evaluated a 10-mm gap in the rat sciatic nerve repaired with sili­ cells and SCs contained similar levels of miR-21 and miR-222mi, cone conduits containing SHED-CM or control medium. The group with whereas R-18a and miR-182 expression was higher in differentiated SHED-CM showed improved reinnervation of target muscles and func­ adipose derived EV. Several studies have demonstrated that EVl miRNAs tional recovery, and higher number of regenerate axons after 12 weeks (including miR-199b, miR-218, miR-148a, miR-135b and miR-221) of nerve transection. The SHEDs medium contained NGF, BDNF, NT-3, isolated from MSC cultures can influence neuronal differentiation and CNTF, GDNF, VEGF and HGF that enhance peripheral nerve regenera­ axonal outgrowth (Qing et al., 2018). tion. On the other hand, the authors showed that factors secreted from Addition of CM obtained from cultured MSCs as well as from Neural SHED may also influence SCs proliferation and production of extracel­ Crest precursor cells increased viability of primary sensory neurons of lular matrix components. However, it should be considered that the the DRG after oxygen-glucose deprivation, and enhanced neurite growth effect of a fluid medium inside the conduit had likely a short time (Shi et al., 2019). MSC-CM also promoted survival and proliferation of persistence. In order to maintain a longer release, (Kano et al., 2017) cultured SCs, and increased the expression of NGF, BDNF and bFGF by implanted a collagen graft soaked with SHED-CM within the nerve gap the cultured SCs (Yang et al., 2009). These effects were related to the created by transection of the facial nerve in rats. The combination of contents of trophic factors in the CM, including epidermal growth factor anti-inflammatory M2 macrophage inducers, MCP-1 and secreted ecto­ (EGF), platelet-derived growth factor alpha (PDGFα), ciliary neuro­ domain of sialic acid-binding Ig-like lectin-9 (sSiglec-9) was found trophic factor (CNTF) and VEGF alpha (VEGFα). In addition, MSCs play essential for SHED-CM mediated functional recovery after facial nerve an immunomodulatory role regulating immune cells through direct injury. Notably, MCP-1/sSiglec-9 induced the polarization of M2 mac­ cellular contact and the release of cytokines (Brini et al., 2017). Indeed, rophages, which antagonized the pro-inflammatory M1 conditions MSCs obtained from adipose tissue and from bone marrow were found to associated with nerve injury, promoted proliferation and migration of express a similar profile of neurotrophic factors secretion (Hsiao et al., endogenous SCs, and enhanced extension of the peripheral nerve. On the 2012), and most of the trophic factors detected in those cells secretome other hand, (Tsuruta et al., 2018) established a novel model for superior are known to promote axonal regeneration from in vitro and in vivo laryngeal nerve (SLN) injury following trauma, that causes delay in the studies (Allodi et al., 2011). A comparative study of MSCs derived from onset of the swallowing reflex and gain of laryngeal residue in the human adult bone marrow, adipose tissue and Wharton's jelly, focusing pharynx. Systemic intravenous administration of 1 ml SHED-CM in rats on gene expression and secretome content and neurotrophic properties, after a SLN injury improved swallowing function and increased the revealed that, despite the differences in growth factor secretion, the MSC density of regenerated myelinated fibers, associated to M2 macrophage secretome derived from all cell sources had significant potential to polarization and neovascularization, suggesting that SHED-CM may stimulate neurite outgrowth of DRG neurons and to reduce oxidative cell provide therapeutic benefits for patients with such injury. The potential death (Petrenko et al., 2020). of stem cells from dental sources was pointed out by the comparative The development of artificial nerve grafts, composed of a study of the secretome composition of MSCs from dental apical papilla 7 E. Contreras et al. Experimental Neurology 354 (2022) 114069 (SCAPs) and from bone marrow; it was found that SCAPs secreted Adilakshmi, T., Sudol, I., Tapinos, N., 2012. Combinatorial action of miRNAs regulates transcriptional and post-transcriptional gene silencing following in vivo PNS injury. significantly larger amounts of chemokines and neurotrophins than PLoS One 7, e39674. https://doi.org/10.1371/journal.pone.0039674. bone marrow derived MSCs (BMSCs; (Yu et al., 2016). Allodi, I., Guzmán-Lenis, M.S., Hernàndez, J., Navarro, X., Udina, E., 2011. In vitro comparison of motor and sensory neuron outgrowth in a 3D collagen matrix. J. Neurosci. Methods 198, 53–61. https://doi.org/10.1016/j.jneumeth.2011.03.006. 3.3. Use of MSC secretome in neuropathic pain Allodi, I., Udina, E., Navarro, X., 2012. Specificity of peripheral nerve regeneration: interactions at the axon level. Prog. 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In contrast, gabapentin used as control treatment induced Greensmith, L., Behrens, A., Raivich, G., Mirsky, R., Jessen, K.R., 2012. C-Jun reprograms Schwann cells of injured nerves to generate a repair cell essential for only short-lasting antinociception. The effects could be related with regeneration. Neuron 75, 633–647. https://doi.org/10.1016/j.neuron.2012.06.021. changes in cytokine levels; IL-1β, TNF-α, and IL-6 were reduced, and IL- Arthur-Farraj, P.J., Morgan, C.C., Adamowicz, M., Gomez-Sanchez, J.A., Fazal, S.V., 10 was increased in nerve and spinal cord by treatment with CM and Beucher, A., Razzaghi, B., Mirsky, R., Jessen, K.R., Aitman, T.J., 2017. Changes in the coding and non-coding transcriptome and DNA methylome that define the MSCs (Gama et al., 2018). Interestingly, it has been reported that Schwann cell repair phenotype after nerve injury. 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