Amniotic fluid stem cell conditioned medium’s role in Schwann cell proliferation, survival, and cellular antioxidant activity under normative and oxidative stress conditions

Introduction

Peripheral nerve injuries present a significant clinical challenge for care providers and often result in significant morbidity and incomplete recovery of function despite timely intervention (1-3). Autograft repair remains the gold-standard treatment modality for peripheral nerve injuries unable to be addressed with direct end-to-end repair. Unfortunately, its use is often limited by factors like donor site morbidity and restricted graft availability (4,5). While alternative methods, such as allografts and tissue-engineered nerve grafts, exist, they fail to achieve optimal functional recovery for many patients, with up to 50% experiencing ongoing dysfunction, pain, or dysesthesias (6).

Peripheral nerve recovery following injury is a complex physiological process initiated by Wallerian degeneration, which involves the breakdown of the axon and myelin sheath distal to the injury site (7-10). This is followed by the recruitment of inflammatory cells, such as macrophages and neutrophils, which clear debris and release factors that support nerve repair. Schwann cells are crucial in this regenerative process. They dedifferentiate and proliferate to form the Bands of Büngner, a structure that guides the regrowth of axons. Additionally, Schwann cells secrete neurotrophic factors and modify the extracellular matrix, thus providing both structural and biochemical support for nerve regeneration (11-13).

The ability of Schwann cells to survive and proliferate in the inflammatory microenvironment post-injury is critical, as exposure to higher levels of oxidative stress can impair Schwann cell function (14). Previous studies have revealed that Schwann cell senescence, secondary to oxidative stress, can be a culprit for incomplete recovery after peripheral nerve injury (15-17). As such, enhancing Schwann cell resilience is essential for successful nerve regeneration. Emerging therapeutic strategies utilizing cell-based approaches have been developed to enhance Schwann cell function and modulate the inflammatory response, potentially improving the efficacy of nerve repair techniques.

Schwann cell transplantation techniques aim to facilitate peripheral nerve regeneration after injury (18). These techniques involve harvesting Schwann cells from healthy peripheral nerves, subsequent in-vitro expansion, and then reimplanting donor Schwann cells to peripheral nerve injury sites in adjunct with a nerve scaffold. While this may offer some clinical benefits, its use is limited by the costs and lead time required to acquire, expand, and reimplant native Schwann cells to the injury site. Nonetheless, cell-based therapies offer promise in facilitating peripheral nerve recovery. The regenerative nature of stem cell transplantation and other cell-based therapies have been attributed to the paracrine effects of transplanted stem cells on local tissue environments, specifically in creating a favorable redox environment conducive to tissue regeneration (19,20). As such, increasing interest has been in using highly concentrated stem cell conditioned medium in regenerative medicine (19). Human amniotic fluid-derived stem cells can be conveniently collected after childbirth and harvested for therapeutic use. Additionally, amniotic fluid stem cell conditioned medium (AFS-CM) may serve as a valuable option due to the high passage capacity of amniotic fluid stem cells. Furthermore, the lack of immunogenic and tumorigenic potential and the practical applicability of AFS-CM pose it as a promising adjunct treatment for peripheral nerve injury (19).

The present study assesses the effects of AFS-CM on Schwann cell viability and proliferation. More specifically, we explore the role of AFS-CM on Schwann cell survival and proliferation under normative and oxidative stress conditions, preventing oxidative stress-induced premature senescence of Schwann cells in vitro and maintaining cellular redox homeostasis. We present this article in accordance with the ARRIVE reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-24-107/rc).

Methods

Isolation and culture of primary rat Schwann cells

The experiments of the present study were approved by the Institution of Animal Care and Use Committee (IACUC) at Atrium Wake Forest Baptist Health (IACUC # A19-101), in compliance with institutional guidelines for the care and use of animals. A protocol was prepared for this study without registration. Schwann cells were obtained and isolated from adult male Lewis rats obtained from Charles Rivers Laboratories using a protocol previously described by Haastert et al. (21). Briefly, the sciatic nerve from male Lewis rats was harvested in a pre-degenerated state and prepared for Schwann cell isolation. Sciatic nerve epineurium was dissected from the nerve harvest, and the resulting epineural-free nerve was transferred in Dulbecco’s Modified Eagle Medium (DMEM) with 1% penicillin/streptomycin (pen/strep; Thermo Fisher Scientific, Waltham, MA, USA, Catalog number 15140122) (22) to a sterile workstation. Nerve tissue was washed in DMEM and cultured onto non-coated wells on a six-well plate with DMEM + 10% fetal bovine serum (FBS; Thermo Fischer Scientific, Catalog number A5256801) (23) + 1% pen/strep. Schwann cells were subsequently dissociated from the tissue by adding 0.125% collagenase + 1.25 U mL⁻¹ dispase to create a dissociation medium. The tissue was then incubated with the dissociation medium for 20 hours at 37 ℃ with 5% carbon dioxide (CO₂). The resulting tissue was separated mechanically by gentle trituration until a homogenous solution was obtained, after which samples were centrifuged at 235 g for 5 min at 21 ℃. A pellet was formed after centrifugation and re-suspended in DMEM + 10% FBS + 1% pen/strep and 1% glutamate. The cell pellet was expanded in laminin + poly-L-ornithine (Sigma Aldrich, Milwaukee, WI, USA) coated flasks until ~70–80% confluence. The resultant cell passage was passed and maintained in DMEM + 10% dimethyl sulfoxide (DMSO) solution and stored in liquid nitrogen until further use. All cell stocks were thawed and passed once before being used for in-vitro experiments.

Verification of Schwann cell purity

Primary cultured rat Schwann cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS, Thermo Fisher Scientific) at 37 ℃ for 15 min. Fixed cells were washed with PBS and permeabilized using PBS containing 0.3% Triton X-100 then blocked with 10% rabbit serum for 30 min at 37 ℃. Cells then were incubated at 4 ℃ overnight with primary S100 antibody (1:500, Abcam, Cambridge, UK) then Alexa Fluor 488-conjugated secondary antibody for 30 min at 37 ℃ (Thermo Fisher, Waltham, MA, USA). Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (1:5,000, Thermo Fisher). Cells were imaged using a fluorescent microscope (Olympus, Japan). Confirmation of Schwann cell phenotype can be seen in Figure 1.

Figure 1 Immunofluorescence analysis of primary cultured rat Schwann cells. (A) A bright-field image displaying primary cultured rat Schwann cells before staining. The image reveals the characteristic spindle-shaped morphology of Schwann cells, with processes extending from the cell bodies (magnification ×200). (B) Fluorescence image showing the nuclei of Schwann cells stained with DAPI (blue). The DAPI staining highlights the nuclear localization within the cells, confirming the presence of Schwann cells in the culture (magnification ×200). (C) Fluorescence image showing Schwann cells stained with an anti-S100 antibody (green), a specific marker for Schwann cells. The green fluorescence indicates successful labeling of the S100 protein within the Schwann cells’ cytoplasm. The overlay with DAPI staining (blue) in this panel demonstrates the colocalization of S100 protein within the cells, confirming their identity as Schwann cells (magnification ×200). DAPI, 4',6-diamidino-2-phenylindole.

Formulation and characterization of AFS-CM

Amniotic fluid stem cells were obtained from Nutech Inc. (Birmingham, AL, USA) and cultured in DMEM (Corning, NY, USA; Product number 15-018-CV) with 20% FBS in a T175 flask (Thermo Fisher Scientific) until ~80% confluence, after which cells were maintained in serum-free DMEM for 24 hours. Passage two to four cultured amniotic fluid stem cells were used in this study. The conditioned medium was then collected and centrifuged at 4 ℃ at 3,000 g for 5 min. After that, the centrifuged medium was re-centrifuged with ultra-centrifugal filters (Thermo Fisher Scientific) (24) with a semipermeable membrane of 100kDa at 4 ℃, 3,000 g for 2 hours. This was done to purify the AFS-CM formulation.

The resulting supernatant solution was collected and stored at −80 ℃ until further use. According to the manufacturer’s instructions, protein content was measured using a Bradford protein assay kit (Thermo Fisher Scientific, Product number A55866) (25). Three separate Bradford protein assay sample measurements were used to quantify the mean protein amount in each AFS-CM batch. The cytokines and growth factors of the AFS-CM were analyzed using a multiplex customized Quantibody human enzyme-linked immunosorbent assay (ELISA) array (Raybiotech, peachtree Corners, GA, USA) that detected 39 different cytokines and growth factors. The cytokines with the top 5 greatest concentrations included, insulin-like growth factor binding protein (IGFBP), bone morphogenetic protein (BMP), transforming growth factor beta (TGFβ), basic fibroblast growth factor (bFGF) and insulin-like growth factor (IGF) (level of detection: 793.8±69.6, 313.3±52.1, 188.3±33.6, 145.7±30.2, 124.6±31.1 pg/mL, respectively).

Schwann cell proliferation test

96 well plates were used with wells pre-coated with laminin + poly-L-ornithine (Sigma Aldrich, Milwaukee, WI, USA). The well-coating procedure was previously described by Ma et al. (26). Primary rat Schwann cells (passages 3 to 5) were plated (4×10³) on a 96 well plate in DMEM (Corning, Product number 15-018-CV) + 10% FBS medium + 1% glutamax (Thermo Fisher Scientific, Catalog number: A1286001) (25) + 0.1% forskolin (100 µL) until 50–60% confluence. Medium was then exchanged with DMEM + 1% FBS with varying concentrations of AFS-CM (0, 1, 10, and 20 µg/mL). Cell proliferation was assessed at one day, three days, and five days after using a Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan, product number: CK04) (27) according to the manufacturer’s instructions. Briefly, 10ul of cell proliferation reagent water-soluble tetrazolium-1 (WST-1) was added to each cell culture well at the previously stated time points and incubated for 1 hour. WST-1 is a stable tetrazolium salt cleaved by nicotinamide adenine dinucleotide phosphate [NAD(P)H] in viable cells. Once reduced, WST-1 emits a formazan dye. The amount of formazan die emitted directly correlates to the number of viable cells. Absorbance was measured at 450 nm after 1 hour using a standard microplate reader.

Schwann cell viability under oxidative stress

A lethal dose curve was generated to ascertain the hydrogen peroxide (H₂O₂) concentration necessary to reduce primary Schwann cell culture (4×10³) by 50% on a pre-coated 96-well plate (Thermo Fisher Scientific) (28). H₂O₂ concentrations used were 0, 20, 50, 80, 100, 500 µM, and 1 mM. The ascertained dose was 80 µM of H₂O₂. Primary Schwann cells were then plated (4×10³) on a 96-well pre-coated plate and incubated until 60–70% confluence in DMEM + 10% FBS medium (100 µL). The medium was then exchanged with DMEM + 1% FBS (100 µL) with varying concentrations of AFS-CM (0, 1, 10, and 20 µg/mL) for twelve hours, after which cell culture was exposed to 80 µM of H₂O₂ for 1 hour. Medium was subsequently exchanged to DMEM + 10% FBS medium (100 µL). Cell culture medium was exchanged with DMEM + 1% FBS + varying concentrations of AFS-CM (0, 1, 5, 10, 20 µg/mL) every 24 hours, thereby ensuring the continued delivery of AFS-CM. Cell viability was assessed at 24 and 48 hours using a CCK-8 (Dojindo, product number: CK04) (27) according to the manufacturer’s instructions.

Reactive oxygen species (ROS) generation

6-well plates were pre-coated with laminin + poly-L-ornithine then plated with primary Schwann cells (4×10³; passages 3 to 5) in DMEM + 1% FBS medium (100 µL) + varying concentrations of AFS-CM (0, 1, 10, and 20 µg/mL) until 60–70% confluency. Cells were washed in phosphate-buffered saline (PBS; Thermo Fisher Scientific, Catalog number: 10010023) and treated with 0 µM, 100 µM, 1 mM, or 4 mM H₂O₂ for 15 min in DMEM + 1% FBS medium. Cellular ROS were quantified for the cell culture using the cellular ROS detection kit (Abcam, product number: ab186027) (29) according to the manufacturer’s instructions. The fluorescence signal was read by a fluorescence microplate reader (Thermo Fisher Scientific) at Ex/Em =520/605 nm (30). An analysis of variance (ANOVA) was conducted to assess the fold change difference between groups.

Quantifying Schwann cell senescence

Six-well plates were coated with laminin + poly-L-ornithine and then plated with primary Schwann cells (1×10⁴; passages 3 to 5) in DMEM + 10% FBS medium + 1% penicillin/streptomycin + 1% glutamine until 60–70% confluency. Cells were then washed in PBS, and the medium was exchanged to DMEM + 10% FBS medium with varying concentrations of AFS-CM (0, 1, 10, and 20 µg/mL). Oxidative stress-induced senescence was initiated using an adapted protocol from Wang et al. (31). Briefly, cells were exposed to 20 µM of H₂O₂ for 15 min in DMEM + 1% FBS medium then washed with PBS after H₂O₂ exposure, and medium was exchanged with DMEM + 10% FBS + varying concentrations of AFS-CM (0, 1, 10, and 20 µg/mL) for 24 hours. The process was then repeated with 10 µM of H₂O₂ for another 24 hours to assess the protective effects of AFS-CM on cell survival against oxidative stress. Cells were then stained for S-beta-galactosidase using the senescence b-galactosidase staining kit (Abcam, product number: ab102534) (32) according to the manufacturer’s instructions. Positive and negatively stained cells were counted. An ANOVA was performed to compare the percent difference in cells staining positive when compared to the control group (no H₂O₂ and no AFS-CM).

Superoxide dismutase (SOD) and catalase measurement

Primary Schwann cells (passages 3 to 5) were then plated (4×10³) on a 96-well plate and incubated until 60–70% confluence in DMEM + 10% FBS medium. The medium was then exchanged with DMEM + 1% FBS with varying concentrations of AFS-CM (0, 1, 10, and 20 µg/mL) for 12 hours. The catalase activity assay kit (Abcam, product number: ab83464) (33) was used to measure catalase activity at an optical density (OD) 570 nm colorimetrically according to the manufacturer’s instructions. In this assay, a proprietary probe reacts with unconverted H₂O₂ to produce a product that can be measured colorimetrically. Thus, the signal intensity is inversely proportional to catalase activity. SOD activity was calculated using an SOD activity assay kit (Abcam, product number: ab65354) (34) which measures the inhibition of xanthine oxidase by SOD. This assay kit was used according to the manufacturer’s instructions. An ANOVA was conducted to assess the fold change difference between groups.

Statistical analysis

Continuous data was analyzed using a one-way ANOVA. An ANOVA post-hoc Tukey’s honestly significant difference (HSD) was performed to assess multiple comparisons of mean diff differences between groups. Statistical significance was established at a P value threshold of less than 0.05. All statistical tests were two-sided and performed using Python’s SciPy and Pandas libraries. Data visualization was facilitated through the Matplotlib and Seaborn libraries.

Results

Protein concentration of AFS-CM

AFS-CM samples (n=28 from four cell lines) were examined utilizing Bradford protein analysis to estimate protein quantities. The mean protein concentration of the AFS-CM samples was 1,918.33 µg/mL, with a standard deviation of 909.74 µg/mL. The protein concentration was highly variable, with our Bradford protein analysis revealing a range in protein content from 305.90 to 4,276.62 µg/mL. To account for this variability, all AFS-CM samples were pooled and used at the indicated concentrations for the experiments, minimizing the potential for technical batch effects from different cultures.

AFS-CM promotes Schwann cell proliferation under normative conditions and optimizes Schwann cell viability under oxidative stress conditions

Schwann cell proliferation capability is critical in peripheral nerve recovery after injury. We sought to explore the mitogenic effect of AFS-CM on primary rat Schwann cell culture using a CCK-8 analysis. CCK-8 analysis revealed that AFS-CM caused a nearly dose-dependent increase in Schwann cell proliferation (Figure 2), indicating a mitogenic effect on primary rat Schwann cells. Our findings demonstrate that the optimal proliferative response to AFS-CM in cell culture was achieved at 10 µg/mL on both days 3 and 5. Additionally, the data indicate increased variability in the proliferative effects as the dosage of AFS-CM increased, suggesting a dose-dependent ceiling to its therapeutic efficacy.

Figure 2 CCK-8 assay demonstrating Schwann cell proliferation. Each cell culture was treated with varying AFS-CM doses. The graphs represent the relative OD reading compared to cell culture pre-treated with 0 µg/mL of AFS-CM. A total of seven experiments were conducted. Graphs are as follows: fold change difference in Schwan cell viability at (A) 1 day (P<0.001); (B) 3 days (P<0.001); and (C) 5 days (P<0.001). Error bars signify a 95% confidence interval. ANOVA test assessed mean group differences. A post-hoc Tukey’s HSD was performed to assess multiple comparisons of mean differences between groups. A “*” indicates a post-hoc Tukey HSD P value of <0.05. CCK-8, Cell Counting Kit-8; AFS-CM, amniotic fluid stem cell conditioned medium; OD, optical density; ANOVA, analysis of variance; HSD, honestly significant difference.

Peripheral nerve Schwann cells must resist an inflammatory mediate oxidative burst that occurs soon after peripheral nerve injury to facilitate nerve recovery. We discovered that AFS-CM minimized the harmful effect of H₂O₂-mediated oxidative stress on primary Schwann cells. Our CCK-analysis revealed an AFS-CM mediated increase in fold change difference in Schwann cells in an oxidative stressed environment at the 24- and 48-hour period (Figure 3).

Figure 3 Fold change difference in Schwann cell proliferation after oxidative stress challenge with 80 µM of H₂O₂ for 1 hour. Cell cultures were treated with varying doses of AFS-CM (0, 1, 5, 10, 20 µg/mL) for 12 hours before the oxidative stress challenge. A positive control (“media only”) denotes cell culture in which cells did not undergo oxidative stress challenge. The graphs represent the relative OD reading compared to positive control in which cells did not undergo oxidative stress. Graphs are as follows: (A) change difference at 24 hours (P<0.001); (B) change difference at 48 hours (P<0.001). Error bars denote a 95% confidence interval. ANOVA test assessed mean group differences. A post-hoc Tukey’s HSD was performed to assess multiple comparisons of mean differences between groups. A “*” indicates a post-hoc Tukey HSD P value of <0.05. AFS-CM, amniotic fluid stem cell conditioned medium; OD, optical density; ANOVA, analysis of variance; HSD, honestly significant difference.

AFS-CM reduces cellular ROS via upregulation of catalase and SOD enzymes

ROS can serve as critical secondary messengers in normal cellular processes; however, its dysregulation secondary to external stressors can lead to cell damage and dysfunction. Accordingly, we sought to explore the effect of AFS-CM on cellular ROS when cells were exposed to increasing concentrations of H₂O₂. Cellular ROS levels were obtained for our primary rat Schwann cell culture after a 15-minute challenge of varying concentrations of H₂O₂. Our initial experiment revealed that H₂O₂ elicited a dose-dependent increase in cellular ROS from primary Schwann cells (Figure 4A). AFS-CM decreased cellular ROS levels when cells were challenged with 0 µM, 100 µM, 1 mM, and 4 mM of H₂O₂. Cells treated with AFS-CM exhibited statistically significant differences in ROS at varying concentrations of H₂O₂ (Figure 4B) with an increased concentration of AFS-CM. Interestingly, AFS-CM’s attenuating effect on ROS generation was only observed at higher concentrations (4 mM) of H₂O₂. There was an increase in cell ROS generation with Schwann cells challenged with 0 µM of H₂O₂ pre-treated with an AFS-CM dose of 5 µg/mL and higher.

Figure 4 The following illustration represents the relative production of cellular ROS for primary Schwann cells pre-treated with varying concentrations of AFS-CM. The X-axis denotes the concentration of H₂O₂ for the oxidative stress challenge. (A) The positive control, which indicates an H₂O₂-related increase in cellular ROS species generation. (B) The fold change difference in cell reactive oxygen species generation compared to control (0 µg/mL of AFS-CM) at 100 µM, 1 mM, and 4 mM of H₂O₂. ANOVA test assessed mean group differences was performed. Error bars denote a 95% confidence interval. A post-hoc Tukey’s HSD was performed to assess multiple comparisons of mean differences between groups. A “*” indicates a post-hoc Tukey HSD P value of <0.05. ROS, reactive oxygen species; AFS-CM, amniotic fluid stem cell conditioned medium; ANOVA, analysis of variance; HSD, honestly significant difference.

We sought to explore the effect of AFS-CM on facilitating cellular redox homeostasis in two key enzymes: catalase and SOD. These enzymes are critical for the Schwann cell after injury by mitigating oxidative stress and improving function, viability, and nerve recovery. SOD functions to convert superoxide free radicals to H₂O₂, whereas catalase converts H₂O₂ to H₂O and oxygen (O₂). Further experimentation revealed increased catalase and SOD activity with the administration of AFS-CM. Our analysis revealed increased catalase activity (as denoted by a decreased OD score) for cell cultures treated with 5 µg/mL of AFS-CM or more (Figure 5A). Schwann cell SOD activity was significantly increased with AFS-CM doses of 10 and 20 µg/mL (Figure 5B).

Figure 5 The following experiment results demonstrate the role of conditioned media on catalase and superoxide dismutase activity of primary rat Schwann cells. Here, non-converted H₂O₂ interacts with a proprietary probe, releasing a signal that can be measured colorimetrically. Catalase activity is inversely proportional to the OD signal. Superoxide dismutase activity is measured by SOD inhibition of xanthine oxidase according to the manufacturer’s instructions. We report the fold change difference (in percent) of the OD signaling for Schwann cell cultures treated with varying concentrations of AFS-CM compared to control (medium only without H₂O₂). (A) The percent fold change difference in catalase activity when treated with varying concentrations of AFS-CM (P value <0.001) compared to the control. (B) The percent fold change difference in superoxide dismutase activity when treated with varying concentrations of AFS-CM (P value <0.001) compared to the control. Error bars represent a 95% confidence interval. Asterisk “*” denotes Tukey’s HSD P value <0.05. OD, optical density; SOD, superoxide dismutase; AFS-CM, amniotic fluid stem cell conditioned medium; HSD, honestly significant difference.

AFS-CM prevents stress-induced senescence in primary rat Schwann cells

Schwann cells run a risk of transforming into their stress-induced senescence phenotype after prolonged oxidative stress. This phenomenon is increasingly recognized as a key player in incomplete peripheral nerve recovery after injury (35). We found that AFS-CM developed a protective effect against stress-induced senescence in primary rat Schwann cells when challenged with H₂O₂. Our results reveal a lower proportion of senescent positive cells for cultures treated with 5, 10 and 20 µg/mL of AFS-CM when compared to 0 µg/mL of AFS-CM (Figure 6).

Figure 6 The following experiment results highlight the protective effect of AFS-CM against H₂O₂-mediated Schwann cell senescence. Briefly, Schwann cell culture was treated with varying doses of AFS-CM (0, 1, 5, 10, 20 µg/mL) and initially treated with 20 µM of H₂O₂ for 15 min followed by 10 µM of H₂O₂ for 15 min every 24 hours. Error bars represent 95% confidence intervals. (A) Schwann cell staining with S-beta galactosidase and senesced cells staining blue green. (B) Data are presented as the percent difference of cells staining positive for S-beta galactosidase compared to control (no H₂O₂ challenge). ANOVA test assessed mean group differences was performed (P<0.001). A post-hoc Tukey’s HSD was performed. Asterisk (*) represents a P<0.05. Image scale bars =400 µm. “CTRL” denotes the control group. AFS-CM, amniotic fluid stem cell conditioned medium; ANOVA, analysis of variance; HSD, honestly significant difference.

Discussion

Peripheral nerve injury confers significant morbidity for the afflicted, wherein most patients will be unable to attain pre-injury function (36,37). The Schwann cell is a critical component of peripheral nerve regeneration as it is vital in all parts of the regenerative process (38). Despite the innate regenerative capacity of peripheral nerves, ischemia, and the inflammatory process to clear out the debris from damage that occurs soon after peripheral nerve injury can lead to an unfavorable oxidative stressed environment that is cytotoxic to Schwann cells and hinder nerve regeneration. In this study, we found that AFS-CM has therapeutic effects to facilitate rat Schwann cell proliferation and survival under normative and oxidative stress conditions, reduce ROS production, and prevent cellular senescence under oxidative stress in vitro.

Our study revealed a near dose-dependent increase in cell proliferation with increasing doses of AFS-CM. However, the cyto-proliferative effect of stem cell conditioned medium on Schwann cells is not unique to our study. Guo et al. (39) assessed the paracrine effects of human umbilical cord mesenchymal stem cells on primary rat Schwann cells. The authors reported an increase in Schwann cell viability at 24- and 48-hour time points when compared to control (P<0.01) and an increased rate of Schwann cell proliferation at 48 hours (P<0.01). Similarly, we observed increased cell viability and proliferation at one, three, and five-day time points with increasing concentrations of AFS-CM. Of note, our results suggested an optimal proliferative effect of AFS-CM at 10 µg/mL at days 3 and 5, with higher doses conferring no additional benefit suggesting a ceiling effect with increment of dosages.

Interestingly, our findings reveal that AFS-CM had a protective effect against H₂O₂-induced oxidative stress at the 24- and 48-hour period. This may be due to the upregulation of cellular catalase and superoxide enzymes during the treatment of Schwann cell culture with AFS-CM. Free O₂⁻ radicals are produced during cellular respiration by the mitochondria and nitric oxide enzymes during both homeostasis and periods of intense inflammation (40). Catalase and SOD are antioxidants critical for the cellular defense against oxidative stress. SOD catalyzes the cell’s dismutation of free O₂ radicals to molecular O₂ and H₂O₂ (41). Catalase in turn breaks down H₂O₂ to form O₂ and H₂O. The increased activity of these antioxidants noted in our experiments may be mediated by activation of the Nrf2/KEAP/ARE pathway as suggested by other investigators. Yan et al. (42) discovered that human placental mesenchymal stem cell conditioned medium protected against H₂O₂-induced oxidative damage and cellular apoptosis of fetal alveolar epithelial cells via the activation of Nrf2/KEAP1/ARE signaling pathway. This upregulation in antioxidants may cause a reduction in cellular ROS, thus ameliorating the excessive ROS-induced cellular damage. Interestingly, in our experiment, the protective effect of AFS-CM on reducing cellular ROS was more prominent when Schwann cells were exposed to higher levels of H₂O₂ (4 mM), suggesting that AFS-CM may only trigger the signaling cascades to restore the cellular homeostasis when the damage is beyond the cells’ self-repair under oxidative stress. It is important to note that we saw an increase in cellular ROS when exposed to AFS-CM doses of 5 µg/mL in cells not challenged with H₂O₂. We speculate this to be due to an increased cell activity secondary to AFS-CM mitogenic effect on Schwann cells, as evidenced in Figure 2. To our knowledge, no previous studies have attempted to quantify the oxidative stress during peripheral nerve injury in vivo. Future research looking to explore the therapeutic potential of AFS-CM on peripheral nerve injury and regeneration should consider this magnitude of oxidative stress as this may play a role in the therapeutic efficacy of AFS-CM.

Still, it is important to note the AFS-CM’s role in mitigating oxidative stress-induced senescence in our study. There is increasing evidence that Schwann cell senescence may be a culprit in failed and/or suboptimal peripheral nerve regeneration (17,43). Our findings reveal that AFS-CM mitigated the effects of repeated sublethal administration of H₂O₂ toward Schwann cells. ROS burst is an expected physiologic response soon after peripheral nerve injury (44,45) and can harm peripheral nerve regeneration. Büttner et al. (46) revealed that injury-induced inflammatory response was dramatically upregulated, resulting in a persistent hyper-inflammatory state up to 8 weeks after peripheral nerve injury. Several studies reveal that the neuroprotective effects of stem cells conditioned medium against oxidative stress via the upregulation of cellular antioxidants. Liu et al. (47) reported ROS-savaging activities of human-derived adipose mesenchymal stem cells on H₂O₂ treated rat Schwann cells. The authors highlight several studies demonstrating the role of exosomes in combating oxidative stress via the adaptive regulation of the nuclear factor erythroid 2-related factor (Nrf-2) defense system (48-50). Activation of the Nrf-2 defense system plays a role in preventing cellular stress-induced senescence. Shin et al. (51) demonstrated the role of Nrf-2 in preventing stress-induced senescence among H₂O₂-exposed human mesenchymal stem cells. The authors demonstrated a lower rate of stress-induced senescence with activation of Nrf2, a finding that was not seen among Nrf-2 knock-out mesenchymal stem cells. Thus, it can be suggested that the early activation of the Nrf2/KEAP1/ARE pathway may prevent stress-induced senescence of Schwann cells after peripheral nerve injury. Further experiments are warranted to investigate whether the effects of AFS-CM on cellular senescence were exerted through the activation of the Nrf-2 pathway.

Our study has limitations. We analyzed the therapeutic effect of AFS-CM in vitro only. It’s important to note the intricate nature of multiple systems involved in peripheral nerve injuries in vivo, which may contribute to the therapeutic effect of AFS-CM. For example, Schwann cells’ innate regenerative potential may vary based on host age, glucose metabolism (52), and baseline microenvironment before peripheral nerve injury. Additionally, our study assesses one-time AFS-CM delivery after oxidative stress challenge on rat Schwann cells. The optimal timing for treatment following injury needs further exploration as it may be a key factor in understanding the translational therapeutic capability of AFS-CM to in-vivo models. Nonetheless, our study demonstrates the proliferative effect of AFS-CM on Schwann cells without affecting the cell viability over time. AFS-CM also demonstrates its ability to improve Schwann cell survival under oxidative stress via the upregulation of cellular antioxidants and inhibition of cell senescence. Our lab is currently investigating the in vivo therapeutic effect of AFS-CM on peripheral nerve regeneration after transection injury.

Conclusions

In conclusion, this study demonstrates the therapeutic potential of AFS-CM in promoting Schwann cell proliferation and enhancing cellular resilience against oxidative stress in peripheral nerve injuries. The observed protective effects, particularly the upregulation of antioxidant enzymes, suggest that AFS-CM may facilitate peripheral nerve recovery after injury.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-24-107/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-24-107/dss

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-24-107/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-24-107/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The experiments of the present study were approved by the Institution of Animal Care and Use Committee (IACUC) at Atrium Wake Forest Baptist Health (IACUC # A19-101), in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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