Comparative Analysis of In Vitro Neuroprotection and Neuritogenesis on Axotomised Retinal Ganglion Cells by Small Extracellular Vesicles Derived from Various Mesenchymal Stem Cells

Comparative Analysis of In Vitro Neuroprotection and Neuritogenesis on Axotomised Retinal Ganglion Cells by Small Extracellular Vesicles Derived from Various Mesenchymal Stem Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comparative Analysis of In Vitro Neuroprotection and Neuritogenesis on Axotomised Retinal Ganglion Cells by Small Extracellular Vesicles Derived from Various Mesenchymal Stem Cells Matyas Kutnyanszky, Phil Stephens, Ben Mead This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8603346/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract The paracrine neuroprotective effects of Mesenchymal Stem Cells (MSC) are well known, and our understanding of the underlying mechanisms is expanding by the day. From among their diverse secretome, extracellular vesicles (EVs) have gained particular interest lately, owing to their good safety profile, stability, and relative ease of use as a cell-free therapy. The cargo and thus the potential effect of these nano-sized lipid membrane-enclosed vesicles is highly dependent on the type, age, and environment of the donor cells. Therefore, it is paramount to know which cell types are best utilised in any given situation. Glaucoma is a chronic progressive optic neuropathy that is the most common cause of irreversible blindness worldwide, characterised by the loss of Retinal Ganglion Cells (RGC) whose axons make up the optic nerve. Preservation of these neurons via the administration of the right EVs represents a promising approach for slowing down or halting disease progression, thereby preventing vision loss. Here, we evaluate the neuroprotective and neuritogenic potential of EVs isolated from five different cell types using a rodent in vitro model of RGC degeneration. Our findings showed that Adipose Mesenchymal Stem Cells (ADSC) release the most potent EVs, with Bone Marrow (BMSC) being a close second. EVs released by cells of the Umbilical Cord (UCSC), Dental Pulp (DPSC), Dermal Fibroblasts (DF), and an Oral Mucosal Lamina Propria-Progenitor cells (OMLP-PCs) did not have an observable benefit. Thus, our study provides greater insight into how the efficacy of different EVs compare to each other. Biological sciences/Cell biology Biological sciences/Neuroscience Biological sciences/Stem cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Glaucoma refers to a set of ocular diseases characterised by the gradual loss of Retinal Ganglion Cells (RGCs), the neurons responsible for relaying visual stimuli to the brain through their axons, which make up the optic nerve (1,2). The retina, on account of being an outgrowth of the central nervous system, possesses minimal regenerative capabilities (3). Thus, the loss of these neurons and the subsequent ablation in eye-to-brain communication lead to significant and permanent visual impairment. Indeed, Glaucoma is currently considered the leading cause of irreversible blindness worldwide (4,5). Mesenchymal Stem Cells (MSCs) are stromal cells capable of self-renewal and multilineage differentiation situated in mesenchymal tissues such as bone marrow (BMSCs), adipose tissue (ADSCs), umbilical cord (UCSCs), and dental pulp (DPSCs) (6,7). As interest in the field of stem cell-based therapies is rising, more and more attempts are being made to harvest the power of MSC for the treatment of various diseases (8–10). One way MSCs operate is by releasing various neurotrophic and neuroprotective factors in a paracrine fashion, thereby encouraging the survival and regeneration of neighbouring cells (11,12). Extracellular Vesicles (EVs) are part of this protective secretome, lipid-bound nano-sized particles our body utilises for cell-to-cell communication by transporting proteins, nucleic acids, and other biomolecules (13–16). Following their formation and loading, these vesicles are released into the intracellular space from where they can be taken up by other cells, altering their gene expression profiles and internal biochemistry (17,18). EVs are released from almost every cell in our body and can be easily isolated from biological fluids, or conditioned media in the case of in vitro cell cultures. Based on biogenesis, EVs can be further subdivided into Exosomes, Microvesicles, and Apoptotic Bodies (19,20). Due to the lack of specific markers, however, confirming the route of production is difficult; thus, vesicles are often separated based on their size instead. Exosomes, which appear to be mostly responsible for the beneficial paracrine effects of MSCs, occupy the 30-150 nm size range, while microvesicles are classified as 100-1000 nm in size. Apoptotic bodies tend to be even larger. A simple and commonly used approach for separating smaller vesicles from larger ones is filtration with a 220 nm cutoff point, creating a mixed population of microvesicles and exosomes, which from now on will be referred to as small Extracellular Vesicles (sEVs) (21–23). To date, several studies have been published utilising EVs sourced from different MSCs on various in vivo and in vitro models of ocular diseases (24). The most well-studied are BMSC-EVs, which have been shown to protect trabecular meshwork cells from oxidative stress (25) and encourage the growth of cortical neuron axons (26) in vitro . When injected intravitreally, these vesicles were able to reduce inflammation and oxidative stress in diabetic retinal injury models (27) and reduce RGC loss in various animal glaucoma models (13,14). Similarly, both ADSC-EVs and UCSC-EVs were successfully used to attenuate RGC death and retinal degeneration in animal models of glaucoma (28–30) or retinal neuropathy (31–36), with UCSC-EVs achieving similar success in vitro on human retinal vascular endothelial cells (HRECs) and rat retinal neurons cultured in hyperglycemic conditions (37,38). For the other cell types used here, although the use of DPSC-EVs in ocular conditions remains underexplored, some studies have already shown intravitreal injection of the stem cells themselves to be neuroprotective in rodent glaucoma models (39,40), and their EVs to improve gait in a rat Parkinson’s disease model (41). Unlike MSC-EVs, Fibroblast-derived vesicles have been shown not to be neuroprotective in a wide variety of retinal injury models, and were included to serve as a negative control (13,14,29). Finally, EVs from an immortalised Oral Mucosal Lamina Propria-Progenitor cells (OMLP-PCs; referred to as OMPCs from now on) were also included for their ability to encourage cell proliferation and wound repopulation in vitro (42). Although all of these cell types have demonstrated some degree of success, not all vesicles are created equal. Exosomes are selectively loaded, and cargo composition is influenced by several factors, including the type, age, and differentiation stage of the progenitor cells, as well as other internal and external stimuli (21,43,44). As a result, sEVs of different origins may elicit a varying degree of neuroprotective responses in recipient cells. Addressing this, the present study aimed to compare the neuroprotective efficacy of sEVs in vitro sourced from four commonly utilised MSCs (Adipose, Bone marrow, Umbilical cord, and Dental pulp mesenchymal stem cells), Dermal Fibroblasts, and from an Oral Progenitor Cell line. Results 3.1. Mesenchymal Stem Cells secrete small vesicles with Exosome surface markers All 6 cell types release small Extracellular Vesicles, visualised and measured using a ZetaView Nanoparticle Tracking Analyser and Time-resolved Fluorescence (TRF). Although concentrations varied, the size distribution was similar among vesicles originating from different cell types. Particle sizes ranged between 50-300 nm with modes of 130-150 nm for each sample (Figure 1B). There were a number of observable differences, mostly arising from hDF-sEVs preferentially clustering in the lower size brackets, however, this did not affect which brackets contained the highest and lowest number of particles. Either CD81 or CD9 were detectable in each population, although in different concentrations (Figure 1C). Notably, hDF-sEVs did not appear to possess CD81 markers. 3.2. sEVs released by hADSC and hBMSC improved overall retinal cell survival Treatment with either hADSC or hBMSC sEVs successfully increased the number of DAPI-stained cells observed in heterogeneous retinal cell cultures. The best results were achieved with 3e9 sEVs, in both cases elevating the number of surviving cells 3.93 +/- 0.24-fold (p: 0.0015) and 2.69 +/- 0.71-fold (p: 0.0466), respectively (Figure 3). At the higher dose of 1.5e10 particles per well, neuroprotection was still observable, albeit to a lesser extent. Vesicles sourced from hUCSC and hDPSC had no observable influence on retinal cell survival, while those originating from hOMPCs appeared to slightly decrease cell survival rates at all concentrations used. Notably, delivering hDF-sEVs at higher doses of 3e9 and 1.5e10 particles significantly decreased the number of surviving cells, by 0.59 +/- 0.02 fold (p:0.0402) and 0.4 +/- 0.13 fold (p: 0.0041) respectively. 3.3. sEVs released by hADSC and hBMSC improved RGC survival The number of RGC (identified by their morphology and βIII-tubulin positive staining) was noticeably higher in wells treated with hADSC or hBMSC sEVs. The best results were again achieved using 3e9 vesicles per well, where the number of RGC was 5.36 +/- 2.29 (p: 0.2928) times higher for hADSC-sEV treatment and 2.70 +/- 0.93 (p: 0.0965) times higher for hBMSC-sEV treatment compared to PBS-treated controls. hUCSC sEVs did not seem to elicit any RGC neuroprotection, while hDPSC sEVs trended towards an increased survival rate (1.39 +/- 0.28; p: 0.4108) when 1.4e10 vesicles were applied. Similarly to overall cell survival rates, hOMPC and hDF sEVs appeared to have a slightly and moderately detrimental effect, respectively, with the most noticeable being 1.5e10 hDF sEVs leading to a 0.48 +/- 0.19 fold change (p:0.0856). 3.4. sEVs released by hADSC and hBMSC improved RGC neurite length and number To measure the neuritogenic effect of sEVs, first, the number of neurites observed in each well was counted and normalised to per 100 RGC, then, the five longest neurites were measured and averaged (Figure 4.). Cell cultures treated with 1.5e9 or 1.5e10 hADSC sEVs showed a significant increase in neuritogenesis, reaching 9.92 +/- 4.14 (p:0.0405) and 11.93 +/- 1.135 (p: 0.0138) neurites per 100 RGCs respectively, compared to 0.79 +/- 0.79 in PBS treated controls. Although 3e9 sEVs were the optimal amount for overall cell or RGC survival, here they did not have a significant influence on neuritogenesis, reaching 6.84 +/- 1.88 (p:0.2059) neurites per 100 RGCs. Similarly, 1.5e9 hAMSC sEVs achieved the best results in the analysis of neurite length, reaching 103.9 +/- 23.9 (p: 0.0277) μm on average compared to 93.12 +/- 20.01 μm (p: 0.0508) for 3e9 particles, 75.6 +/- 13.7 μm (p: 0.1334) for 1.5e10 particles, and 8.94 +/- 8.94 μm for PBS. Akin to what the data showed for RGC survival rates, hBMSC sEV treatment failed to achieve significant results. However, administering 3e9 sEVs noticeably elevated the number of neurites per 100 RGCs to 14.00 +/- 4.94 (p: 0.0633) while the PBS control group had 0.64 neurites per 100 RGCs on average. A great increase in the mean neurite length was also observed for the same treatment group of 3e9 hBMSC sEVs, this time reaching the significance level (28.81 +/- 7.49; p: 0.0176) compared to the 2.35 μm average length in the control group. Although neither of the 4 remaining cell types showed significant influence on neuritogenesis, it is worth noting that hUCSC sEVs increased the number of neurites per 100 RGCs by around 1.8-fold and average length by around 1.2-fold when administered at concentrations of 1.4e9 or above. Overall, results suggest the optimal concentration of sEVS to be 3e9 for both hADSC and hBMSC Vesicles, while sEVs from the other cell types are unlikely to have a positive effect. Discussion In this study, we compared the neuroprotective and regenerative potential of the sEVs produced by four types of Mesenchymal Stem Cells, one Fibroblast and one Oral progenitor cell line in vitro . Among these, hADSC-sEVs were the most and hOMPC-sEVs the least effective at promoting RGC survival and neuritogenesis in mixed retinal cultures, with hBMSC-sEVs also showing a modest positive effect. Thus, sEVs originating from hADSCs and hBMSCs would be worth further investigating as a potential therapy for glaucomatous nerve damage. A common feature of all forms of Glaucoma is the damage suffered by RGCs, disrupting communication between the retina and the brain and thus eventually leading to blindness. There are numerous systems available for studying retinal neuroprotection in vitro (45). Among these, RGC cultures are a simple, effective and often-used method that offers a good balance between complexity and throughput (46). Although the structure of the retina and, therefore, a lot of context and potentially relevant cell-cell interactions are lost in this model, unlike retinal explants, RGC cultures allow for dividing the isolated cells into multiple treatment groups, reducing the number of animals required. The results shown here are in line with previous studies that showed ADSC-derived EVs to be protective of RGCs and other kinds of neurons. In vitro , these sEVs were previously demonstrated to reduce the percentage of apoptotic cells and increase the number of surviving RGCs in retinal cultures subjected to increased hydrostatic pressure (47). Similar to how their parental cells have done so in co-culture (48). In another experiment, filtered (220 nm cutoff) FBS-free conditioned media collected from mouse ADSCs successfully alleviated H2O2 or light-induced damage to 661W RGC precursor-like cell cultures (49,50). However, the researchers attributed the positive outcomes to the secreted immune protein Progranulin (PGRN) despite the inevitable presence of sEVs. Similarly, positive results have been reported in vivo by Ji and colleagues (30), who tested human ADSC-derived sEVs in a mouse model of glaucoma. Here the researchers showed improvements in RGC survival rates, function, and retinal thickness two weeks after intravitreal injection of sEVs. The group also found microglia-associated neuroinflammatory responses to be reduced by sEVs injection, adding another facet to their neuroprotective repertoire. In other retinal and Peripheral or Central Nervous System (PNS/CNS) conditions, the EVs of various ADSC (mouse, rabbit rat) were found to be neuroprotective in streptozotocin (STZ)-induced diabetic retinopathy (31,32) and spinal cord damage (51). Furthermore, in a rat sciatic nerve crush model, injection of sEVs immediately after damage reduced functional decline and improved recovery, albeit not as well as injections of the parental cells themselves (52). BMSCs and their sEVs are the most thoroughly investigated among the cells used in this current study. Given their popularity, many times their potential has been proven both in vitro and in vivo , although most of these studies focused on utilising MSCs and their entire secretomes, rather than the EVs they release. Indeed, multiple studies have achieved positive results involving human (39,53) or rodent (12,54,55) BMSC in various rodent glaucoma models. There have even been cases of hBMSCs used in clinical trials for the treatment of macular oedema, hereditary retinal dystrophy, and other degenerative retinal disorders (56–58). Among the studies focusing on EVs, the research group of Zheng et al . (47) showed BMSC-sEVs to protect RGCs from hydrostatic pressure-caused stress in vitro , although to a slightly lesser extent than ADSC-sEVs. In other cases, these vesicles were able to encourage the growth of cortical neuron axons (26) and save trabecular meshwork cells from H2O2-induced oxidative stress (25). In vivo , intravitreal injections of BMSC-EVs have been shown to protect RGC in various models of glaucoma, including microbead-, laser-, or pressure transducer-induced OHT (14,59), Optic nerve crush (ONC) (13), and DBA/2J genetic mice models (60). Similar to hADSC-sEVs, the vesicles produced by BMSCs were also able to reduce inflammation and protect retinal cells in STZ-induced diabetic retinopathy (27). Notably, the regenerative benefits of BMSC EVs are not limited to neurons or related cells, as they have also been shown to promote skeletal muscle regeneration (61), protect cardiac myocytes from apoptosis following an infarct (62), and even increase overall survival rates following experimentally induced sepsis in mice (63). As with their progenitor cells, BMSC-EVs are also being explored in the clinics. Multiple phase I and phase I/II clinical trials have been cited by two recent reviews (64,65), albeit very few explored ophthalmological diseases. Our findings have shown them to increase cell survival and promote neurite growth while having no measurable benefit on RGCs or the number of neurites they sprout. It should be noted, however, that their benefit on both of these metrics was comparable to that of the hADSC-sEVs, and the observed non-significant results could be explained by the low power and high internal variance of the results. Alternatively, these vesicles might elicit their beneficial effects by targeting the surrounding supporting cells rather than the RGCs themselves, which would, of course, be lost in less complex models, such as ours, while still showing in vivo . This might explain why our and Zheng’s group found ADSC-sEVs more potent than BMSC-sEVs in vitro, while most report the opposite in vivo. Nonetheless, our observations showed that hBMSC-sEVs could be beneficial in vitro if applied at the right concentration, which is in line with what has previously been reported in the literature. Although multiple studies have been conducted investigating MSCs of the Umbilical Cord and their secretomes, evaluating the results of these might be more difficult than those of the other cell types. Many different cell types can be found there, including but not limited to Cord blood, the endothelium of cord arteries or veins, and the connective Wharton’s Jelly (WJ) (66,67), each with slightly different surface marker expression and secretome profiles (68). Since different research groups might not refer to the same group of cells when talking about UCSCs, with pure WJ and a mix of every cell type being the two most common, special care must be taken when comparing results. With that said, our results deviate from what has been reported by others. Here, we found no significant benefits on cell survival rates or neurite length, and although the number of neurites per RGC-like cells nearly doubled on average, the results were calculated to be non-significant. As with BMSC-sEVs, this might be due to the low power and high internal variance of our results or may be explained by UCSC-sEVs acting primarily on supporting cells to encourage RGC survival indirectly. Although not models of Glaucoma, in vitro UCSC-sEVs have been shown to protect human retinal vascular endothelial cells (HRECs) (37) and rat retinal neurons (38) when cultured in hyperglycemic conditions to simulate diabetic retinopathy. Modelling similar conditions in vivo , these vesicles were able to reduce inflammation, prevent vascular damage and reduce retinal thickening in STZ-induced diabetic retinopathy in rats and mice (33–36). When applied to glaucomatous nerve damage, UCSC-EVs or the stem cells themselves were shown to reduce lesion areas and the number of TUNEL-positive cells in mice subjected to laser-induced retinal damage (29). In rat ONC models of glaucoma, these vesicles have been reported to be protective of RGCs, without promoting axon regeneration (28), the opposite of what we observed in vitro . However, yet another study found them to be rather ineffective at preserving RGCs, while also showing their parental cells to be not only protective but also to promote the regeneration of the crushed optic nerve axons (69). Both of these studies used very similar amounts of EVs and similar techniques to prepare tissue samples and to analyse their results, highlighting the complexity of UCSCs and their secretomes. The EVs of DPSCs, unlike the other MSCs discussed so far, remained relatively unexplored for nervous system or ophthalmological disorders, with one study finding nasal administration to improve gait in a rodent model of Parkinson’s disease (41). Their progenitor cells, on the other hand, are well known for their protective and regenerative abilities. In vitro , co-culturing human DPSCs with rat retinal isolates was shown to greatly enhance the survival rates of RGCs and to do so in a similar or greater degree than BMSCs or ADSCs, respectively (48). Our research group not only observed similar benefits in vitro with DPSCs of rat origin but also tested these cells in vivo in a rat optic nerve crush model and found them to preserve retinal thickness, reduce RGC death and promote axon regeneration 21 days after injury (40). In studies evaluating them in the context of CNS and PNS diseases, DPSC transplantation was shown to aid the regeneration of the transected sciatic nerve in rats (70,71). Furthermore, in spinal cord injury models, cells grafted to the injury site increased the survival of motor neurons and promoted functional recovery in rats (72,73). Despite the promising showing of DPSCs in the existing literature, in the current study, their EVs failed to improve survival rates or axon regeneration. One possible explanation for this discrepancy could lie in the complex secretome of these cells. The aforementioned studies comparing their neuroprotective efficacy with ADSCs and BMSCs found DPSCs to secrete a greater variety of neurotropic factors. In other words, their potential may be reliant on the proteins they release rather than the vesicles and their cargo. DF-derived EVs were included due to multiple studies showing them not to be neuroprotective in various glaucoma models, including laser-induced retinal injury (29), optic nerve crush (13), and elevated intraocular pressure (14). However, others found them to promote neurite growth in vitro, particularly via activating Wnt10b signalling (74). In our present study, Fibroblast sEVs performed the worst out of all. Administering these particles appeared to significantly decrease cell survival rates while having a more neutral effect on neurite growth. This may suggest that the introduction of DF-sEVs resulted in the suppression of pro-survival and/or the activation of pro-death pathways, although further experiments are required to confirm this hypothesis. Overall, our data support the ineffectiveness of these particles for treating nerve injuries. Finally, our interest in the EVs of OMPC arose from the research of Knight and colleagues showing these vesicles to be beneficial for wound healing and to do so more efficiently than hBMSC-sEVs (42). Although anti-scar-forming properties are less relevant in the context of retinal or neurological diseases, their ability to encourage cell proliferation was considered to be worth exploring. Unfortunately, not only were OMPC-sEVs observed to be ineffective in promoting neuritogenesis, but they also appeared to be slightly detrimental to cell survival. The simplest explanation for this is that these vesicles and their cargo probably do not target the biochemical circuits relevant to neuron survival and neurite outgrowth. Although the results shown here are promising, given the limitations of this experimental design, further investigation needs to be carried out. As mentioned above, the digestion of retinae eliminates some much-needed interactions between RGCs and their neighbouring supporting cells and removes RGCs from the context of an organised tissue. We also did not investigate how certain sEVs achieved their beneficial effects, and why some were better than others. EVs carry a wide variety of cargo molecules and surface proteins, all of which can affect the internal biochemistry of the recipient cell (17,18). Among these, miRNA garnered special interest, as interfering with their action has been shown to diminish the beneficial effects of EVs (13–16). Isolating and comparing the miRNA cargo of the EV presented here could lead to a better understanding of their mechanism of action. Furthermore, investigating the other components of MSC secretomes would provide better insight into why certain EVs do not share the neuroprotective capabilities of their progenitor cell. In conclusion, after comparing the neuroprotective and neuritogenic ability of sEVs from five different cell types in vitro , we found those isolated from hADSCs and hBMSCs to be the most effective. Moreover, concentrations of 1.5e9 - 3e9 vesicles per 125.000 cells tended to achieve the best results on both of these metrics. Thus, further investigations may be worth conducting, focusing on the in vivo efficacy of ADSC and BMSC sEVs, alongside the mechanism of action responsible for the observed benefits. Methods 5.1. Mesenchymal Stem Cell Cultures Human Mesenchymal Stem Cells from Bone Marrow (MSC-031, RoosterBio), Adipose tissue (C46001AD, RoosterBio), Umbilical Cord (V-hUCSC, Newmarket Scientific), or Dental Pulp (PT-5025, Lonza), Human Dermal Fibroblasts, adult (C0135C, Gibco), and human Oral Mucosal Progenitor Cells were seeded into T175 flasks (10127340; Thermo Fisher) at ~1.2e5 cells/cm2 and incubated at 37°C and 5% CO2. 35 ml Dulbecco's Modified Eagle Medium (DMEM; 61965059; Thermo Fisher) or alpha Minimum Essential Medium (αMEM; 22571038; Thermo Fisher) was provided, supplemented with 10% EV-depleted fetal bovine serum (FBS; 15434573; Thermo Fisher) and 1% penicillin/streptomycin. For hUCSCs, aMEM was further supplemented with Basic fibroblast growth factor (bFGF, 78003.1; STEMCELL Technologies). The medium was replaced every 2-3 days, and cells were passaged when over 80% confluent using 0.25% trypsin/ EDTA (11590626; Thermo Fisher). Except for the hOMPC cell line, cells were used at passages 2-5 post-acquisition. 5.2. Extracellular Vesicle Isolation and Storage sEVs were isolated using differential ultracentrifugation. Briefly: Culture medium was sequentially centrifuged at 300xg for 10 minutes, 2,000xg for 10 minutes and 10,000xg for 30 minutes, before being forced through a 0.22 μm pore size Millex®-OR Filter Unit (SLGP033RS; Sigma-Aldrich). The supernatant was collected, and pellets were discarded in each step. Filtered supernatant was spun at 100,000xg for 70 minutes. Pellets were collected, pooled, and washed in large amounts of sterile PBS before being spun down a second time at 100,000xg for 70 minutes. A constant temperature of 4°C was maintained for each spin. Pellets were collected in 1-2 ml sterile PBS and condensed down to less than 100 μl using Amicon™ Ultra-4 Centrifugal Filter Units (UFC8100; Millipore) equilibrated with Milli-Q water prior to use as advised by the manufacturer. Isolates were stored at -80°C. 5.3. Extracellular Vesicle Quantification and Quality Control For quantification, a ZetaView® Nanoparticle Tracking Analyser (Particle Metrix GmbH, Ammersee, Germany) was used. Particle size and concentration were measured at 11 positions over 1 cycle (Laser Wavelength: 488 nm; Filter Wavelength: Scatter; Sensitivity: 80.0; Shutter: 100). Time-resolved fluorescence TRF was used for further confirmation, targeting the known exosomal markers CD81 and CD9, as well as IgG for a negative control. Protein concentration of sEVs samples was measured using a Micro BCA™ Protein Assay Kit (23235; Thermo Scientific™) according to the manufacturer’s instructions. Equal amounts of protein (1μg/well) were transferred to a high-binding, flat-bottom 96-well plate (655097; Greiner), topped up to 300μl total volume with PBS, sealed, and incubated overnight at 4°C. The following day, the samples were blocked in 10% milk diluted in PBS + 0.1% Tween-20 (P1379; Sigma) for 2 hours, and incubated with 1μg/well 1ry (CD81: 10630D; Invitrogen, CD9: MAB1880; R&D Systems, IgG:14-4724-85; Thermo Fischer), and then 0.2μg/ml biotin-conjugated 2ry antibodies (31800; Invitrogen) for 1 hour (Buffer: Reagent Dilutent Concentrate 2 diluted 1:5 in distilled water, DY995; R&D Systems). This was followed by a further 45-minute incubation with Europium-labelled Streptavidin (1244-36; DELFIA) diluted to 0.1μg/ml in RED Buffer (42-02; Uniogen Oy) and another 10 minutes in a Fluorescence enhancer solution (1244-105; DELFIA). Samples were washed 3 times between each step and 6 times before the fluorescent enhancer was added (Wash Buffer: Reagent Dilutent Concentrate 2 diluted 1:100 in distilled water, DY995; R&D Systems). All incubation steps were carried out at room temperature with shaking while the plates were sealed. Fluorescence was measured using a microplate reader (FLUOstar Omega; BMG Labtech). 5.4. Animals Animals were kept and euthanised according to local guidelines. Ethical approval was obtained from the Cardiff University's Animal Welfare Ethical Review Body (AWERB). All protocols and methods were conducted in accordance with local guidelines and pre-approved standard operating procedures. Adult, female, Sprague Dawley Rats weighing 100-150 g were supplied by Charles River Laboratories. Constant surveillance was provided by trained staff under conditions of 21°C, 55% humidity and 12-12-hour light and dark cycle, with food/water given ad libitum . Euthanasia was carried out via rising concentrations of CO2, without prior anaesthesia. 100% CO2 gas was delivered at a rate of 10L/min for 4 mins and 14L/min for a further 4 mins. Death was then confirmed via cervical dislocation and harvested tissues were immediately processed. 5.5. Retinal Ganglion Cell Cultures Retinal cells were isolated using a Papain Dissociation kit (PDS/LK003150; Worthington Biochemical). Briefly: retinae were dissected, minced and incubated in a solution of Papain and DNase diluted in EBSS at 37°C for 90 minutes with constant motion. Digested tissue was then centrifuged for 6 minutes at 300xg, the supernatant was discarded, and the pellet was resuspended in a mixture of 1.35 ml EBSS, 150 μl Albumin Ovomucoid inhibitor, and 75ul DNase. This suspension was carefully transferred onto 2ml Albumin Ovomucoid inhibitor, creating a density gradient, and centrifuged at 70xg for 6 minutes. The supernatant was discarded and pelleted cells were resuspended in 1ml supplemented Neurobasal Plus medium (Neurobasal-A [A3582901; Thermo Fisher] 2% B27 supplement [17504044; Gibco], 0.5 mM L-glutamine [25030081; Thermo Fisher] and 50 mg/ml of gentamycin [15710064; Thermo Fisher]). 125,000 retinal cells were seeded in 300 μl Supplemented Neurobasal Plus medium in each of eight wells of Millicell® EZ Slides (PEZGS0816; Millipore) at ~1.8e5 cells/cm2 pre-coated with 100 mg/ml poly-D-lysine (A3890401; Thermo Fisher) for 60 minutes and 20 mg/ml laminin (10267092; Fisher Scientific Ltd) for 30 minutes. Cultures were immediately treated with sEVs resuspended in 50ul PBS or PBS alone (Final volume: 350ul per well) and incubated for 3 days under conditions of 37°C and 5% CO 2 . 5.6. Immunocytochemistry Retinal cell cultures were fixed in 4% Polyformaldehyde (PFA) for 10 minutes and blocked in 3% Bovine Serum Albumin and 0.1% Triton-X 100 (T9284-500ML; Sigma-Aldrich) solution for 20 minutes. Fixed cultures were then incubated for 1h at room temperature with either primary (anti-β-III-Tubulin, T8578-200UL; Merck; diluted 1:400) or secondary (Alexa Fluor® 488 Goat anti-mouse IgG, A32723; Invitrogen; diluted 1:500) antibodies in 3% Bovine Serum Albumin, 0.05% Tween 20 (P1379-500ml; Sigma) solutions and mounted in VECTASHIELD® Antifade Mounting Medium containing DAPI (H-1200-10; 2B Scientific). Samples were washed in PBS for 10 minutes, 3 times between each step, after the primary antibodies were added. Mounted slides were left to dry overnight, shielded from light and stored at 4°C. 5.7. Fluorescent Microscopy Samples were visualised using a Leica fluorescent microscope system (Leica DM6000 body, DFC350 FX monochrome Digital Camera, EL6000 External light source) at 600x magnification with excitation and emission wavelengths of 358/461 nm and 493/519 nm for DAPI and AlexaFluor488, respectively. DAPI-stained cells, RGCs, and neurites were counted manually. Here, RGCs were defined as elliptical cells where β-III-antibody forms a halo around the nucleus, with a pole of stronger clustering also observable. 5.8. Data Analysis Images were collected of 10-15 neurites per well (where possible) and their length was determined using the Fiji Neuroanatomy Plugin as described by Durmaz and colleagues (75). Data was analysed and visualised using GraphPad Prism 10 Software. Treatment groups were compared to PBS control using one-way ANOVA with Dunnett test. (* p > 0.05; ** p < 0.01; *** p < 0.005). Post-hoc Power calculation was carried out using R Studio’s pwr package ( pwr.anova.test(k = 5, f = Effect_size, sig.level = 0.05, n = 3 ). Effect size (Cohen’s f) was determined using a free online calculator (Effect Size Calculators; Partial eta-squared (Fixed effects); https://effect-size-calculator.herokuapp.com/#partial-eta-squared-fixed-effects ). Declarations Competing Interests Statement The Authors declare no conflict of interest. Author Contribution All experiments and statistical analyses were carried out by M. K. with the help and guidance of B. M. All drafts and the final version of the manuscript were written by M.K. and proofread / corrected by B. M. and P. S. Data Availability All data created and examined throughout this investigation are contained within this article. Upon request, the corresponding author will provide any datasets used or analyzed during the current work. References https://www.nhs.uk/conditions/glaucoma/ . nhs.uk. 2017 [cited 2023 July 2]. 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Ischemic Preconditioning Potentiates the Protective Effect of Stem Cells through Secretion of Exosomes by Targeting Mecp2 via miR-22. PLOS ONE . 9 (2), e88685 (2014). Wang, X. et al. Exosomal miR-223 Contributes to Mesenchymal Stem Cell-Elicited Cardioprotection in Polymicrobial Sepsis. Sci. Rep. 8 (1), 13721 (2015 Sept). Kou, M. et al. Mesenchymal stem cell-derived extracellular vesicles for immunomodulation and regeneration: a next generation therapeutic tool? Cell. Death Dis. 2022 July 4 ;13(7):1–16 . Sanz-Ros, J. et al. Extracellular Vesicles as Therapeutic Resources in the Clinical Environment. Int. J. Mol. Sci. 24 (3), 2344 (2023). Li, L. et al. Human umbilical cord mesenchymal stem cells in diabetes mellitus and its complications: applications and research advances. Int J Med Sci. Sept 11;20(11):1492–507. (2023). Davies, J. E., Walker, J. T. & Keating, A. Concise Review: Wharton’s Jelly: The Rich, but Enigmatic, Source of Mesenchymal Stromal Cells. Stem Cells Transl Med. 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Dental Pulp Cells Produce Neurotrophic Factors, Interact with Trigeminal Neurons in Vitro , and Rescue Motoneurons after Spinal Cord Injury. Dev. Biol. 238 (1), 120–132 (2001). Tassew, N. G. et al. Exosomes Mediate Mobilization of Autocrine Wnt10b to Promote Axonal Regeneration in the Injured CNS. Cell. Rep. 5 (1), 99–111 (2017 July). Durmaz, E., Kutnyanszky, M. & Mead, B. Isolation and Culture of Primary Retinal Ganglion Cells from Rodent Retina. Methods Mol. Biol. Clifton NJ . 2708 , 1–10 (2023). Additional & Information. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8603346","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":606173156,"identity":"109a93bc-32f5-44bf-932f-b10463b8f894","order_by":0,"name":"Matyas Kutnyanszky","email":"","orcid":"","institution":"Cardiff University","correspondingAuthor":false,"prefix":"","firstName":"Matyas","middleName":"","lastName":"Kutnyanszky","suffix":""},{"id":606173157,"identity":"a1a4bee1-57a0-4082-aead-7bf3e3aa99aa","order_by":1,"name":"Phil Stephens","email":"","orcid":"","institution":"Cardiff University","correspondingAuthor":false,"prefix":"","firstName":"Phil","middleName":"","lastName":"Stephens","suffix":""},{"id":606173158,"identity":"1b147338-e35b-4941-a90a-7d33ad9c7a45","order_by":2,"name":"Ben Mead","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBACgwMMDNIfKkDMxAYIzcCGX4slSIvEGbCWxhlniNFiD9LC2wZiJjASp8XsePPD25Lz7OTN25MbGw4w2MkzSKQl4Ndy5pix9cdtyYZzzjwEaUk2bJBIO4Bfy40EM2nJbQcYZ0gktj/+wMCcwCCR3oBXi8H959+keeccsAdqAdlST4SWGzxm0rwNBxKhWg4DtRBwmMGZnGJriWPJyTN4QH4xOG7YxvMsAb+W48c33v5QY2c7gz39YcOBimp5fvY0A7xa0E0gGCujYBSMglEwCogBAMajTcb8Zm26AAAAAElFTkSuQmCC","orcid":"","institution":"Cardiff University","correspondingAuthor":true,"prefix":"","firstName":"Ben","middleName":"","lastName":"Mead","suffix":""}],"badges":[],"createdAt":"2026-01-14 15:38:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8603346/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8603346/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104797147,"identity":"65917ada-19d5-4ab8-84fd-5d3a24baf689","added_by":"auto","created_at":"2026-03-17 09:48:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":149536,"visible":true,"origin":"","legend":"\u003cp\u003eSmall Extracellular Vesicle (sEV) secretion by five different Mesenchymal Stem Cells. \u003cstrong\u003eA)\u003c/strong\u003e Representative images of ZetaView results showing the size distribution of sEVs. \u003cstrong\u003eB)\u003c/strong\u003e Size distribution of sEVs isolated from five different MSCs. Data was compiled from multiple isolations (~8). \u003cstrong\u003eC)\u003c/strong\u003e Time-resolved Fluorescence (TRF) of CD81, CD9, and IgG of sEVs samples. hADSC: human Adipose Stem Cell, hBMSC: Human Bone Marrow Stem Cell, hDPSC: human Dental Pulp Stem Cells, hUCSC: human Umbilical Cord Stem Cell, hDF: human Dermal Fibroblast, hOMPC: human Oral Mucosal Progenitor Cell.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8603346/v1/c69dea3e0b100934b7a8f1f2.png"},{"id":104808753,"identity":"20ae54cc-28e5-4cc9-8c07-2a32a55a6643","added_by":"auto","created_at":"2026-03-17 12:39:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":997911,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of rat heterogeneous retinal Cell cultures treated with 3e9 small extracellular vesicles (sEVs). Images are representative of the entire culture. Cells were stained against βIII-tubulin (green; retinal ganglion cells) and with DAPI (blue), n=3 with two technical repeats. hADSC: human Adipose Stem Cell, hBMSC: Human Bone Marrow Stem Cell, hDPSC: human Dental Pulp Stem Cells, hUCSC: human Umbilical Cord Stem Cell, hDF: human Dental Fibroblast hOMPC: human Oral Mucosal Progenitor Cell.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8603346/v1/1f26abf031990b9bfa8fa257.png"},{"id":104797150,"identity":"3c2bf77f-b64c-446d-bd43-c71f14f5a197","added_by":"auto","created_at":"2026-03-17 09:48:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":422360,"visible":true,"origin":"","legend":"\u003cp\u003eCell survival in heterogeneous retinal cell cultures treated with small extracellular vesicles (sEVs) from various cell types. Overall cell survival rates (top) and RGC survival rates (bottom) adjusted to PBS controls are shown. Treatment groups are labelled as the number of sEVs per 350 μl culture medium with error bars showing the standard error of the mean (n=3). Comparisons were carried out using ANOVA. hADSC: human Adipose Stem Cell, hBMSC: Human Bone Marrow Stem Cell, hDPSC: human Dental Pulp Stem Cells, hUCSC: human Umbilical Cord Stem Cell, hDF: human Dermal Fibroblasts, hOMPC: human Oral Mucosal Progenitor Cell, PBS: Phosphate Buffered Saline ** p\u0026lt;0.01; * p\u0026lt;0.05, ns non-significant.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8603346/v1/e84ab07127984717913b37ee.png"},{"id":104797149,"identity":"b72efa4d-11d2-40cc-963f-ca49c5a14542","added_by":"auto","created_at":"2026-03-17 09:48:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":415945,"visible":true,"origin":"","legend":"\u003cp\u003eNeuritogenesis in heterogeneous retinal cell cultures treated with small extracellular vesicles (sEVs) from various cell types. Presented here are the number of neurites observed in each well, normalised to 100 cells with RGC-like morphology (top), and the mean length of the 5 longest neurites displayed in μm (bottom). Treatment groups are labelled as the number of sEVs per 350 μl culture medium with error bars showing the standard error of the mean (n=3). Comparisons were carried out using ANOVA. hADSC: human Adipose Stem Cell, hBMSC: Human Bone Marrow Stem Cell, hDPSC: human Dental Pulp Stem Cells, hUCSC: human Umbilical Cord Stem Cell, hDF: human Dermal Fibroblasts, hOMPC: human Oral Mucosal Progenitor Cell, PBS: Phosphate Buffered Saline ** p\u0026lt;0.01; * p\u0026lt;0.05, ns non-significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8603346/v1/604ebce9e1c42c57c2041710.png"},{"id":104835471,"identity":"a3a62c85-2e6e-43c2-8a31-e488c4f97e5d","added_by":"auto","created_at":"2026-03-17 17:45:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2469655,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8603346/v1/b0024678-ad96-4e5b-9154-9a7dad4424c4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Analysis of In Vitro Neuroprotection and Neuritogenesis on Axotomised Retinal Ganglion Cells by Small Extracellular Vesicles Derived from Various Mesenchymal Stem Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlaucoma refers to a set of ocular diseases characterised by the gradual loss of Retinal Ganglion Cells (RGCs), the neurons responsible for relaying visual stimuli to the brain through their axons, which make up the optic nerve (1,2). The retina, on account of being an outgrowth of the central nervous system, possesses minimal regenerative capabilities (3). Thus, the loss of these neurons and the subsequent ablation in eye-to-brain communication lead to significant and permanent visual impairment. Indeed, Glaucoma is currently considered the leading cause of irreversible blindness worldwide (4,5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMesenchymal Stem Cells (MSCs) are stromal cells capable of self-renewal and multilineage differentiation situated in mesenchymal tissues such as bone marrow (BMSCs), adipose tissue (ADSCs), umbilical cord (UCSCs), and dental pulp (DPSCs) (6,7). As interest in the field of stem cell-based therapies is rising, more and more attempts are being made to harvest the power of MSC for the treatment of various diseases\u0026nbsp;(8–10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOne way MSCs operate is by releasing various neurotrophic and neuroprotective factors in a paracrine fashion, thereby encouraging the survival and regeneration of neighbouring cells (11,12). Extracellular Vesicles (EVs) are part of this protective secretome, lipid-bound nano-sized particles our body utilises for cell-to-cell communication by transporting proteins, nucleic acids, and other biomolecules\u0026nbsp;(13–16). Following their formation and loading, these vesicles are released into the intracellular space from where they can be taken up by other cells, altering their gene expression profiles and internal biochemistry\u0026nbsp;(17,18). EVs are released from almost every cell in our body and can be easily isolated from biological fluids, or conditioned media in the case of in vitro cell cultures. Based on biogenesis, EVs can be further subdivided into Exosomes, Microvesicles, and Apoptotic Bodies\u0026nbsp;(19,20). Due to the lack of specific markers, however, confirming the route of production is difficult; thus, vesicles are often separated based on their size instead. Exosomes, which appear to be mostly responsible for the beneficial paracrine effects of MSCs, occupy the 30-150 nm size range, while microvesicles are classified as 100-1000 nm in size. Apoptotic bodies tend to be even larger. A simple and commonly used approach for separating smaller vesicles from larger ones is filtration with a 220 nm cutoff point, creating a mixed population of microvesicles and exosomes, which from now on will be referred to as small Extracellular Vesicles (sEVs)\u0026nbsp;(21–23).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo date, several studies have been published utilising EVs sourced from different MSCs on various \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models of ocular diseases (24). The most well-studied are BMSC-EVs, which have been shown to protect trabecular meshwork cells from oxidative stress (25) and encourage the growth of cortical neuron axons (26) \u003cem\u003ein vitro\u003c/em\u003e. When injected intravitreally, these vesicles were able to reduce inflammation and oxidative stress in diabetic retinal injury models (27) and reduce RGC loss in various animal glaucoma models (13,14). Similarly, both ADSC-EVs and UCSC-EVs were successfully used to attenuate RGC death and retinal degeneration in animal models of glaucoma\u0026nbsp;(28–30)\u0026nbsp;or retinal neuropathy\u0026nbsp;(31–36), with UCSC-EVs achieving similar success in vitro on human retinal vascular endothelial cells (HRECs) and rat retinal neurons cultured in hyperglycemic conditions\u0026nbsp;(37,38).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the other cell types used here, although the use of DPSC-EVs in ocular conditions remains underexplored, some studies have already shown intravitreal injection of the stem cells themselves to be neuroprotective in rodent glaucoma models (39,40), and their EVs to improve gait in a rat Parkinson’s disease model (41). Unlike MSC-EVs, Fibroblast-derived vesicles have been shown not to be neuroprotective in a wide variety of retinal injury models, and were included to serve as a negative control (13,14,29).\u0026nbsp;Finally, EVs from an immortalised Oral Mucosal Lamina Propria-Progenitor cells (OMLP-PCs; referred to as OMPCs from now on) were also included for their ability to encourage cell proliferation and wound repopulation \u003cem\u003ein vitro\u003c/em\u003e (42).\u003c/p\u003e\n\u003cp\u003eAlthough all of these cell types have demonstrated some degree of success, not all vesicles are created equal. Exosomes are selectively loaded, and cargo composition is influenced by several factors, including the type, age, and differentiation stage of the progenitor cells, as well as other internal and external stimuli (21,43,44). As a result, sEVs of different origins may elicit a varying degree of neuroprotective responses in recipient cells. Addressing this, the present study aimed to compare the neuroprotective efficacy of sEVs \u003cem\u003ein vitro\u003c/em\u003e sourced from four commonly utilised MSCs (Adipose, Bone marrow, Umbilical cord, and Dental pulp mesenchymal stem cells), Dermal Fibroblasts, and from an Oral Progenitor Cell line. \u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. \u0026nbsp;Mesenchymal Stem Cells secrete small vesicles with Exosome surface markers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 6 cell types release small Extracellular Vesicles, visualised and measured using a ZetaView Nanoparticle Tracking Analyser and Time-resolved Fluorescence (TRF). Although concentrations varied, the size distribution was similar among vesicles originating from different cell types. Particle sizes ranged between 50-300 nm with modes of 130-150 nm for each sample (Figure 1B). There were a number of observable differences, mostly arising from hDF-sEVs preferentially clustering in the lower size brackets, however, this did not affect which brackets contained the highest and lowest number of particles. Either CD81 or CD9 were detectable in each population, although in different concentrations (Figure 1C). Notably, hDF-sEVs did not appear to possess CD81 markers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. \u0026nbsp;sEVs released by hADSC and hBMSC improved overall retinal cell survival\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreatment with either hADSC or hBMSC sEVs successfully increased the number of DAPI-stained cells observed in heterogeneous retinal cell cultures. The best results were achieved with 3e9 sEVs, in both cases elevating the number of surviving cells 3.93 +/- 0.24-fold (p: 0.0015) and 2.69 +/- 0.71-fold (p: 0.0466), respectively (Figure 3). At the higher dose of 1.5e10 particles per well, neuroprotection was still observable, albeit to a lesser extent. Vesicles sourced from hUCSC and hDPSC had no observable influence on retinal cell survival, while those originating from hOMPCs appeared to slightly decrease cell survival rates at all concentrations used. Notably, delivering hDF-sEVs at higher doses of 3e9 and 1.5e10 particles significantly decreased the number of surviving cells, by 0.59 +/- 0.02 fold (p:0.0402) and 0.4 +/- 0.13 fold (p: 0.0041) respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. \u0026nbsp;sEVs released by hADSC and hBMSC improved RGC survival\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe number of RGC (identified by their morphology and \u0026beta;III-tubulin positive staining) was noticeably higher in wells treated with hADSC or hBMSC sEVs. The best results were again achieved using 3e9 vesicles per well, where the number of RGC was 5.36 +/- 2.29 (p: 0.2928) times higher for hADSC-sEV treatment and 2.70 +/- 0.93 (p: 0.0965) times higher for hBMSC-sEV treatment compared to PBS-treated controls. hUCSC sEVs did not seem to elicit any RGC neuroprotection, while hDPSC sEVs trended towards an increased survival rate (1.39 +/- 0.28; p: 0.4108) when 1.4e10 vesicles were applied. Similarly to overall cell survival rates, hOMPC and hDF sEVs appeared to have a slightly and moderately detrimental effect, respectively, with the most noticeable being 1.5e10 hDF sEVs leading to a 0.48 +/- 0.19 fold change (p:0.0856).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. \u0026nbsp;sEVs released by hADSC and hBMSC improved RGC\u003c/strong\u003e \u003cstrong\u003eneurite length and number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo measure the neuritogenic effect of sEVs, first, the number of neurites observed in each well was counted and normalised to per 100 RGC, then, the five longest neurites were measured and averaged (Figure 4.). Cell cultures treated with 1.5e9 or 1.5e10 hADSC sEVs showed a significant increase in neuritogenesis, reaching 9.92 +/- 4.14 (p:0.0405) and 11.93 +/- 1.135 (p: 0.0138) neurites per 100 RGCs respectively, compared to 0.79 +/- 0.79 in PBS treated controls. Although 3e9 sEVs were the optimal amount for overall cell or RGC survival, here they did not have a significant influence on neuritogenesis, reaching 6.84 +/- 1.88 (p:0.2059) neurites per 100 RGCs. Similarly, 1.5e9 hAMSC sEVs achieved the best results in the analysis of neurite length, reaching 103.9 +/- 23.9 (p: 0.0277) \u0026mu;m on average compared to 93.12 +/- 20.01 \u0026mu;m (p: 0.0508) \u0026nbsp;for 3e9 particles, 75.6 +/- 13.7 \u0026mu;m (p: 0.1334) for 1.5e10 particles, and 8.94 +/- 8.94 \u0026mu;m for PBS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAkin to what the data showed for RGC survival rates, hBMSC sEV treatment failed to achieve significant results. However, administering 3e9 sEVs noticeably elevated the number of neurites per 100 RGCs to 14.00 +/- 4.94 (p: 0.0633) while the PBS control group had 0.64 neurites per 100 RGCs on average. A great increase in the mean neurite length was also observed for the same treatment group of 3e9 hBMSC sEVs, this time reaching the significance level (28.81 +/- 7.49; p: 0.0176) compared to the 2.35 \u0026mu;m average length in the control group.\u003c/p\u003e\n\u003cp\u003eAlthough neither of the 4 remaining cell types showed significant influence on neuritogenesis, it is worth noting that hUCSC sEVs increased the number of neurites per 100 RGCs by around 1.8-fold and average length by around 1.2-fold when administered at concentrations of 1.4e9 or above. Overall, results suggest the optimal concentration of sEVS to be 3e9 for both hADSC and hBMSC Vesicles, while sEVs from the other cell types are unlikely to have a positive effect.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we compared the neuroprotective and regenerative potential of the sEVs produced by four types of Mesenchymal Stem Cells, one Fibroblast and one Oral progenitor cell line \u003cem\u003ein vitro\u003c/em\u003e. Among these, hADSC-sEVs were the most and hOMPC-sEVs the least effective at promoting RGC survival and neuritogenesis in mixed retinal cultures, with hBMSC-sEVs also showing a modest positive effect. Thus, sEVs originating from hADSCs and hBMSCs would be worth further investigating as a potential therapy for glaucomatous nerve damage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA common feature of all forms of Glaucoma is the damage suffered by RGCs, disrupting communication between the retina and the brain and thus eventually leading to blindness. There are numerous systems available for studying retinal neuroprotection \u003cem\u003ein vitro\u003c/em\u003e (45). Among these, RGC cultures are a simple, effective and often-used method that offers a good balance between complexity and throughput (46). Although the structure of the retina and, therefore, a lot of context and potentially relevant cell-cell interactions are lost in this model, unlike retinal explants, RGC cultures allow for dividing the isolated cells into multiple treatment groups, reducing the number of animals required.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results shown here are in line with previous studies that showed ADSC-derived EVs to be protective of RGCs and other kinds of neurons. \u003cem\u003eIn vitro\u003c/em\u003e, these sEVs were previously demonstrated to reduce the percentage of apoptotic cells and increase the number of surviving RGCs in retinal cultures subjected to increased hydrostatic pressure (47). Similar to how their parental cells have done so in co-culture (48). In another experiment, filtered (220 nm cutoff) FBS-free conditioned media collected from mouse ADSCs successfully alleviated H2O2 or light-induced damage to 661W RGC precursor-like cell cultures (49,50). However, the researchers attributed the positive outcomes to the secreted immune protein Progranulin (PGRN) despite the inevitable presence of sEVs. Similarly, positive results have been reported \u003cem\u003ein vivo\u003c/em\u003e by Ji and colleagues (30), who tested human ADSC-derived sEVs in a mouse model of glaucoma. Here the researchers showed improvements in RGC survival rates, function, and retinal thickness two weeks after intravitreal injection of sEVs. The group also found microglia-associated neuroinflammatory responses to be reduced by sEVs injection, adding another facet to their neuroprotective repertoire. In other retinal and Peripheral or Central Nervous System (PNS/CNS) conditions, the EVs of various ADSC (mouse, rabbit rat) were found to be neuroprotective in streptozotocin (STZ)-induced diabetic retinopathy (31,32) and spinal cord damage (51). Furthermore, in a rat sciatic nerve crush model, injection of sEVs immediately after damage reduced functional decline and improved recovery, albeit not as well as injections of the parental cells themselves (52).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBMSCs and their sEVs are the most thoroughly investigated among the cells used in this current study. Given their popularity, many times their potential has been proven both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, although most of these studies focused on utilising MSCs and their entire secretomes, rather than the EVs they release. Indeed, multiple studies have achieved positive results involving human (39,53) or rodent (12,54,55) BMSC in various rodent glaucoma models. There have even been cases of hBMSCs used in clinical trials for the treatment of macular oedema, hereditary retinal dystrophy, and other degenerative retinal disorders\u0026nbsp;(56–58). Among the studies focusing on EVs, the research group of Zheng \u003cem\u003eet al\u003c/em\u003e.\u0026nbsp;(47)\u0026nbsp;showed BMSC-sEVs to protect RGCs from hydrostatic pressure-caused stress \u003cem\u003ein vitro\u003c/em\u003e, although to a slightly lesser extent than ADSC-sEVs. In other cases, these vesicles were able to encourage the growth of cortical neuron axons\u0026nbsp;(26)\u0026nbsp;and save trabecular meshwork cells from H2O2-induced oxidative stress\u0026nbsp;(25). \u003cem\u003eIn vivo\u003c/em\u003e, intravitreal injections of BMSC-EVs have been shown to protect RGC in various models of glaucoma, including microbead-, laser-, or pressure transducer-induced OHT\u0026nbsp;(14,59), Optic nerve crush (ONC)\u0026nbsp;(13), and DBA/2J genetic mice models\u0026nbsp;(60). Similar to hADSC-sEVs, the vesicles produced by BMSCs were also able to reduce inflammation and protect retinal cells in STZ-induced diabetic retinopathy\u0026nbsp;(27). Notably, the regenerative benefits of BMSC EVs are not limited to neurons or related cells, as they have also been shown to promote skeletal muscle regeneration\u0026nbsp;(61), protect cardiac myocytes from apoptosis following an infarct\u0026nbsp;(62), and even increase overall survival rates following experimentally induced sepsis in mice\u0026nbsp;(63). As with their progenitor cells, BMSC-EVs are also being explored in the clinics. Multiple phase I and phase I/II clinical trials have been cited by two recent reviews\u0026nbsp;(64,65), albeit very few explored ophthalmological diseases. Our findings have shown them to increase cell survival and promote neurite growth while having no measurable benefit on RGCs or the number of neurites they sprout. It should be noted, however, that their benefit on both of these metrics was comparable to that of the hADSC-sEVs, and the observed non-significant results could be explained by the low power and high internal variance of the results. Alternatively, these vesicles might elicit their beneficial effects by targeting the surrounding supporting cells rather than the RGCs themselves, which would, of course, be lost in less complex models, such as ours, while still showing \u003cem\u003ein vivo\u003c/em\u003e. This might explain why our and Zheng’s group found ADSC-sEVs more potent than BMSC-sEVs in vitro, while most report the opposite in vivo. Nonetheless, our observations showed that hBMSC-sEVs could be beneficial in vitro if applied at the right concentration, which is in line with what has previously been reported in the literature.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough multiple studies have been conducted investigating MSCs of the Umbilical Cord and their secretomes, evaluating the results of these might be more difficult than those of the other cell types. Many different cell types can be found there, including but not limited to Cord blood, the endothelium of cord arteries or veins, and the connective Wharton’s Jelly (WJ) (66,67), each with slightly different surface marker expression and secretome profiles (68). Since different research groups might not refer to the same group of cells when talking about UCSCs, with pure WJ and a mix of every cell type being the two most common, special care must be taken when comparing results. With that said, our results deviate from what has been reported by others. Here, we found no significant benefits on cell survival rates or neurite length, and although the number of neurites per RGC-like cells nearly doubled on average, the results were calculated to be non-significant. As with BMSC-sEVs, this might be due to the low power and high internal variance of our results or may be explained by UCSC-sEVs acting primarily on supporting cells to encourage RGC survival indirectly. Although not models of Glaucoma, \u003cem\u003ein vitro\u003c/em\u003e UCSC-sEVs have been shown to protect human retinal vascular endothelial cells (HRECs) (37) and rat retinal neurons (38) when cultured in hyperglycemic conditions to simulate diabetic retinopathy. Modelling similar conditions \u003cem\u003ein vivo\u003c/em\u003e, these vesicles were able to reduce inflammation, prevent vascular damage and reduce retinal thickening in STZ-induced diabetic retinopathy in rats and mice\u0026nbsp;(33–36). When applied to glaucomatous nerve damage, UCSC-EVs or the stem cells themselves were shown to reduce lesion areas and the number of TUNEL-positive cells in mice subjected to laser-induced retinal damage\u0026nbsp;(29). In rat ONC models of glaucoma, these vesicles have been reported to be protective of RGCs, without promoting axon regeneration\u0026nbsp;(28), the opposite of what we observed \u003cem\u003ein vitro\u003c/em\u003e. However, yet another study found them to be rather ineffective at preserving RGCs, while also showing their parental cells to be not only protective but also to promote the regeneration of the crushed optic nerve axons\u0026nbsp;(69). Both of these studies used very similar amounts of EVs and similar techniques to prepare tissue samples and to analyse their results, highlighting the complexity of UCSCs and their secretomes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe EVs of DPSCs, unlike the other MSCs discussed so far, remained relatively unexplored for nervous system or ophthalmological disorders, with one study finding nasal administration to improve gait in a rodent model of Parkinson’s disease (41). Their progenitor cells, on the other hand, are well known for their protective and regenerative abilities.\u003cem\u003e\u0026nbsp;In vitro\u003c/em\u003e, co-culturing human DPSCs with rat retinal isolates was shown to greatly enhance the survival rates of RGCs and to do so in a similar or greater degree than BMSCs or ADSCs, respectively (48). Our research group not only observed similar benefits \u003cem\u003ein vitro\u003c/em\u003e with DPSCs of rat origin but also tested these cells \u003cem\u003ein vivo\u003c/em\u003e in a rat optic nerve crush model and found them to preserve retinal thickness, reduce RGC death and promote axon regeneration 21 days after injury (40). In studies evaluating them in the context of CNS and PNS diseases, DPSC transplantation was shown to aid the regeneration of the transected sciatic nerve in rats (70,71). Furthermore, in spinal cord injury models, cells grafted to the injury site increased the survival of motor neurons and promoted functional recovery in rats (72,73). Despite the promising showing of DPSCs in the existing literature, in the current study, their EVs failed to improve survival rates or axon regeneration. One possible explanation for this discrepancy could lie in the complex secretome of these cells. The aforementioned studies comparing their neuroprotective efficacy with ADSCs and BMSCs found DPSCs to secrete a greater variety of neurotropic factors. In other words, their potential may be reliant on the proteins they release rather than the vesicles and their cargo.\u003c/p\u003e\n\u003cp\u003eDF-derived EVs were included due to multiple studies showing them not to be neuroprotective in various glaucoma models, including laser-induced retinal injury (29), optic nerve crush (13), and elevated intraocular pressure (14). However, others found them to promote neurite growth in vitro, particularly via activating Wnt10b signalling (74). In our present study, Fibroblast sEVs performed the worst out of all. Administering these particles appeared to significantly decrease cell survival rates while having a more neutral effect on neurite growth. This may suggest that the introduction of DF-sEVs resulted in the suppression of pro-survival and/or the activation of pro-death pathways, although further experiments are required to confirm this hypothesis. Overall, our data support the ineffectiveness of these particles for treating nerve injuries.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, our interest in the EVs of OMPC arose from the research of Knight and colleagues showing these vesicles to be beneficial for wound healing and to do so more efficiently than hBMSC-sEVs (42). Although anti-scar-forming properties are less relevant in the context of retinal or neurological diseases, their ability to encourage cell proliferation was considered to be worth exploring. Unfortunately, not only were OMPC-sEVs observed to be ineffective in promoting neuritogenesis, but they also appeared to be slightly detrimental to cell survival. The simplest explanation for this is that these vesicles and their cargo probably do not target the biochemical circuits relevant to neuron survival and neurite outgrowth.\u003c/p\u003e\n\u003cp\u003eAlthough the results shown here are promising, given the limitations of this experimental design, further investigation needs to be carried out. As mentioned above, the digestion of retinae eliminates some much-needed interactions between RGCs and their neighbouring supporting cells and removes RGCs from the context of an organised tissue. We also did not investigate how certain sEVs achieved their beneficial effects, and why some were better than others. EVs carry a wide variety of cargo molecules and surface proteins, all of which can affect the internal biochemistry of the recipient cell (17,18). Among these, miRNA garnered special interest, as interfering with their action has been shown to diminish the beneficial effects of EVs\u0026nbsp;(13–16). Isolating and comparing the miRNA cargo of the EV presented here could lead to a better understanding of their mechanism of action. Furthermore, investigating the other components of MSC secretomes would provide better insight into why certain EVs do not share the neuroprotective capabilities of their progenitor cell.\u003c/p\u003e\n\u003cp\u003eIn conclusion, after comparing the neuroprotective and neuritogenic ability of sEVs from five different cell types \u003cem\u003ein vitro\u003c/em\u003e, we found those isolated from hADSCs and hBMSCs to be the most effective. Moreover, concentrations of 1.5e9 - 3e9 vesicles per 125.000 cells tended to achieve the best results on both of these metrics. Thus, further investigations may be worth conducting, focusing on the \u003cem\u003ein vivo\u003c/em\u003e efficacy of ADSC and BMSC sEVs, alongside the mechanism of action responsible for the observed benefits.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e5.1. \u0026nbsp;Mesenchymal Stem Cell Cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman Mesenchymal Stem Cells from Bone Marrow (MSC-031, RoosterBio), Adipose tissue (C46001AD, RoosterBio), Umbilical Cord (V-hUCSC, Newmarket Scientific), or Dental Pulp (PT-5025, Lonza), Human Dermal Fibroblasts, adult (C0135C, Gibco), and human Oral Mucosal Progenitor Cells were seeded into T175 flasks (10127340; Thermo Fisher) at ~1.2e5 cells/cm2 and incubated at 37\u0026deg;C and 5% CO2. 35 ml Dulbecco\u0026apos;s Modified Eagle Medium (DMEM; 61965059; Thermo Fisher) or alpha Minimum Essential Medium (\u0026alpha;MEM; 22571038; Thermo Fisher) was provided, supplemented with 10% EV-depleted fetal bovine serum (FBS; 15434573; Thermo Fisher) and 1% penicillin/streptomycin. For hUCSCs, aMEM was further supplemented with Basic fibroblast growth factor (bFGF, 78003.1; STEMCELL Technologies). The medium was replaced every 2-3 days, and cells were passaged when over 80% confluent using 0.25% trypsin/ EDTA (11590626; Thermo Fisher). Except for the hOMPC cell line, cells were used at passages 2-5 post-acquisition. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.2. \u0026nbsp;Extracellular Vesicle Isolation and Storage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003esEVs were isolated using differential ultracentrifugation. Briefly: Culture medium was sequentially centrifuged at 300xg for 10 minutes, 2,000xg for 10 minutes and 10,000xg for 30 minutes, before being forced through a 0.22 \u0026mu;m pore size Millex\u0026reg;-OR Filter Unit (SLGP033RS; Sigma-Aldrich). The supernatant was collected, and pellets were discarded in each step. Filtered supernatant was spun at 100,000xg for 70 minutes. Pellets were collected, pooled, and washed in large amounts of sterile PBS before being spun down a second time at 100,000xg for 70 minutes. A constant temperature of 4\u0026deg;C was maintained for each spin. Pellets were collected in 1-2 ml sterile PBS and condensed down to less than 100 \u0026mu;l using Amicon\u0026trade; Ultra-4 Centrifugal Filter Units (UFC8100; Millipore) equilibrated with Milli-Q water prior to use as advised by the manufacturer. Isolates were stored at -80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.3. \u0026nbsp;Extracellular Vesicle Quantification and Quality Control\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor quantification, a ZetaView\u0026reg; Nanoparticle Tracking Analyser (Particle Metrix GmbH, Ammersee, Germany) was used. Particle size and concentration were measured at 11 positions over 1 cycle (Laser Wavelength: 488 nm; Filter Wavelength: Scatter; Sensitivity: 80.0; Shutter: 100). Time-resolved fluorescence TRF was used for further confirmation, targeting the known exosomal markers CD81 and CD9, as well as IgG for a negative control. Protein concentration of sEVs samples was measured using a Micro BCA\u0026trade; Protein Assay Kit (23235; Thermo Scientific\u0026trade;) according to the manufacturer\u0026rsquo;s instructions. Equal amounts of protein (1\u0026mu;g/well) were transferred to a high-binding, flat-bottom 96-well plate (655097; Greiner), topped up to 300\u0026mu;l total volume with PBS, sealed, and incubated overnight at 4\u0026deg;C. The following day, the samples were blocked in 10% milk diluted in PBS + 0.1% Tween-20 (P1379; Sigma) for 2 hours, and incubated with 1\u0026mu;g/well 1ry (CD81: 10630D; Invitrogen, CD9: MAB1880; R\u0026amp;D Systems, IgG:14-4724-85; Thermo Fischer), and then 0.2\u0026mu;g/ml biotin-conjugated 2ry antibodies (31800; Invitrogen) for 1 hour (Buffer: Reagent Dilutent Concentrate 2 diluted 1:5 in distilled water, DY995; R\u0026amp;D Systems). This was followed by a further 45-minute incubation with Europium-labelled Streptavidin (1244-36; DELFIA) diluted to 0.1\u0026mu;g/ml in RED Buffer (42-02; Uniogen Oy) and another 10 minutes in a Fluorescence enhancer solution (1244-105; DELFIA). Samples were washed 3 times between each step and 6 times before the fluorescent enhancer was added (Wash Buffer: Reagent Dilutent Concentrate 2 diluted 1:100 in distilled water, DY995; R\u0026amp;D Systems). All incubation steps were carried out at room temperature with shaking while the plates were sealed. Fluorescence was measured using a microplate reader (FLUOstar Omega; BMG Labtech).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.4. \u0026nbsp;Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals were kept and euthanised according to local guidelines. Ethical approval was obtained from the Cardiff University\u0026apos;s Animal Welfare Ethical Review Body (AWERB). All protocols and methods were conducted in accordance with local guidelines and pre-approved standard operating procedures. Adult, female, Sprague Dawley Rats weighing 100-150 g were supplied by Charles River Laboratories. Constant surveillance was provided by trained staff under conditions of 21\u0026deg;C, 55% humidity and 12-12-hour light and dark cycle, with food/water given\u003cem\u003e\u0026nbsp;ad libitum\u003c/em\u003e. Euthanasia was carried out via rising concentrations of CO2, without prior anaesthesia. 100% CO2 gas was delivered at a rate of 10L/min for 4 mins and 14L/min for a further 4 mins. Death was then confirmed via cervical dislocation and harvested tissues were immediately processed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.5. \u0026nbsp;Retinal Ganglion Cell Cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRetinal cells were isolated using a Papain Dissociation kit (PDS/LK003150; Worthington Biochemical). Briefly: retinae were dissected, minced and incubated in a solution of Papain and DNase diluted in EBSS at 37\u0026deg;C for 90 minutes with constant motion. Digested tissue was then centrifuged for 6 minutes at 300xg, the supernatant was discarded, and the pellet was resuspended in a mixture of 1.35 ml EBSS, 150 \u0026mu;l Albumin Ovomucoid inhibitor, and 75ul DNase. This suspension was carefully transferred onto 2ml Albumin Ovomucoid inhibitor, creating a density gradient, and centrifuged at 70xg for 6 minutes. The supernatant was discarded and pelleted cells were resuspended in 1ml supplemented Neurobasal Plus medium (Neurobasal-A [A3582901; Thermo Fisher] 2% B27 supplement [17504044; Gibco], 0.5 mM L-glutamine [25030081; Thermo Fisher] and 50 mg/ml of gentamycin [15710064; Thermo Fisher]).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e125,000 retinal cells were seeded in 300 \u0026mu;l Supplemented Neurobasal Plus medium in each of eight wells of Millicell\u0026reg; EZ Slides (PEZGS0816; Millipore) at ~1.8e5 cells/cm2 pre-coated with 100 mg/ml poly-D-lysine (A3890401; Thermo Fisher) for 60 minutes and 20 mg/ml laminin (10267092; \u0026nbsp;Fisher Scientific Ltd) for 30 minutes. Cultures were immediately treated with sEVs resuspended in 50ul PBS or PBS alone (Final volume: 350ul per well) and incubated for 3 days under conditions of 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.6. \u0026nbsp;Immunocytochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRetinal cell cultures were fixed in 4% Polyformaldehyde (PFA) for 10 minutes and blocked in 3% Bovine Serum Albumin and 0.1% Triton-X 100 (T9284-500ML; Sigma-Aldrich) solution for 20 minutes. Fixed cultures were then incubated for 1h at room temperature with either primary (anti-\u0026beta;-III-Tubulin, T8578-200UL; Merck; diluted 1:400) or secondary (Alexa Fluor\u0026reg; 488 Goat anti-mouse IgG, A32723; Invitrogen; diluted 1:500) antibodies in 3% Bovine Serum Albumin, 0.05% Tween 20 (P1379-500ml; Sigma) solutions and mounted in VECTASHIELD\u0026reg; Antifade Mounting Medium containing DAPI (H-1200-10; 2B Scientific). Samples were washed in PBS for 10 minutes, 3 times between each step, after the primary antibodies were added. Mounted slides were left to dry overnight, shielded from light and stored at 4\u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.7. \u0026nbsp;Fluorescent Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples were visualised using a Leica fluorescent microscope system (Leica DM6000 body, DFC350 FX monochrome Digital Camera, EL6000 External light source) at 600x magnification with excitation and emission wavelengths of 358/461 nm and 493/519 nm for DAPI and AlexaFluor488, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDAPI-stained cells, RGCs, and neurites were counted manually. Here, RGCs were defined as elliptical cells where \u0026beta;-III-antibody forms a halo around the nucleus, with a pole of stronger clustering also observable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.8. \u0026nbsp;Data Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImages were collected of 10-15 neurites per well (where possible) and their length was determined using the Fiji Neuroanatomy Plugin as described by Durmaz and colleagues (75).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData was analysed and visualised using GraphPad Prism 10 Software. Treatment groups were compared to PBS control using one-way ANOVA with Dunnett test. (* p \u0026gt; 0.05; ** p \u0026lt; 0.01; *** p \u0026lt; 0.005).\u003c/p\u003e\n\u003cp\u003ePost-hoc Power calculation was carried out using R Studio\u0026rsquo;s pwr package (\u003cstrong\u003epwr.anova.test(k = 5, f = Effect_size, sig.level = 0.05, n = 3\u003c/strong\u003e). Effect size (Cohen\u0026rsquo;s f) was determined using a free online calculator (Effect Size Calculators; Partial eta-squared (Fixed effects); \u003cu\u003ehttps://effect-size-calculator.herokuapp.com/#partial-eta-squared-fixed-effects\u003c/u\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests Statement\u003c/h2\u003e \u003cp\u003eThe Authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll experiments and statistical analyses were carried out by M. K. with the help and guidance of B. M. All drafts and the final version of the manuscript were written by M.K. and proofread / corrected by B. M. and P. S.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data created and examined throughout this investigation are contained within this article. Upon request, the corresponding author will provide any datasets used or analyzed during the current work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.nhs.uk/conditions/glaucoma/\u003c/span\u003e\u003cspan address=\"https://www.nhs.uk/conditions/glaucoma/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. nhs.uk. 2017 [cited 2023 July 2]. Glaucoma. Available from: https://www.nhs.uk/conditions/glaucoma/\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJonas, J. B. et al. \u003cem\u003eGlaucoma Lancet\u003c/em\u003e ;\u003cb\u003e390\u003c/b\u003e(10108):2183\u0026ndash;2193. (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahar, M. \u0026amp; Cavalli, V. Intrinsic mechanisms of neuronal axon regeneration. \u003cem\u003eNat. Rev. 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Clifton NJ\u003c/em\u003e. \u003cb\u003e2708\u003c/b\u003e, 1\u0026ndash;10 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdditional \u0026amp; Information.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8603346/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8603346/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The paracrine neuroprotective effects of Mesenchymal Stem Cells (MSC) are well known, and our understanding of the underlying mechanisms is expanding by the day. From among their diverse secretome, extracellular vesicles (EVs) have gained particular interest lately, owing to their good safety profile, stability, and relative ease of use as a cell-free therapy. The cargo and thus the potential effect of these nano-sized lipid membrane-enclosed vesicles is highly dependent on the type, age, and environment of the donor cells. Therefore, it is paramount to know which cell types are best utilised in any given situation. Glaucoma is a chronic progressive optic neuropathy that is the most common cause of irreversible blindness worldwide, characterised by the loss of Retinal Ganglion Cells (RGC) whose axons make up the optic nerve. Preservation of these neurons via the administration of the right EVs represents a promising approach for slowing down or halting disease progression, thereby preventing vision loss. Here, we evaluate the neuroprotective and neuritogenic potential of EVs isolated from five different cell types using a rodent in vitro model of RGC degeneration. Our findings showed that Adipose Mesenchymal Stem Cells (ADSC) release the most potent EVs, with Bone Marrow (BMSC) being a close second. EVs released by cells of the Umbilical Cord (UCSC), Dental Pulp (DPSC), Dermal Fibroblasts (DF), and an Oral Mucosal Lamina Propria-Progenitor cells (OMLP-PCs) did not have an observable benefit. Thus, our study provides greater insight into how the efficacy of different EVs compare to each other.","manuscriptTitle":"Comparative Analysis of In Vitro Neuroprotection and Neuritogenesis on Axotomised Retinal Ganglion Cells by Small Extracellular Vesicles Derived from Various Mesenchymal Stem Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-17 09:47:55","doi":"10.21203/rs.3.rs-8603346/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-08T05:42:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-07T11:15:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T20:10:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148391830989132040402514879030281836425","date":"2026-03-17T17:16:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5234494536222681622697269758277315608","date":"2026-03-17T16:11:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-16T12:56:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39471845473131765190218660667306702026","date":"2026-03-14T17:28:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80604993266541363721615275108105749258","date":"2026-03-13T08:40:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210744043254026870876696177435935830448","date":"2026-03-12T16:39:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-12T16:22:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-12T15:46:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-12T13:45:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-10T03:05:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-10T01:42:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6fdccd96-7022-4178-b771-5136b58adcfb","owner":[],"postedDate":"March 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64510867,"name":"Biological sciences/Cell biology"},{"id":64510868,"name":"Biological sciences/Neuroscience"},{"id":64510869,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-05-14T05:39:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-17 09:47:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8603346","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8603346","identity":"rs-8603346","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}