Therapeutic effects of conditioned medium of immortalized dental pulp stem cells from human exfoliated deciduous teeth against experimental autoimmune neuritis

Abstract

Cell-free therapy using the conditioned medium (CM) of mesenchymal stem cells has attracted great interest in regenerative medicine due to its fewer ethical and safety concerns. Recently, we established an immortalized dental pulp stem cell line from human exfoliated deciduous teeth (SHED). Herein, we have investigated the therapeutic potentials of SHED-CM on the peripheral neuropathy of experimental autoimmune neuritis (EAN). This model was established by immunizing mice with the myelin protein zero peptide. Multiple administrations of SHED-CM from the day reaching around the peak of symptoms ameliorated significantly the clinical score with slightly recovered motor function. The administrations upregulated myelin basic protein (MBP) and a critical transcriptional factor for myelination EGR2 and phosphorylation of HGF receptor c-MET, but conversely downregulated the negative regulator c-JUN in the sciatic nerves, suggesting the augmentation of remyelination. Moreover, SHED-CM augmented the proliferation of mouse Schwann cell line IMS32 cells partly depending upon HGF, neuregulin 1, and bFGF. SHED-CM induced phosphorylation of c-MET and neuregulin 1 receptors ErbB2-4. In primary mouse Schwann cells, SHED-CM upregulated EGR2 and MBP and promoted the EGR2-mediated myelination. SHED-CM has potent therapeutic effects on the peripheral neuropathy of EAN by promoting the remyelination of Schwann cells possibly through EGR2 upregulation.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-22449-6.

Introduction

Guillain-Barré syndrome (GBS) is an autoimmune peripheral demyelinating disease, which is a common cause of acute flaccid paralysis characterized by symmetrical weakness of the limbs, numbness, and hyporeflexia^1–3. GBS typically occurs after an infectious disease, and number of evidence indicates that molecular mimicry exists between microbial antigens and nerve². The immune response generates antibodies that cross-react with gangliosides on nerve membranes, resulting in nerve damage and functional blockade of nerve conduction². To date, there is no effective treatment for GBS and only symptomatic therapies such as intravenous immunoglobulin⁴ and plasma exchange⁵ are available.

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases and has been recognized as an animal model of human immune-mediated neuritis such as GBS^6–8. EAN disease is induced by immunizing mice with neurogenic components of the peripheral nerve system (PNS) such as myelin protein zero (P0) emulsified in adjuvant together with administration of pertussis toxin^9,10. P0 is a major structural component of the myelin sheath in the PNS and is specifically expressed by only Schwann cells¹¹. Schwann cells play a critical role in producing the myelin sheath around neuronal axons of motor and sensory neurons in the PNS for insulation and decreasing membrane capacitance in the axon^12,13. On the onset of neuroinflammation in the PNS, demyelination of the damaged neurons occurs, in which dedifferentiation of Schwann cells plays a central mechanism. The dedifferentiation is induced by downregulation of early growth response 2 (EGR2, also termed KROX20), the master inducer of peripheral myelination¹⁴, and conversely upregulation of c-JUN, the dedifferentiation inducer^15,16. Thereafter, for regeneration of PNS, redifferentiation of Schwann cells through the reverse process of the dedifferentiation is critically important, leading to remyelination^12,16,17. In the EAN, Schwann cells are a major target of autoimmune responses mainly via P0-specific antibody-dependent macrophage-mediated cytotoxicity².

Mesenchymal stem cells (MSCs) are adult stem cells with self-renewal and multipotent differentiation capacity^18–20. Owing to their tissue repair and regeneration potential and immunomodulatory ability, they have attracted great interest in the field of regenerative medicine and have shown potent therapeutic effects on a variety of diseases including neurological disorders²¹. Although MSCs have multipotent differentiation potential even in vivo, numbers of evidence revealed that the therapeutic effects of MSCs highly rely on paracrine effects by secretion of a variety of bioactive factors including cytokines, growth factors, neurotrophic factors, and exosomes^22–24. Therefore, the therapy using only the conditioned medium (CM) or MSC secretome in place of MSC transfer is currently considered to have several advantages as a safer cell-free therapy; the therapy has much fewer concerns regarding ethical regulations and safety risks such as immunogenicity, embolism, thrombosis²⁵, or tumor progression^26,27. With the aim of translating this into practical medicines, we recently established an immortalized dental pulp stem cell line from human exfoliated deciduous tooth (SHED), which constantly secrets abundant cytokines²⁸. We demonstrated that the SHED-CM has potent therapeutic effects on the pressure ulcer formation by promoting angiogenesis and oxidative stress resistance through vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF)²⁸.

Dental pulp develops embryologically from ectomesenchyme of neural crest origin, which is the same origin as cells of the PNS²⁹. This property differs greatly from those of other MSCs, including bone marrow-derived MSCs, which originate from the mesenchyme. Therefore, dental pulp stem cells have high potential to differentiate into various types of cells, especially those of the ectodermal lineage, such as neurons, Schwann cells, and glial cells, which cells are crucial for nerve function^30,31. Dental pulp cells have a high capacity to secrete neurotrophic factors, which are critical for nerve cell survival, growth, and repair; nerve growth factor (NGF), brain derived neurotrophic growth factor (BDNF), neurotrophin-3 (NT-3), glia derived neurotrophic factor (GDNF), VEGF, HGF, neuregulin 1 (NRG1) and basic fibroblast growth factor (bFGF)^32,33. Additionally, SHED secretes exosomes, which are an emerging and effective therapeutic strategy for promoting nerve regeneration and functional recovery. Moreover, the PNS has a higher regenerative capacity than the central nerve system (CNS), which is mostly dependent on the high plasticity of Schwann cells. These unique properties make SHED more suitable for treating peripheral neuropathy. Therefore, SHED-CM could be effective for treatment of the peripheral neuropathy^30,31. Accumulating evidence have revealed that the cell transfer therapy using MSCs including SHED, as well as cell-free therapy using MSC-CM have therapeutic effects on various neurodegenerative disease models including Alzheimer’s and Parkinson’s disease, spinal cord injury, ischemic brain injury, peripheral neuropathy, and so on^27,34. However, to date, no therapeutic effects of MSCs or their CM on the EAN have been reported yet. In the present study, therefore, we have investigated the therapeutic potentials of SHED-CM on the EAN established by immunizing C57BL/6 mice with P0_180−199 peptide^9,10. The present results suggested that SHED-CM has potent therapeutic effects on the peripheral neuropathy of EAN by promoting the remyelination of Schwann cells possibly through EGR2 upregulation.

Materials and methods

Cell culture

Immortalized SHED cell line (Clone # B-2), which was established by inducing genes of human telomerase reverse transcriptase, human papillomavirus type 16 E6 and E7, and human B cell-specific Moloney murine leukemia virus integration site 1²⁸ (Cysay Corporation), was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen) at 37°C in an atmosphere of 5% CO₂/95% air. Mouse Schwann cell line IMS32 (Catalogue # SWN-IM32C, Cosmo Bio) cells³⁵ were cultured in its specific medium (Catalogue # SWNMM, Cosmo Bio) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C in an atmosphere of 5% CO₂/95% air. SHED and IMS32 cells were passaged using trypsin once at the end of the week, after the medium had been changed once in the middle of the week. The cells were split at ratios of approximately 1:10 and 1:5, respectively. Passaging was generally performed when cell confluence exceeded 90%.

Preparation of SHED-CM

Typically, SHED that had undergone 15 to 25 passages were routinely used to prepare CM. After SHED reached 70–80% confluency, they were washed twice with PBS and once with serum-free DMEM containing 4,500 mg/L high glucose but no glutamate, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, phenol red and sodium pyruvate (Gibco) and then cultured in serum-free DMEM for 72 h. The culture supernatants were collected, centrifuged at 1,750 × g for 10 min to remove the cell debris, and filtered through a 0.22 μm filter. The culture supernatants were used as the CM and stored at 4°C or − 80°C. Ten-fold (10×) concentrated CM was prepared using a 10 kDa Amicon Ultra-0.5 mL Centrifugal Filters (Merck Millipore) according to the manufacturer’s instruction. Briefly, 0.5 mL of SHED-CM was applied into the Amicon Filter device and centrifuged at 14,000 × g for 15 min. To recover the concentrated SHED-CM, the Amicon filter device was then placed upside down in a clean microcentrifuge tube and centrifuged at 1,000 × g for 2 min. There was no significant decrease in SHED cell viability when cultured for 72 h under serum-free conditions for SHED-CM preparation (Fig. S1). High consistency in cytokine concentration was observed across different batches of SHED-CM preparation (Fig. S2).

Mice

C57BL/6 male mice, 6 ~ 7 weeks old, were purchased from Sankyo Labo Service. All mice were maintained under pathogen-free conditions, and all animal experiments were approved by the President and by the Institutional Animal Care and Use Committee of Tokyo Medical University (Approval numbers: R4-107, R5-100, and R6-012) and performed in accordance with institutional, science community, and national guidelines for animal experimentation and the Animal Research: Reporting of In Vivo Experiments guidelines.

EAN mouse model

EAN mouse model was established as reported^7,9,10,36. Briefly, 50 µL of 2 mg/mL of P0_180−199 peptide (SSKRGRQTPVLYAMLDHSRS, Biologica) emulsified in an equal volume of Freund’s complete adjuvant (Becton Dickinson) containing 20 mg/mL heat killed Mycobacterium tuberculosis H37Ra (List Biological Laboratories) was injected subcutaneously into the hind limbs of the mouse on day 0 and day 7. On the day before the first immunization, 400 ng pertussis toxin (List Biological Laboratories), and on day 1 and 3 after the first immunization, 300 ng pertussis toxin were given intraperitoneally. Mice were scored for clinical signs: 0, no clinical signs; 1, less lively; 2, tail paresis or mild limb paresis; 3, limb paresis or mild ataxia; 4, severe ataxia. Although the clinical scores were not assessed completely blindly, a set of objective scoring criteria was meticulously applied to minimize personal bias. The work has been reported in line with the ARRIVE guidelines 2.0. Mice were euthanized by cervical dislocation before dissection of tissues.

Treatment with SHED-CM

After immunization with the P0 peptide two times at one week interval, mice gradually showed paralysis of tail and limbs and the clinical score greatly increased with reducing the running ability. The mice were randomly divided into two groups (n = 5): SHED-CM or control medium treatment. Starting on day 15, when the clinical score approached the plateau of clinical score, 200 µL of SHED-CM or control medium was administered intraperitoneally into the EAN mice multiple times as indicated consecutively every two or three days.

Histological analysis

Sciatic nerves were removed and subjected to analyses with the transmission electron microscopy (TEM) analysis. After pre-fixation in Karnofsky’s solution, the tissue was washed with 0.1 M phosphate buffer and post-fixed overnight with osmium tetroxide. The fixed tissue was dehydrated with ethanol, replaced with QY-1 (Nisshin-EM), and an Epon inclusion specimen was prepared using Quetol 812 (Nisshin-EM). Ultrathin sections of the inclusion specimens were prepared with an ultramicrotome and double stained with uranyl acetate and lead acetate and evaluated by TEM using a 1400 Flash (JEOL). Digital electron micrographs of sciatic nerve sections were analyzed to determine the G-ratio, which is defined as the ratio of axon diameter (excluding myelin) to myelinated fiber diameter (including myelin), and the diameter of the axons. Using Gration software (http://gratio.efil.de), which was integrated with ImageJ ((https://imagej.net/ij/) for analysis, the outer and inner diameters of the myelin sheath were measured from 50 randomly selected fibers per mouse.

Motor function assessment

Functional running ability was tested using a treadmill (Exer 3/6, Columbus Instrument) as reported⁹. Briefly, mice were set to complete a 36-s run at a treadmill speed of 15 cm/s. Running times in each mouse were measured every two or three days, and mice that cannot successfully complete the running task for the 36-s interval were considered to fail.

Cytokine measurement

Content of cytokines in the CM was comprehensively analyzed using Human Cytokine Antibody Array G Series 4000 (RayBiotech) at Filgen Inc. This array is a multiplex enzyme-linked immunoassay (ELISA) array that can semi-quantitatively determine the expression of 274 different cytokines (Table S1). The intensity of each signal was determined by laser scanning using GenePix 4400 A (Molecular Devices). Cytokines expressed in SHED-CM at levels 1.5-fold or greater than those in control medium background is considered significant, according to the manufacturer’s instruction. The concentrations of insulin-like growth factor binding protein-4 (IGFBP-4), VEGF, HGF, bFGF, and NRG1 were determined with either unconcentrated or 10× concentrated SHED-CM using respective sandwich ELISA kits (R&D Systems), following the manufacturers’ instructions.

Western blot analysis

Sciatic nerves and cauda equina nerves were isolated and lysed in RIPA buffer (50 mM Tris-HCl pH7.6 containing 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) containing protease inhibitors (Sigma-Aldrich) and phosphatase inhibitors (Nacalai) with sonication, and centrifuged at 20,400 × g for 30 min at 4 °C. The supernatant was collected and the protein concentration was measured using the BCA Protein Assay kit (Takara). IMS32 cells were stimulated with 10-fold concentrated SHED-CM or control medium, recombinant (r)NRG1 (100 ng/mL, Biolegend), rHGF (50 ng/mL, Biolegend), and incubated at 37 °C. After 5 min, 1 mL of cold PBS was added to stop the stimulation and samples were centrifuged at 1,750 × g for 5 min at 4 °C. Resultant precipitates were lysed in RIPA buffer containing protease and phosphatase inhibitors, followed by sonication. The supernatants were then collected by centrifugation at 20,400 × g for 30 min at 4 °C, and used as samples for western blot. Concentration of SHED-CM or control medium was performed using 10 kDa Amicon Ultra-0.5 mL Centrifugal Filters (Merck Millipore) according to the manufacturer’s instructions. The samples were separated by SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membrane (Merck Millipore). The membrane was then blocked, and probed with antibodies against the following molecules: MBP (Catalog # ABN912, Merck Millipore), EGR2 (Clone # EPR4004, abcam), c-JUN (Clone # G-4, Santa Cruz), phospho-MET (Tyr1234/1235, Catalog # 3126, Cell Signaling), MET (Ab-1003, Catalog # SAB4300599, Merck Millipore), phospho-p44/42 MAPK ERK1/2 (Thr202/Tyr204, Catalog #9101, Cell Signaling), ERK1/2 (Clone #137F5, Cell signaling), phospho-AKT (Ser473, Catalog #9271, Cell Signaling), AKT (Catalog # 9272, Cell Signaling), phospho-ErbB2 (Tyr1196, Clone # D66B7, Cell Signaling), phospho-ErbB3 (Tyr1289, Clone # 21D3, Cell Signaling), phospho-ErbB4 (Tyr984, Catalog # 3790, Cell Signaling), or β-actin (Clone # C4, Santa Cruz). Then, the membrane was incubated with an appropriate secondary antibody conjugated to horseradish peroxidase, and visualized with the enhanced chemiluminescence detection system (Cytiva) according to the manufacturer’s instructions. Immunoreactive bands were detected with an iBright FL1500 Imaging System (Thermo Fisher Scientific). Each band intensity was quantified using iBright Analysis Software (Thermo Fisher Scientific).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from the sciatic nerves using RNeasy Mini Kit (Qiagen). cDNA was prepared using oligo(dT) primer and SuperScript IV Reverse Transcriptase (Invitrogen). qPCR was performed using KAPA SYBR Fast qPCR Kit (Kapa Biosystems) and the Thermal Cycler Dice Real-Time System according to the manufacturer’s instructions (Takara). Hypoxanthine phosphoribosyl transferase (HPRT) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to normalize mRNA. Relative expression of qPCR products was determined using the ΔΔCt method to compare mRNA expression of the target and housekeeping genes. Following specific mouse primer pairs were used (Takara): HPRT, 5′-TTGTTGTTGGATATGCCCTTGACTA-3′ and 5′-AGGCAGATGGCCACAGGACTA-3′; GAPDH, 5′-TGTGTCCGTCGTGGATCTGA-3′ and TTGCTGTTGAAGTCGCAGGAG-3′; TNF-α, 5’-TATGGCCCAGACCCTCACA-3’ and 5’-GGAGTAGACAAGGTACAACCCATC-3’; IFN-γ, 5’-CATTGAAAGCCTAGAAAGTCTG-3’ and 5’-CTCATGAATGCATCCTTTTTCG-3’; IL-17 A 5’-CTGATCAGGACGCGCAAAC-3’ and 5’-TCGCTGCTGCCTTCACTGTA− 3’; EGR2, 5’-GCACTTTAATGGCTTGGGACTGA-3’ and 5’-TTAAGGGTTGAGATGGCCAGAGA-3’; myelin basic protein (MBP), 5’-ATCCAAGTACCTGGCCACAG-3’ and 5’-TGTGTGAGTCCTTGCCAGAG-3’.

Cell proliferation assay

IMS32 cells (1 × 10³ cells/200 µL) were cultured in serum-free DMEM containing SHED-CM or control medium (0, 1, 3, 10, 30%) for 72 h, and cell proliferative activity was determined using CellTiter-Glo 2.0 Cell Viability Assay (Promega) and measured with GloMax Discovery Microplate Reader (Promega) according to the manufacturer’s instructions. To examine the effects of HGF, NRG1 and bFGF in SHED-CM on the cell proliferation, IMS32 cells were similarly cultured with SHED-CM (30%) that had been preincubated with neutralizing antibodies against HGF (Clone # 24612, R&D Systems) or NRG1 (Catalog # AF-396-NA, R&D Systems) or bFGF (Clone # BHFB15, Biolegend) or respective control antibodies for 30 min, and cell proliferative activity was determined as described above.

Exosome preparation

SHED-CM was separated into two fractions by ultracentrifugation using the Beckman Optima MAX-XP Ultracentrifuge at 186,000 × g for 120 min at 4°C with TLA-55 rotor (Beckman Coulter): the upper fraction contained SHED-CM without exosomes and the lower fraction contained precipitated exosomes from SHED-CM as described²⁸. The lower fraction was resuspended in the same volume of control medium before ultracentrifugation. The content of exosomes was analyzed by the NanoSight NS300 Nanoparticle Tracking Analysis System (Malvern Panalytical).

Isolation and culture of primary mouse Schwann cells

Mice were euthanized and their sciatic nerves were dissected out and placed into PBS supplemented with penicillin/streptomycin. Epineurium, connective tissue, blood vessels were stripped off with fine forceps. The nerves were cut into small pieces and collected into 15 mL conical tube containing DMEM. Enzymatic digestion was performed with 5 mL of 0.05% collagenase in DMEM at 37°C for 2 h. The enzymatic activity was then stopped by adding 5 mL of DMEM containing 10% FBS and centrifuged at 440 × g for 5 min. The cells were gently resuspended by pipetting in 2 mL of DMEM containing 10% normal horse serum (Serana), 1 µM forskolin (Wako) and 10 ng/mL NRG1. The cells were then seeded into poly-L-lysine-coated wells in 24-well plates at a volume of 0.5 mL per well. The culture medium was replaced every 3 days with 0.5 mL of fresh medium containing the same constituents until the cells became confluent. Cells at passages 2 to 4 were used in all experiments described in the text. To induce myelination of Schwann cells, it was replaced with DMEM containing 1% FBS, 1% N2 Supplement (Wako), 50 µg/mL L-ascorbic acid and stimulated with or without SHED-CM (50%) for 5 days.

Immunofluorescence staining

Cells were fixed with 4% paraformaldehyde for 20 min. After washing in PBS, cells were blocked with 1% BSA and permeabilized with 0.2% Triton X-100 in PBS for 30 min. Subsequently, cells were incubated with primary antibodies against EGR2 (Catalog # BS-8368R, Bioss) and MBP (Clone # F-6, Santa Cruz), followed by incubation with anti-rabbit IgG Alexa Fluor 594 (Biolegend) and anti-mouse IgG Alexa Fluor 488 (abcam). Hoechst33258 (Dojindo) was used for counterstaining. Cells were captured under EVOS FL (Thermo Fischer Scientific).

Statistical analyses

Data are expressed as the mean ± standard deviation (SD) of the mean for each group. Statistical analyses were performed using the unpaired, two-tailed Student’s t-test for comparisons of two groups and one-way analysis of variance with the Tukey or Dunnett multiple comparison test for comparing more than three groups using GraphPad Prism 9 (GraphPad Software). P < 0.05 was considered statistically significant.

Results

Multiple administrations of SHED-CM ameliorate the development of EAN with remyelination and slight recovery of motor function

After two times immunization with P0 peptide, the clinical symptom of the EAN mice increased and reached the peak of the clinical score on day around 15, and then SHED-CM or control medium was intraperitoneally administered every two or three days multiple times. Thereafter, the clinical score significantly decreased by administrations of SHED-CM compared with those of control medium (P < 0.05 during days 20 ~ 31, Fig. 1A). On day 31 the sciatic nerves were removed and subjected to histological analyses. The administrations of SHED-CM significantly reduced the G-ratio (P < 0.05) and tended to increase the thickness of myelin compared with those of control medium (Fig. 1B,C). MBP is a major constituent of the myelin sheath of Schwann cells in the PNS¹¹. Western blot analysis revealed that the expression of MBP at protein level was significantly increased in the sciatic nerves by administrations of SHED-CM compared with those of control medium (P < 0.05, Fig. 1D,E). Similar increase in the expression of MBP at protein level by administrations of SHED-CM was also observed in the cauda equina nerves (P < 0.01, Fig. S3, S10). Consistent with these results, SHED-CM greatly augmented the expression of EGR2 (P < 0.01) but conversely reduced the expression of c-JUN (P < 0.05) in the sciatic nerves (Fig. 1D,E)¹⁴.

Fig. 1.

Multiple administrations of SHED-CM ameliorate the development of EAN with remyelination. (A) C57BL/6 mice were immunized twice with P0_180−189 peptide, and from day 15 SHED-CM or control medium (200 µL) was intraperitoneally administered into the EAN mice every two or three days as indicated. Clinical symptom score was measured with time. (B) On day 31, the sciatic nerves were removed, and their cross sections were stained with toluidine blue and analyzed with a light microscopy and TEM. Representative images are shown. (C) The G-ratio and axon diameter were determined from digital electron micrographs. (D) Tissue lysates were prepared from the sciatic nerves for western blot analysis using antibodies against MBP, EGR2, c-JUN, and β-actin. Full-length blots/gels are presented in Fig. S4. (E) Each band intensity was quantitated and the relative intensity to that of β-actin was calculated and compared. Data are shown as the mean ± SD (n = 5) and are representative of more than two independent experiments. P values were determined using unpaired two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Next, the effects of SHED-CM on the motor function were assessed using a treadmill. The motor function of EAN mice started to decline progressively after around the onset of peripheral neuropathy on day 7. The administrations of SHED-CM slightly recovered the motor defect compared with those of control medium as well (P < 0.05 on days 17, 21 and 29, Fig. 2). These results suggest that the multiple administrations of SHED-CM to EAN mice significantly ameliorate the development of EAN with remyelination and slight recovery of motor function.

Fig. 2.

Multiple administrations of SHED-CM slightly recover the motor function of EAN mice. C57BL/6 mice were immunized twice with P0_180−189 peptide, and from day 15 SHED-CM or control medium (200 µL) was intraperitoneally administered into the ENA mice every two or three days as indicated. Clinical symptom score (A) and functional running ability (B) were measured with time. Data are shown as the mean ± SD (n = 5) and are representative of two independent experiments. P values were determined using unpaired two-tailed Student’s t-test. *P < 0.05, **P < 0.01.

SHED-CM contains a variety of cytokines and growth factors, which have potential to show protective effects on the peripheral neuropathy

To determine the concentrations of cytokines and growth factors in SHED-CM, we used the Human Cytokine Antibody Array G Series 4000 that can semi-quantitatively determine the expression of 274 different cytokines (Table S1). As reported²⁸, SHED-CM contains a variety of cytokines and growth factors (Fig. 3A), including those which have ability to induce peripheral nerve regeneration; HGF, NGF, BDNF, VEGF, NRG1, and so on^37–39. Among them, NRG1, a member of the transmembrane and secreted epidermal growth factor (EGF)-like growth factors signaling through ErbB tyrosine kinase receptors, plays critical roles in neuronal differentiation and myelination⁴⁰. The concentration of NRG1 in the SHED-CM was determined using 10× concentrated SHED-CM to be approximately 7.4 pg/mL (Fig. S2). Consistent with the results, western blot analysis using antibody against NRG1 detected the band slightly in 1× and greatly in 10× concentrated SHED-CM (Fig. 3B). The concentrations of HGF and bFGF were determined to be approximately 7.1 ng/mL and 270 pg/mL, respectively (Fig. S2). Thus, SHED-CM contains a variety of cytokines and growth factors, which have potential to show protective effects on the peripheral neuropathy.

Fig. 3.

SHED-CM contains a variety of cytokines and growth factors which have potential to show protective effects on the peripheral neuropathy. (A) The content of cytokines in SHED-CM was comprehensively analyzed using the cytokine antibody array, and the concentrations of 274 different cytokines were semi-quantitatively determined (Table S1). Cytokines expressed in SHED-CM at levels 1.5-fold or greater than those in control medium are shown. (B) The 1× and 10× concentrated SHED-CM and control medium were analyzed for expression of NRG1 by western blot using antibody against NRG1. Full-length blots/gels are presented in Fig. S5.

Administrations of SHED-CM augment the expression of phosphorylated c-MET and suppress the expression of proinflammatory cytokines in the sciatic nerves

Because HGF and its receptor c-MET were demonstrated to play critical roles in repair and regeneration of Schwann cells^41,42, tissue lysates of the sciatic nerves were prepared from the EAN mice administered with SHED-CM or control medium on day 23 (Fig. 4A). The western blot analysis revealed that SHED-CM enhanced more phosphorylation of c-MET than control medium (P < 0.01, Fig. 4B,C). In addition, RT-qPCR analysis revealed that SHED-CM reduced the mRNA expression of proinflammatory cytokines such as TNF-α (P < 0.05), IFN-γ (P < 0.05), and IL-17 A (P < 0.001) in the sciatic nerves (Fig. 4D). Thus, these results suggest that SHED-CM activates c-MET and reduces the inflammation in the sciatic nerves.

Fig. 4.

Administrations of SHED-CM augment the expression of phosphorylated c-MET and suppress the expression of proinflammatory cytokines in the sciatic nerves. C57BL/6 mice were immunized twice with P0_180−189 peptide, and from day 15, SHED-CM or control medium (200 µL) was intraperitoneally administered every two or three days as indicated. Clinical symptom score was measured with time (A). On day 23 the sciatic nerves were removed and their tissue lysates were subjected to western blot analysis using antibodies against phosphorylated c-MET together with β-actin (B). Full-length blots/gels are presented in Fig. S5. Each band intensity was quantified, and the relative intensity to that of β-actin was calculated and compared (C). Total RNAs were also extracted from the sciatic nerves and subjected to RT-qPCR to detect the expression of inflammatory cytokines, TNF-α, IFN-γ, and IL-17 A (D). Data are shown as the mean ± SD (n = 4 ~ 5) and are representative of two independent experiments. P values were determined using unpaired two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

SHED-CM augments proliferation of mouse Schwann cell line IMS32 cells independently of exosomes

Next, to investigate the possible molecular mechanism underlying the therapeutic effects of SHED-CM on EAN, mouse Schwann cell line IMS32 was used in vitro, and effects of SHED-CM on their proliferation were examined. Correlating with increasing percentages of SHED-CM, IMS32 cells became firmly adhered onto the plate, extended, and expanded (Fig. 5A). Consistent with these morphological changes, SHED-CM dose-dependently augmented the proliferation of IMS32 cells (P < 0.001 at 10 and 30%, Fig. 5B).

Fig. 5.

SHED-CM augments proliferation of mouse Schwann cell line IMS32 cells independently of exosomes. Mouse Schwann cell line IMS32 cells were cultured with different percentages of SHED-CM or control medium for 72 h. Representative photographs of the cell appearance taken under microscopy are shown (A). Proliferative activity was determined using CellTiter-Glo (B). SHED-CM was separated into two fractions by ultracentrifugation: the upper fraction contained SHED-CM without exosomes and the lower fraction contained precipitated exosomes from SHED-CM. The precipitate was resuspended in the same volume of control medium before ultracentrifugation. The content of exosomes was analyzed with scanning electron microscopy analysis (C) and nanoparticle tracking analysis (D) using NanoSight, together with resultant concentrations of exosomes (E). IMS32 cells were cultured in the presence of each fraction together with total SHED-CM for 3 days and proliferative activity was determined using CellTiter-Glo (F). Data are shown as the mean ± SD (n = 3) and are representative of two independent experiments. P values were determined using one-way analysis of variance with the Dunnett (B) or Tukey (E, F) multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

SHED-CM mainly consists of proteins such as cytokines and extracellular vesicles such as exosomes^24,26. Exosomes play critical roles in transmission of disease states as a tool for cell-cell communication, and therefore exosomes derived from MSCs have been applied as a cell-free therapy alternative to MSCs in a variety of disease models⁴³. We next examined the role of exosomes in SHED-CM on the augmentation of proliferation of IMS32 cells. SHED-CM was separated into two fractions by ultracentrifugation: the upper fraction contained mainly proteins and was depleted of exosomes, and the lower fraction contained precipitates of exosomes derived from the SHED-CM. The precipitate was resuspended in the same volume of control medium as before the ultracentrifugation. The concentration and purity of exosomes in each fraction was determined by the NanoSight (P < 0.001 between upper and lower fractions, Fig. 5C–E). Then, the effects of each fraction on the augmentation of proliferation of IMS32 cells were examined. The upper fraction of SHED-CM depleted of exosomes similarly augmented the proliferation of IMS32 cells to total SHED-CM, whereas the lower fraction containing exosomes derived from SHED-CM showed much less enhancing effect (P < 0.05 between upper and lower fractions, Fig. 5F). These results suggest that SHED-CM induces proliferation of IMS32 Schwann cells independently of exosomes.

SHED-CM augments proliferation of IMS32 cells in a party HGF-, NRG1-, and bFGF-dependent manners with increased expression of EGR2.

To further examine the molecular mechanism, we next examined which molecules in the SHED-CM are important for augmentation of proliferation of IMS32 cells. To address this question, IMS32 cells were cultured with several recombinant cytokines and growth factors which are possibly present in the SHED-CM, and their proliferative activities were determined. Among them, HGF (P < 0.05), NRG1 (P < 0.001), and bFGF (P < 0.001) significantly augmented the proliferation of IMS32 cells (Fig. 6A). To confirm their contribution, the effect of neutralizing antibodies against them were examined. These antibodies partially but significantly inhibited the SHED-CM-induced proliferative activity compared with control antibodies (P < 0.01 with ant-HGF, P < 0.01 wit anti-NRG1, P < 0.01 with anti-bFGF, Fig. 6B). Consistent with these results, SHED-CM augmented the phosphorylation of HGF receptor, c-MET, and NRG1 receptors, ErbB2, 3, and 4 (Fig. 6C). ErbB3 or 4 forms a heterodimer with ErbB2, an orphan receptor lacking a ligand-binding domain but having a signal transduction pathway as a co-receptor⁴⁴. The SHED-CM also induced phosphorylation of their downstream signaling molecules such as ERK1/2 and AKT important for cell proliferation (Fig. 6C). Moreover, consistent with the in vivo results (Fig. 1C), SHED-CM, as well as HGF, NRG1, and bFGF increased the expression of EGR2 in IMS32 cells (Fig. 6D). These results suggest that SHED-CM augments proliferation of IMS32 Schwann cells in a partially HGF-, NRG1-, and bFGF-dependent manner with enhanced phosphorylation of their receptors and downstream signaling molecules including c-MET, ErbB2, 3, and 4, ERK1/2, and AKT, and increased expression of EGR2.

Fig. 6.

SHED-CM augments proliferation of IMS32 cells in a partially HGF-, NRG1- and bFGF-dependent manner with increased expression of EGR2. (A) IMS32 cells were cultured with several recombinant cytokines and growth factors which are present in the SHED-CM as indicated for 72 h and their proliferative activities were determined using CellTiter-Glo. (B) IMS32 cells were cultured with 30% SHED-CM in the presence of antibodies against HGF, NRG1, and bFGF together with their respective control antibodies for 72 h, and proliferative activity was determined using CellTiter-Glo. (C) IMS32 cells were incubated with 50% of 10× concentrated SHED-CM, HGF (50 ng/mL) or NRG1 (100 ng/mL) in serum-free medium for 5 min, and subjected to western blot analysis using antibodies against phosphorylated c-MET, ErbB2, 3, and 4, ERK1/2, AKT, and their respective total molecules or β-actin. Full-length blots/gels are presented in Fig. S6. 7. (D) Cell lysates were prepared from the samples obtained in (A) and subjected to western blot analysis using antibodies against EGR2 and β-actin. Full-length blots/gels are presented in Fig. S8. Each band intensity was quantified and the relative intensity to that of β-actin was calculated. Data are shown as the mean ± SD (n = 3, A, B) and are representative of three independent experiments. P values were determined using one-way analysis of variance with the Dunnett multiple comparison test (A) or unpaired two-tailed Student’s t-test (B). *P < 0.05, **P < 0.01, ***P < 0.001.

SHED-CM augments EGR2 expression in primary Schwann cells and promotes their myelination in EGR2-dependent manner

Finally, the effects of SHED-CM on primary Schwann cells obtained from mouse sciatic nerves were examined. Similar to the results obtained using IMS32 cells, SHED-CM but not control medium slightly augmented proliferation of primary Schwann cells in dose-dependent manner (P < 0.05 at 50%, Fig. 7A). Immunofluorescence staining analysis revealed that SHED-CM greatly enhanced the expression of both EGR2 and MBP (Fig. 7B). Consistent with these results, western blot analysis also showed that SHED-CM greatly enhanced the expression of EGR2 (P < 0.001) and MBP (P < 0.01) compared with control medium (Fig. 7C,D). To further examine the role of EGR2 in the SHED-CM-induced myelination of primary Schwann cells, the effects of knockdown of EGR2 expression by its specific siRNA were next examined. The knockdown of EGR2 expression (P < 0.05) in primary Schwann cells significantly reduced their myelination with decreased expression of MBP (P < 0.001 between EGR2 and control siRNAs) compared with control siRNA treatment (Fig. 7E–G). These results suggest that SHED-CM augments EGR2 expression in primary Schwann cells and promotes their myelination in EGR2-dependent manner.

Fig. 7.

SHED-CM augments EGR2 expression in primary Schwann cells and promotes their myelination in EGR2-dependent manner. Primary Schwann cells obtained from mouse sciatic nerves were cultured with SHED-CM (0, 10, 30, and 50%) in DMEM medium containing 1% FBS for 3 days (A) or 5 days (B–D). Proliferative activity was determined using CellTiter-Glo (A). Immunofluorescence staining analysis of primary Schwann cells cultured with 50% SHED-CM or control medium was performed using antibodies against EGR2 and MBP (B). Cell lysates of primary Schwann cells cultured with 50% SHED-CM or control medium were prepared and subjected to western blot analysis using antibodies against EGR2, MBP, and β-actin (C). Full-length blots/gels are presented in Fig. S9. Each band intensity of EGR2, MBP, and β-actin was quantitated and the relative intensity to that of β-actin was calculated and compared (D). Primary Schwann cells obtained from mouse sciatic nerves were transfected with EGR2-specific siRNA or control siRNA and cultured for 3 days and subsequently cultured with 50% SHED-CM or control medium for further 5 days. Immunofluorescence staining analysis was performed using antibodies against EGR2 and MBP (E). RT-qPCR was performed to measure the mRNA expression of EGR2 and MBP before culture with SHED-CM (F) and after culture with SHED-CM for further 5 days (G). Data are shown as the mean ± SD (n = 3) and are representative of three independent experiments. P values were determined using one-way analysis of variance with the Dunnett (A) or Tukey (G) multiple comparison test or unpaired two-tailed Student’s t-test (D, F). *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

In the present study, we have investigated the therapeutic potentials of SHED-CM on the peripheral neuropathy of EAN. Multiple intraperitoneal administrations of SHED-CM to EAN mice starting on day 15 significantly ameliorated the clinical score with increased myelination. The rationale for choosing day 15 as the start point is that the clinical score is high enough in the early phase to be sensitive to subsequent treatment. In the sciatic nerves, the administrations of SHED-CM augmented the phosphorylation of c-MET, a receptor for HGF, and the expression of the critical transcriptional factor for myelination, EGR2¹⁴. In contrast, the administrations conversely suppressed the expression of the negative regulator of myelination, c-JUN, which drives demyelinating Schwann cells back to the immature state^15,16, and inhibited the expression of inflammatory cytokines. In vitro SHED-CM dose-dependently induced the proliferation of mouse Schwann cell line IMS32 in a partially HGF-, NRG1-, and bFGF-dependent manner. SHED-CM induced phosphorylation of c-MET, ErbB2, 3, 4, and their downstream signaling molecules ERK1/2 and AKT. Notably, SHED-CM also augmented the expression of EGR2 and MBP in primary Schwann cells and promoted the myelination in EGR2-dependent manner. Taken together, the present results suggest that SHED-CM has therapeutic effects on the peripheral neuropathy of EAN by promoting the regeneration of Schwann cells possibly through EGR2. This is consistent with that EGR2 is known to have rather suppressive activity in T cell and macrophages^45,46.

For the peripheral nerve regeneration, important roles of a variety of secreted factors including NRG1, HGF, bFGF, GDNF, BDNF, VEGF, nerve growth factor, and so on have been reported^37–39. These growth factors are highly expressed in injured nerves and activate neuronal cells and Schwann cells for repairing and regenerating the injured nerves. Of note, most of these growth factors are likely present in the SHED-CM, although the concentrations are largely varied depending on individual factors. NRG1 plays the most important role in Schwann cell development, survival and myelination for peripheral nerve regeneration^40,47,48. HGF is a multifunctional protein promoting angiogenesis cell survival, cell migration and anti-inflammation in a variety of cell types through c-MET, and also inducing neurotrophic effects in both the CNS and PNS⁴². HGF was shown to promote the peripheral nerve regeneration by activating Schwann cells and their remyelination in the peripheral nerve crush model⁴¹. bFGF is also known to have ability of facilitating axon growth and nerve regeneration after peripheral nerve injury⁴⁹. GDNF is a survival factor for spinal cord motor neurons and dorsal root ganglion neurons, and protects injured peripheral nerves and promotes axonal regeneration⁵⁰. GDNF was also reported to promote the repair Schwann cell state and inhibits the switch to myelination⁵¹. IGF-1 regulates several functions to promote cell motility, survival, proliferation and myelination of Schwann cells, and also acts directly on neurons to support neurite outgrowth, resulting in regeneration in several different settings⁵². Thus, the therapeutic effects of SHED-CM on EAN are most likely due to the synergistic effects of these molecules, including HGF, NRG1, bFGF and possibly others, as their antibodies only partially inhibited SHED-CM-induced proliferation of IMS32 cells.

Schwann cells are the glia cells, which play an important role in myelinating axon of the PNS^12,53. Immature Schwann cells differentiate into either myelinating or non-myelinating mature Schwann cells by modulating with NRG1^40,48. EGR2 is one of early-immediate response genes induced by multiple stimuli including cytokines, growth factors, and cell stress. In the PNS, EGR2 is expressed in only myelinating Schwann cells among different stages of Schwann cells and plays an important role in transforming the immature Schwann cells into myelinating Schwann cells¹⁴. The regeneration of peripheral nerves highly relies on the plasticity of Schwann cells. Upon nerve injury, fully mature Schwann cells undergo the dedifferentiation towards a cell phenotype resembling different properties of immature Schwann cell stage^12,30,54. During the dedifferentiation, Schwann cells downregulate the factors promoting myelination such as EGR2, and conversely upregulate the dedifferentiation inducer c-JUN¹⁵. Thereafter, Schwann cells start breaking down myelin, and activate a repair program which provides a supportive environment for axonal regrowth. Then, Schwann cells start re-enhancing EGR2 and forming cellular conduits along which axons can regrow and express molecules promoting the regeneration of injured neurons^12,38.

EGR2 is a key transcription factor, which is modulated by axonal contact through membrane-bound NRG1 type III and soluble NRG1, for induction of Schwann cell differentiation and peripheral myelination⁴⁷. Studies of EGR2-decicient mice and mutations in EGR2 gene in patients with peripheral neuropathies revealed that EGR2 is essential for myelination of peripheral nerves^55,56. Induction of EGR2 expression highly depends on NRG1-mediated signaling pathways through ERK1/2 and subsequent Yin Yang 1 during peripheral myelination^57,58. Upregulation of EGR2 by the other factors such as epidermal growth factor, platelet-derived growth factor-BB, platelet-derived growth factor-AA, IL-1β, bFGF was also reported in MSCs and osteoprogenitors^59,60.

Although we have found the protective effects of SHED-CM on EAN by upregulating EGR2 in Schwann cells, intriguingly, EGR2 also plays immunoregulatory roles in T cells and macrophages^45,46. In T cells, EGR2 plays vital roles in the induction of T cell anergy and the suppressive activities of regulatory T cells expressing lymphocyte activation gene 3^61,62. In macrophages, EGR2 is important for polarization into M2 macrophages, which possess tissue repair and anti-inflammatory properties^63,64. Although SHED-CM was also reported to have such immunosuppressive, tissue repair and anti-inflammatory effects via T cells and macrophages^34,65,66, whether SHED-CM-induced upregulation of EGR2 in these cells contributes to the protective effects of SHED-CM on EAN remains to be addressed.

Compared to other MSCs, the likelihood of successfully preparing stem cells from dental pulp tissue is much lower. This is because the dental pulp tissue obtained from a child is very small, and the stem cells are often absorbed too²⁹. Therefore, even if SHEDs are obtained, there are generally only a few stem cells, although SHEDs have a unique potential to highly differentiate into neurons. To make the most effective use of this potential, immortalization is one of good options, particularly if only the CM is used for treatment, as this poses a much lower risk of tumorigenesis. We previously demonstrated that, compared to primary SHED-CM, immortalized SHED-CM contains a concentration of cytokines that is overall ~ 10 times higher²⁸. Therefore, it is highly expected that immortalized SHED-CM will show more potent therapeutic effects than primary SHED-CM, despite the absence of direct data demonstrating this difference. To meet the regulatory requirements for manufacturing and quality control and eventually for implementation in practical human medicine, it is necessary to produce CM that is consistently safe and effective. Immortalized SHED could be an ideal cell source to produce such CM, because it grows infinitely and vigorously, and stably and consistently secretes a variety of cytokines. But rigorous safety tests, such as monitoring for chromosomal abnormalities using karyotyping test, are necessary and critical to ensure safety⁶⁷.

Conclusion

The present results revealed that SHED-CM has potent therapeutic effects on the peripheral neuropathy of EAN possibly through the upregulation of EGR2 and subsequent promotion of EGR2-mediated remyelination of Schwann cells. SHED-CM contains various cytokines that act on Schwann cells. Among these, HGF, NRG1 and bFGF partly contribute to the upregulation of EGR2. Thus, the CM of immortalized SHED could pave an avenue to create a novel type of cell-free regenerative medicine, although further investigation addressing safety concerns, standardizing production methods, and demonstrating consistent therapeutic efficacy is warranted.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

BDNF

Brain derived neurotrophic growth factor

bFGF

Basic fibroblast growth factor

CM

Conditioned medium

CNS

Central nerve system

DMEM

Dulbecco’s modified eagle medium

EAN

Experimental autoimmune neuritis

EGR2

Early growth response gene 2

HGF

Hepatocyte growth factor

HPRT

Hypoxanthine phosphoribosyl transferase

IGFBP-4

Insulin-like growth factor binding protein-4

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

GBS

Guillain-Barré syndrome

GDNF

Glia cell derived neurotrophic factor

MBP

Myelin basic protein

MSCs

Mesenchymal stem cells

NGF

Nerve growth factor

NRG1

Neuregulin 1

NT-3

Neurotrophin-3

P0

Myelin protein zero

PNS

Peripheral nerve system

r

Recombinant

RT-qPCR

Reverse transcription-quantitative polymerase chain reaction

SD

Standard deviation of the mean

SHED

Stem cell line from human exfoliated deciduous teeth

VEGF

Vascular endothelial growth factor

Author contributions

HH designed and performed the experiments, analyzed data, and wrote the original draft. HH, ES, and AW performed the experiments and analyzed the data. NY, EH, JS, MY and SM performed the experiments. SM secured funding. FM, YK and IM reviewed and edited the manuscript. TY directed, conceived, designed, wrote, reviewed and edited the manuscript, and secured funding.

Funding

This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grant No. 22K07504, SM), and Cysay Corporation (TY).

Data availability

All data generated or analyzed during this study are included in this article.

Declarations

Competing interests

The authors declare no competing interests.

Ethic approval and consent to participate

The human cell study was approved by the Institutional Review Board of Tokyo Medical University (Project title: Induction of stem cell differentiation and proliferation and therapeutic applications; Approval number: T2021-0117, Date of approval: 2021.06.30). All animal experiments were approved by the President and by the Institutional Animal Care and Use Committee of Tokyo Medical University and performed in accordance with institutional, science community (Project title: Research on the regulation of neural disorders by cytokines and their related molecules; Approval number: R4-107, Date of approval: 2022.02.26; Approval number: R5-100, Date of approval: 2023.04.06; Approval Number: R6-012, Date of approval: 2024.02.29).

Consent for publication

All authors agreed to the publication of this paper.