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Regulation of autophagy in mesenchymal stem cells modulates therapeutic effects on spinal cord injury
Accepted Manuscript Review Regulation of autophagy in mesenchymal stem cells modulates therapeutic effects on spinal cord injury Fukai Ma, Ronggang Li, Hailiang Tang, Tongming Zhu, Feng Xu, Jianhong Zhu PII: DOI: Article Number: Reference:
S0006-8993(19)30367-1 https://doi.org/10.1016/j.brainres.2019.146321 146321 BRES 146321
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
13 May 2019 20 June 2019 2 July 2019
Please cite this article as: F. Ma, R. Li, H. Tang, T. Zhu, F. Xu, J. Zhu, Regulation of autophagy in mesenchymal stem cells modulates therapeutic effects on spinal cord injury, Brain Research (2019), doi: https://doi.org/10.1016/ j.brainres.2019.146321
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Regulation of autophagy in mesenchymal stem cells modulates therapeutic effects on spinal cord injury Fukai Maa,b,#, Ronggang Lia,c,#, Hailiang Tanga,#, Tongming Zhua, Feng Xua,d,*, and Jianhong Zhua,* aDepartment
of Neurosurgery, Fudan University Huashan Hospital and State Key
Laboratory of Medical Neurobiology, the Institutes of Brain Science, Shanghai Medical College, Fudan University, No.12 Urumqi Mid Road, Shanghai 200040, China. bNeurosurgery
Department, Shanghai Tenth People’s Hospital, Tongji University, No.
301 Middle Yanchang Road, Shanghai 200072, Shanghai, China. cDepartment
of Neurosurgery, Shanghai Public Health Clinical Center, Fudan
University, No. 2901 Caolanggong Road, Shanghai 201508, China. dDepartment
of Neurosurgery, Kashgar Prefecture Second People’s Hospital, No. 1
Jiankang Road, Kashgar 844000, China. *Correspondence: #These
[email protected] (F.Xu), [email protected] (J.Zhu)
authors contributed equally.
Abstract Transplantation with mesenchymal stem cells (MSCs) has shown beneficial effects in treating spinal cord injury. Autophagy is an evolutionarily conserved process of degradation and recycling of cellular components that plays an important role in tissue homeostasis and cellular survival. Whether regulating autophagy in MSCs may affect
their therapeutic potential in spinal cord injury repair has not yet been determined. In this study, autophagy was inhibited in MSCs with lentiviruses expressing short hairpin RNA (shRNA) to knock down Becn-1 expression, and autophagy was upregulated in MSCs under nutrient starvation. These MSCs were then labelled with Hoechst and applied to spinal cord-injured rats to evaluate their therapeutic effects. After transplanting MSCs into rats with spinal cord injuries, functional recovery, immunohistochemistry, and remyelination analyses were performed. After inducing autophagy, the MSCs exhibited an accumulation of LC3-positive autophagosomes in the cytoplasm. The expression levels of neurotrophic factors, including vascular endothelial growth factor and brain derived neurotrophic factor, were significantly higher in autophagic MSCs than normal MSCs. The in vivo study showed that more labelled MSCs migrated to the lesion site after induction of autophagy. Inducing autophagy in MSCs promoted functional recovery after spinal cord injury, whereas functional recovery was weak after inhibiting autophagy in MSCs. In contrast to the autophagy inhibition group, transplanting autophagic MSCs exhibited a greater positive impact on axon regeneration, growth of serotonergic fibers, blood vessel regeneration, and myelination, indicating a multifactorial contribution to spinal cord injury repair. These results suggest that autophagy plays important roles in MSCs during spinal cord injury repair. Regulation of autophagy in MSCs before in vivo transplantation may be a potential therapeutic interventional strategy for spinal cord injury.
Keywords Spinal cord injury, Autophagy, Mesenchymal stem cells, Regeneration
1. Introduction Spinal cord injury (SCI) is a devastating disease that is often caused by contusion, compression, or surgical intervention of the spinal cord (Cully, 2018; Takahashi et al., 2018). After injury, inflammation, ischemia, and apoptosis occur, contributing to spinal cord degeneration (Falnikar et al., 2016; Park et al., 2004). SCI often results in permanent loss of motor and sensory functions due to the inability to regenerate lost spinal cord tissue (Tsenkina et al., 2015; Wilems et al., 2015; Yao et al., 2018). Unlike peripheral nervous system regeneration, spontaneous regeneration after spinal cord injury is limited and incomplete (Comolli et al., 2009; Singh et al., 2018). SCI remains a unique clinical challenge requiring better treatment modalities (Anderson et al., 2018; Zhang et al., 2018). Various approaches have been investigated for restoring or improving regeneration in the hostile lesion environment after SCI (Stower, 2018). Improved therapies are still needed to enhance the long-term effectiveness of SCI treatment. The application of mesenchymal stem cells (MSCs) in regenerative medicine holds great promise for the treatment of various intractable central nervous system (CNS) diseases (Assinck et al., 2017; Khan et al., 2018). Previous studies demonstrated that MSCs are immune-privileged cells for in vivo transplantation due to their ability to regulate immune cell function and immune responses. MSCs exert
anti-inflammatory, neurotrophic, and angiogenic effects in SCI repair (Oliveri et al., 2014; Park, 2018). Many studies have shown that MSCs may restore the function of damaged spinal cord and promote functional recovery after SCI (Kang et al., 2012; Ma et al., 2018). Autophagy is an evolutionarily conserved cellular mechanism involving the formation of autophagosomes, which are responsible for degrading damaged and/or aged cytoplasm and cellular organelles (Yan et al., 2018). Autophagy is a crucial cellular process with important roles in maintaining homeostasis and remodeling during cellular development (Doherty and Baehrecke, 2018; Mizushima and Komatsu, 2011). Mounting evidence indicates that autophagic activation is required for selfrenewal, pluripotency, differentiation, and quiescence in stem cells, while dysfunctional autophagy may be related to various diseases (Hansen and Rubinsztein, 2018). However, the role of autophagy in the function of MSCs on SCI has yet to be explored. In this study, we first increased autophagy or inhibited autophagy in MSCs, and then transplanted the two types of MSCs into injured spinal cords to evaluate their effects on functional recovery.
2. Materials and methods 2.1 MSC isolation and culture All experimental procedures involving animals were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and approved by the Institutional
Animal Care and Use Committee. MSCs were isolated from Sprague-Dawley (SD) rats based on the protocols described in previous studies (Anokhina and Buravkova, 2007; Kim et al., 2009). After the rats were sacrificed, the femurs and tibias of the hind limbs were excised, and adherent tissues were removed. The bones were cut and the bone cavities were flushed with Dulbecco’s Modified Eagles Medium (DMEMF12; Hyclone) to obtain bone marrow. After passing through a 70-μm cell strainer, the marrow was centrifuged at 500 × g for 5 min. The pellet was resuspended in lowglucose DMEM supplemented with 15% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. A total of 106 cells were transferred to a T-25 cell culture flask and cultured at 37°C in 5% CO2. After 2 days, the nonadherent cells were removed, and the attached cells were cultured further. At the fourth or fifth passage, the cells were digested with 0.25% trypsin solution and collected for further experiments. 2.2 Flow cytometry analysis of cultured cells Digested cell aliquots (1 × 106 cells/mL) were placed on ice and stained with the following antibodies: anti-CD44 (BD Biosciences), anti-CD90.2 (Thy1.2; BD Biosciences), anti-stem cell antigen-1 (Sca-1; BD Biosciences), anti-CD34 (BD Biosciences), anti-CD45 (BD Biosciences), anti-CD117 (c-kit; BD Biosciences), antiCD11b (ITGAM; BD Biosciences), and anti-MHC II (eBioscience). Flow cytometry (FACSAria II, BD Biosciences) was used to analyze the data. 2.3 MSC differentiation assay Osteogenic differentiation and adipogenic differentiation were induced in MSCs
based on previously described methods (Pittenger et al., 1999). Briefly, the MSCs were cultured in differentiation medium for three weeks. Adipogenic differentiation was confirmed by Oil Red-O staining (Sigma-Aldrich, St. Louis, MO, USA). Osteogenic differentiation was confirmed with Alizarin Red staining (Sigma-Aldrich). Images were observed under an inverted phase-contrast microscope (Nikon, Tokyo, Japan). 2.4 Autophagic inhibition or induction in MSCs Oligonucleotides for Becn-1 short hairpin RNA (shRNA) (shBecn1) were synthesized by GenePharma. The shBecn1 sequence was as follows: 5′gatccGGAGAAAGGCAAGATTGAAGATTCAAGAGATCTTCAATCTTGCCTTT CTCCTTTTTTg-3′. Lentiviral vectors encoding the shRNA were then constructed to transfect MSCs to knock down expression of the essential autophagy gene Becn1 (Dang et al., 2014). MSCs were transfected with the lentiviral vectors according to the manufacturer’s instructions. Three days after MSC transfection, supernatants were replaced with culture medium as described above. Autophagy was induced in MSCs by serum starvation for 10 h before in vivo transplantation. 2.5 Immunocytochemistry Immunocytochemistry was performed to detect LC3, the cytoplasmic presence of which is used as an indicator of autophagy. The two types of MSCs were cultured in 48-well plates, post-fixed in 4% paraformaldehyde for 30 min and then permeabilized in 0.8% Triton X-100 for 5 min. Nonspecific binding was blocked with 10% normal goat serum for 30 min. The cells were incubated with primary mouse anti-
microtubule-associated protein 1 light chain 3 (MAP1LC3) antibody (Cell Signaling Technology), and then with 488-labeled secondary antibody (Invitrogen, Carlsbad, CA, USA). The cell nuclei were counterstained with Hoechst 33342. The staining was viewed under a confocal laser-scanning microscope (Nikon). 2.6 Enzyme-linked immunosorbent assay (ELISA) measurements The neurotrophins produced by MSCs were examined using ELISA kits. There MSCs were divided into three groups: MSCs in which autophagy was inhibited with shRNA (shBecn1-MSC group), normal MSCs (MSC group), and MSCs in which autophagy was induced (autophagy-MSC group). The cells were cultured in DMEM supplemented with 10% FBS for 18 h. The culture supernatant was collected, and cytokines were quantified using ELISA kits specific for nerve growth factor (R&D Systems), vascular endothelial growth factor (VEGF; R&D Systems), brain derived neurotrophic factor (BDNF; R&D Systems) and neurotrophin 3 (R&D Systems). 2.7 Spinal cord injury surgical procedures Adult female SD rats (200−220 g) were used in this study. Anesthesia was induced with an intraperitoneal injection of 4% chloral hydrate (400 mg/kg body weight). The hair on the back was removed, and the skin and muscle layers were cut open. A thoracic laminectomy at T8−T9 was performed with micro rongeurs to expose the spinal cord segment. Then, a left-sided spinal cord lateral hemisection was made with microscissors, which were plunged vertically into the spinal cord. After washing with phosphate buffered saline (PBS), gauze or gelatin foam was used to control bleeding in the surgical field. The muscles were sutured immediately using a
3-0 Vicryl suture (Ethicon) and the skin was then closed using a 5-0 chromic suture (Ethicon). During the operation, a heat lamp was used to maintain the body temperature at approximately 37°C. 2.8 MSC transplantation After surgical injury, the rats were randomly divided into four treatment groups (n = 8 per group): the PBS group, shBecn1-MSC group, MSC group, and autophagyMSC group. PBS was used as a control. The MSCs were labeled with Hoechst 33342 (5 µg/mL) (Sigma) before transplantation. For the groups treated with MSC transplantation, 1 × 106 cells in 200 µL of culture medium were administered intravenously (i.v.) via the tail vein. Rats were housed in a standard facility with free access to food and water. Four weeks after injury, the rats were sacrificed for histological analysis. 2.9 Functional evaluation Basso, Beattie, and Bresnahan (BBB) open field locomotor scoring was performed weekly by two blinded observers for 4 weeks to evaluate functional recovery, as described in previous studies(Basso et al., 1995). Rats were placed in an open field arena and allowed to move freely over a 4-min period. The BBB scale measures left hind limb movement involving paw placement, weight support, joint movements, and limb movement coordination. A score of 0 indicates no hindlimb movement, whereas the maximum score of 21 indicates the locomotion of a normal, healthy rat. 2.10
Immunohistochemistry
Four weeks following surgery and treatment, rats were perfused transcardially with 4% paraformaldehyde in phosphate buffer. A spinal cord segment containing the injury lesion was dissected and post-fixed in 4% paraformaldehyde overnight at 4°C. The segments were then transferred to 20% sucrose solution for 3 days, embedded in tissue-freezing medium and cut longitudinally in 20-μm sections on a cryostat. Every fifth slice was selected for immunohistochemical analysis. In total, 10 slices per sample were analyzed. Tissues were mounted onto gelatin-coated glass slides and stored at –80°C until use. The longitudinal sections were stained with hematoxylin and eosin to detect the structures. Hoechst 33342-labeled cells were observed under a microscope. Glass slides were fixed in acetone for 10 min, permeabilized with 0.8% Triton X-100 in 0.01 M PBS (pH 7.4) for 5 min and blocked with 10% normal goat serum for 30 min. Then, the slides were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used in this study: rabbit polyclonal antineurofilament (NF) heavy polypeptide antibody (Abcam, Cambridge, UK), 5-HT (serotonin) rabbit antibody (Immunostar), mouse monoclonal anti-S100 antibody (Abcam), and rabbit polyclonal anti-von Willebrand factor (vWF) antibody (Millipore). After washing with PBS, slides were incubated with secondary antibodies for 1 h at room temperature. Donkey anti-mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen) and Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Invitrogen) were used as the secondary antibodies. Cell nuclei were stained with Hoechst 33342. Negative
controls were performed using PBS without primary antibodies, and no positive labeling was observed (data not shown). The staining was examined using a laser scanning confocal microscope (Nikon). Five random fields in the middle of the lesion were selected within the dotted line on each slice at 200× magnification. Positive staining was quantified using the Image-Pro Plus software package (Media Cybernetics). 2.11 Examination of the myelin sheath The ultrastructure of the myelin sheath was observed 4 weeks after treatment. Specimens were post-fixed in 2.5% glutaraldehyde solution in PBS for two days at 4°C. After embedding in epoxy resin, they were cut into 70−90-nm ultrathin sections on a microtome. These sections were placed on a copper mesh and subjected to staining with 1% uranyl acetate and 1% lead citrate. After air-drying, the samples were examined under transmission electron microscopy (TEM; H-7650B, Tokyo, Japan). The thickness of the myelin sheaths was evaluated with the Image-Pro Plus software package. 2.12 Statistical analysis A two-way analysis of variance (ANOVA) was performed to compare the functional evaluation data using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Other data were analyzed by one-way ANOVA. The Student-Newman-Keuls posthoc test was used to calculate significance levels. Results are expressed as means ± standard deviation (SD). P < 0.05 was considered significant.
3. Results 3.1 MSC phenotypes and multipotent differentiation Consistent with typical bone marrow-derived MSCs (BMSCs), flow cytometry confirmed that the cells were positive for Sca-1, CD44, and CD90.2 (Thy1.2), and negative for CD34, CD117 (c-kit), CD45, CD11b, and MHC II (Fig. 1). After the BMSCs were cultured in differentiation medium for three weeks, obvious adipocytes and osteocytes were observed by staining with oil red O and Alizarin Red, respectively. For adipogenic differentiation, fat droplets were visualized by oil red O staining (Fig. 2A). Intracellular mineralized nodules, indicators of osteogenic differentiation, were stained by Alizarin Red (Fig. 2B). These differentiated cells represented the multipotent differentiation capacity of BMSCs. 3.2 Characteristics of MSCs after inducing or inhibiting autophagy Immunofluorescence detection of MAP1LC3 was performed to evaluate whether MSCs undergo autophagy. Cells displayed large numbers of MAP1LC3 dots in the cytoplasm after inducing autophagy (Fig. 2C). After transfecting MSCs with lentiviruses expressing shRNA for Becn1 knockdown, we further induced the cells by serum starvation to assess whether the inhibition was effective, and the results showed no obvious MAP1LC3 dots (Fig. 2D). These data indicate that MSCs underwent autophagy after induction, and that shBecn1 inhibited autophagy. 3.3 ELISA analysis The secretion levels of the four neurotrophins were decreased after inhibition of autophagy in MSCs (Fig. 3D). The secretion levels of VEGF and BDNF were
significantly higher in the autophagy-MSC group than in the normal MSC group. Although the secretion level of nerve growth factor were increased significantly in the autophagy-MSC and normal MSC groups compared with the normal MSC group, there was no significant difference between them (P > 0.05). The level of secreted neurotrophin 3 was not different among the three groups (P > 0.05). 3.4 Improvement of functional recovery MSCs (Fig. 3A) of various types, shBecn1-MSCs, normal MSCs, and autophagyMSCs, were used to treat SCI (Fig. 3B). At 4 weeks post-injury, hematoxylin and eosin staining revealed structures at the lesion site, and the dotted line indicated the boundary of the area analyzed by immunohistochemistry (Fig. 3C). Hoechst-labelled MSCs were present in the autophagy-MSC group, whereas very few labelled MSCs were present in the normal MSC group, and no labeled MSCs were observed in the shBecn1-MSC group (Fig. 4A). Recovery of motor function was assessed weekly by open field locomotor test. Left hindlimb locomotion improved gradually over the 4week observation period. Within the first two weeks, there were no differences among the treatment groups. Functional recovery in the PBS-treated group showed small degrees of improvement, while significant improvement in locomotion was observed over the course of the experiment in the autophagy-MSC group. At weeks 3 and 4, BBB scores were significantly higher in the autophagy-MSC group than in the other groups, but no difference was observed between the shBecn1-MSC and PBS groups (Fig. 4B). 3.5 Quantification of axonal fibers and 5-HT-positive fibers
At 4 weeks post-injury, an NF marker was used to evaluate axonal ingrowth at the injury site (Fig. 5A). As shown in Figure 5B, NF-positive fibers were promoted by treatment with autophagy-MSCs. The rats in the autophagy-MSC group exhibited the greatest axonal regeneration, while the shBecn1-MSC and PBS groups displayed only modest regeneration. There were significantly more NF-positive fibers in the groups treated with MSCs at week four after surgery than in the PBS group. The sprouting of serotonergic (5-HT) fibers from primary descending pathways of moto-sensory modulation in the mammalian spinal cord has been shown to aid recovery of locomotion (Courtine et al., 2008; Courtine et al., 2009). Regenerated 5HT fibers were more evident in the lesion site treated with autophagy-MSCs (Fig. 6A). The density of 5-HT-positive nerve fibers was significantly higher in the normal MSC group, and autophagy-MSC group than in the PBS group. Although 5-HT-positive nerve fibers in the normal MSC group were higher than in the shBecn1-MSC group, the difference was not significant (Fig. 6B). 3.6 Schwann cell regeneration analysis Schwann cells were evaluated by immunofluorescence staining for S100. Schwann cells within the lesioned area regenerated in all treatment groups, but were denser in the autophagy-MSC group than in the other treatment groups (Fig. 7A). A quantitative analysis also showed a greater number of S-100-positive cells in the center of the lesion in the MSC group than in the shBecn1-MSC and PBS groups, but this difference was not significant (Fig. 7B). 3.7 Autophagy-MSCs increased blood vessel formation
In terms of neovascularization, blood vessel formation (vWF staining) was observed four weeks after surgery (Fig. 8A). The blood vessel endothelial cell marker vWF signal in the lesion site in the autophagy-MSC group was higher than in the other treatment groups. Only a few blood vessel processes grew into the lesion site in the shBecn1-MSC and PBS groups. No differences in blood vessel density were found between the shBecn1-MSC and PBS groups (Fig. 8B). 3.8 Remyelination in the lesion zone The TEM images showed the high-resolution structure of myelinated axonal fibers (Fig. 9A). The thickness of the newly formed myelin sheath and remyelination of regenerated axons in the lesion site in the autophagy-MSC group was significantly higher than in the normal MSC and shBecn1-MSC groups. The PBS group showed limited myelination. The regenerated myelin sheath thickness at the SCI site was significantly greater in animals treated with MSCs and shBecn1-MSCs than in PBStreated animals (Fig. 9B). These data indicate that autophagy-MSCs not only increased the regeneration of axons within the lesion site, but also promoted myelination of the regenerated axons.
4. Discussion In this study, MSCs were used to treat SCI, due to their advantages of less immune rejection and fewer ethical concerns (Schira et al., 2012; Spejo et al., 2018). It has been reported that MSCs exert positive effects on axonal sprouting associated with accelerated functional recovery (Takahashi et al., 2018; Vaquero et al., 2017) It
remains unknown whether autophagy plays important roles in MSCs when MSCs are used to treat SCI. shRNA is an effective tool for prolonged knockdown of gene expression at the mRNA level for investigating gene function (Davidson and McCray, 2011). In the present study, lentiviral vectors were used to deliver shRNA to knock down Becn1 expression, which is essential for the autophagy process. The immunocytochemistry results showed significant elevation of MAP1LC3 in MSCs pretreated by induction of autophagy (Fig. 2C). In contrast, few MAP1LC3 immunofluorescence dots were present in MSCs after Becn1 knockdown (Fig. 2D). These results indicated that the inhibition or induction of autophagy in MSCs was successful. The results showed that the Hoechst-labelled MSCs migrated to the lesion site (Fig. 4A). This is consistent with previous reports that multiple chemokines at the injury site recruit transplanted MSCs after an injury (Li et al., 2012; Ramalho et al., 2018). However, we did not detect Hoechst-labelled MSCs in the shBecn1-MSC group, possibly because inhibiting autophagy limited the migration ability or cell survival in the inflammatory microenvironment at the lesion site (Fig. 4A). Autophagic MSCs secreted high levels of VEGF and BDNF, which are important growth factors for neural regeneration (Fig. 3D). Taken together, the homing MSCs exerted neuroprotective effects via the paracrine action of different neurotrophic factors after SCI. Before surgery, all rats exhibited normal locomotion (BBB score = 21). Immediately after surgery, all rats exhibited almost complete loss of left hindlimb
locomotion, indicating successful surgery. However, a significant difference was found between the autophagy-MSC group and the other treatment groups at the fourth week post-surgery, suggesting that inducing autophagy in MSCs further improved functional recovery, and inhibiting autophagy impaired MSC function. Sections of the injury site were stained with anti-NF antibody to evaluate axonal regeneration (Fig. 5A) and anti-5-HT antibody to evaluate newly sprouted serotonin fibers (Fig. 6A). Quantitative analysis revealed that inducing autophagy in MSCs promoted axonal and serotonin fiber regeneration. Serotonin plays an important role in locomotor behavior and in functional improvement after SCI (Ribotta et al., 2000). The survival and proliferation of Schwann cells following SCI were detected using S100 staining. The regeneration and migration of Schwann cells play key roles in successful axonal outgrowth and remyelination. After SCI, the loss of blood vessel integrity at the injury site partially results in cavity formation, cell death, and poor axonal regrowth (McCreedy and SakiyamaElbert, 2012). A previous study showed that autophagy enhances VEGF secretion from MSCs via ERK phosphorylation, thus promoting endothelial cell angiogenesis for cutaneous wound healing (An et al., 2018). In this study, we also found that the autophagic MSCs secreted a high level of VEGF (Fig. 3D). Thus, we stained for vWF to analyze angiogenesis in each group. The results showed enhanced vascularization in the autophagy-MSC group at the injured site after SCI, which is beneficial for nerve regeneration. In addition to axonal regeneration, improving the myelination of axons is an
important process in functional recovery. Previous studies have shown that remyelination of surviving or regenerated axons was beneficial for nerve function recovery of the spinal cord(Duncan et al., 2017). Our TEM data showed that treatment with autophagy-MSCs markedly improved myelination of the regenerated axons, as indicated by myelin sheath thickness. The regenerated axons, proliferation of Schwann cells, neovascularization, and remyelination of axons in the autophagy-MSC group all contributed to final functional recovery in SCI. These results indicate that inhibiting autophagy in MSCs limited the therapeutic effects of MSCs in SCI repair. Previous studies considered autophagy a detrimental factor in brain injury (Gao et al., 2018). Autophagy may have different effects on different cell types, but the specific mechanism needs further study. One study reported that elevated autophagy after irradiation leads to the accumulation of reactive oxygen species (ROS) in MSCs resulted in DNA damage and loss of stemness. Inducing autophagy by starvation reduced ROS accumulationassociated DNA damage and maintained MSC stemness, whereas inhibiting autophagy augmented ROS accumulation and DNA damage, resulting in a loss of stemness in MSCs (Hou et al., 2013). It has also been reported that autophagy may prevent radiation deteriorative processes in MSCs. MSCs exposed to low-dose irradiation showed impaired autophagy, leading to cellular senescence (Alessio et al., 2015). Autophagy also plays a fundamental role in MSC differentiation. Ablation of an essential component of mammalian autophagy led to multiple autophagic defects in osteoblasts and defective osteoblast terminal differentiation in primary bone
marrow, suggesting an important role of autophagy in support of osteoblast differentiation (Liu et al., 2013). Additionally, autophagy maintained cellular homeostasis and had a protective effect against environmental stresses. Enhancing autophagy
in
MSCs
may
elevate
cellular
survival
in
an
inflammatory
microenvironment. Inhibiting autophagy made MSCs more susceptible to cell death (Yang et al., 2016). In this study, the autophagy-enhanced therapeutic effect of MSCs may involve multiple aspects.
5. Conclusion We successfully induced or inhibited autophagy in MSCs and then applied these types of MSCs in a rat T8−T9 SCI model. The in vitro study showed that the secretion of neurotrophic and growth factors from autophagic MSCs was increased. We demonstrated that enhancing autophagy in MSCs resulted in a positive impact on neuronal generation, Schwann cell regeneration, and promotion of angiogenesis. The locomotion recovery results were consistent with the immunohistochemical results. On the other hand, inhibiting autophagy downregulated the utility of MSCs for treating SCI. Our study suggests that autophagy is important for MSC function, and that regulating autophagy may provide a novel strategy for improving the regenerative capacity of MSCs in SCI repair.
Data Availability The data used to support the findings of this study are included within the article.
Acknowledgments This work was supported by grants (2018YFA0107900, 31771491, 81471242, 81601069) from the National Nature Science Foundation and Ministry of Science and Technology of China, Shanghai Sailing Program (19YF1404600) and Shanghai Municipal Government (2019CXJQ01). Many thanks for Dr Congfeng Xu and Dr Jiefang Huang in chinese academy of sciences for providing technical support regarding autophagy inhibiting.
Author Disclosure Statement No competing financial interests exist.
Reference
Alessio, N., Del Gaudio, S., Capasso, S., Di Bernardo, G., Cappabianca, S., Cipollaro, M., Peluso, G., Galderisi, U., 2015. Low dose radiation induced senescence of human mesenchymal stromal cells and impaired the autophagy process. Oncotarget. 6, 8155-66. An, Y., Liu, W.J., Xue, P., 2018. Autophagy promotes MSC-mediated vascularization in cutaneous wound healing via regulation of VEGF secretion. 9, 58. Anderson, M.A., O'Shea, T.M., Burda, J.E., Ao, Y., Barlatey, S.L., Bernstein, A.M., Kim, J.H., James, N.D., Rogers, A., Kato, B., Wollenberg, A.L., Kawaguchi,
R., Coppola, G., Wang, C., Deming, T.J., He, Z., Courtine, G., Sofroniew, M.V., 2018. Required growth facilitators propel axon regeneration across complete spinal cord injury. Nature. 561, 396-400. Anokhina, E.B., Buravkova, L.B., 2007. [Heterogeneity of stromal precursor cells isolated from rat bone marrow]. Tsitologiia. 49, 40-7. Assinck, P., Duncan, G.J., Hilton, B.J., Plemel, J.R., 2017. Cell transplantation therapy for spinal cord injury. 20, 637-647. Basso, D.M., Beattie, M.S., Bresnahan, J.C., 1995. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 12, 1-21. Comolli, N., Neuhuber, B., Fischer, I., Lowman, A., 2009. In vitro analysis of PNIPAAm-PEG, a novel, injectable scaffold for spinal cord repair. Acta Biomater. 5, 1046-55. Courtine, G., Song, B., Roy, R.R., Zhong, H., Herrmann, J.E., Ao, Y., Qi, J., Edgerton, V.R., Sofroniew, M.V., 2008. Recovery of supraspinal control of stepping via indirect propriospinal relay connections after spinal cord injury. Nat Med. 14, 69-74. Courtine, G., Gerasimenko, Y., van den Brand, R., Yew, A., Musienko, P., Zhong, H., Song, B., Ao, Y., Ichiyama, R.M., Lavrov, I., Roy, R.R., Sofroniew, M.V., Edgerton, V.R., 2009. Transformation of nonfunctional spinal circuits into functional states after the loss of brain input. Nat Neurosci. 12, 1333-42. Cully, M., 2018. Spinal cord injury: Ion transporter agonist improves recovery. Nat Rev Drug Discov. 17, 621.
Dang, S., Xu, H., Xu, C., Cai, W., Li, Q., Cheng, Y., Jin, M., Wang, R.X., Peng, Y., Zhang, Y., Wu, C., He, X., Wan, B., Zhang, Y., 2014. Autophagy regulates the therapeutic potential of mesenchymal stem cells in experimental autoimmune encephalomyelitis. Autophagy. 10, 1301-15. Davidson, B.L., McCray, P.B., Jr., 2011. Current prospects for RNA interferencebased therapies. Nat Rev Genet. 12, 329-40. Doherty, J., Baehrecke, E.H., 2018. Life, death and autophagy. Nat Cell Biol. Duncan, I.D., Marik, R.L., Broman, A.T., Heidari, M., 2017. Thin myelin sheaths as the hallmark of remyelination persist over time and preserve axon function. Proc Natl Acad Sci U S A. 114, E9685-e9691. Falnikar, A., Hala, T.J., Poulsen, D.J., Lepore, A.C., 2016. GLT1 overexpression reverses established neuropathic pain-related behavior and attenuates chronic dorsal horn neuron activation following cervical spinal cord injury. Glia. 64, 396-406. Gao, Y., Zhang, M.Y., Wang, T., Fan, Y.Y., Yu, L.S., Ye, G.H., Wang, Z.F., Gao, C., Wang, H.C., Luo, C.L., Tao, L.Y., 2018. IL-33/ST2L Signaling Provides Neuroprotection Through Inhibiting Autophagy, Endoplasmic Reticulum Stress, and Apoptosis in a Mouse Model of Traumatic Brain Injury. Front Cell Neurosci. 12, 95. Hansen, M., Rubinsztein, D.C., 2018. Autophagy as a promoter of longevity: insights from model organisms. 19, 579-593. Hou, J., Han, Z.P., Jing, Y.Y., Yang, X., Zhang, S.S., Sun, K., Hao, C., Meng, Y., Yu,
F.H., Liu, X.Q., Shi, Y.F., Wu, M.C., Zhang, L., Wei, L.X., 2013. Autophagy prevents irradiation injury and maintains stemness through decreasing ROS generation in mesenchymal stem cells. Cell Death Dis. 4, e844. Kang, K.N., Kim, D.Y., Yoon, S.M., Lee, J.Y., Lee, B.N., Kwon, J.S., Seo, H.W., Lee, I.W., Shin, H.C., Kim, Y.M., Kim, H.S., Kim, J.H., Min, B.H., Lee, H.B., Kim, M.S., 2012. Tissue engineered regeneration of completely transected spinal cord using human mesenchymal stem cells. Biomaterials. 33, 4828-35. Khan, I.U., Yoon, Y., Kim, A., Jo, K.R., Choi, K.U., Jung, T., Kim, N., Son, Y., Kim, W.H., Kweon, O.K., 2018. Improved Healing after the Co-Transplantation of HO-1 and BDNF Overexpressed Mesenchymal Stem Cells in the Subacute Spinal Cord Injury of Dogs. Cell Transplant. 27, 1140-1153. Kim, K., Dean, D., Mikos, A.G., Fisher, J.P., 2009. Effect of initial cell seeding density on early osteogenic signal expression of rat bone marrow stromal cells cultured on cross-linked poly(propylene fumarate) disks. Biomacromolecules. 10, 1810-7. Li, L., Yang, M., Wang, C., Zhao, Q., Liu, J., Zhan, C., Liu, Z., Li, X., Wang, W., Yang, X., 2012. Effects of cytokines and chemokines on migration of mesenchymal stem cells following spinal cord injury. Neural Regen Res. 7, 1106-12. Liu, F., Fang, F., Yuan, H., Yang, D., Chen, Y., Williams, L., Goldstein, S.A., Krebsbach, P.H., Guan, J.L., 2013. Suppression of autophagy by FIP200 deletion leads to osteopenia in mice through the inhibition of osteoblast
terminal differentiation. J Bone Miner Res. 28, 2414-30. Ma, Y.H., Zeng, X., Qiu, X.C., Wei, Q.S., Che, M.T., Ding, Y., Liu, Z., Wu, G.H., Sun, J.H., Pang, M., Rong, L.M., Liu, B., Aljuboori, Z., Han, I., Ling, E.A., Zeng, Y.S., 2018. Perineurium-like sheath derived from long-term surviving mesenchymal stem cells confers nerve protection to the injured spinal cord. Biomaterials. 160, 37-55. McCreedy, D.A., Sakiyama-Elbert, S.E., 2012. Combination therapies in the CNS: engineering the environment. Neurosci Lett. 519, 115-21. Mizushima, N., Komatsu, M., 2011. Autophagy: renovation of cells and tissues. Cell. 147, 728-41. Oliveri, R.S., Bello, S., Biering-Sorensen, F., 2014. Mesenchymal stem cells improve locomotor recovery in traumatic spinal cord injury: systematic review with meta-analyses of rat models. Neurobiol Dis. 62, 338-53. Park, E., Velumian, A.A., Fehlings, M.G., 2004. The role of excitotoxicity in secondary mechanisms of spinal cord injury: a review with an emphasis on the implications for white matter degeneration. J Neurotrauma. 21, 754-74. Park, K., 2018. Functional recovery in spinal cord injury using mesenchymal stem cells. J Control Release. 278, 159. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R., 1999. Multilineage potential of adult human mesenchymal stem cells. Science. 284, 143-7.
Ramalho, B.D.S., Almeida, F.M., Sales, C.M., de Lima, S., Martinez, A.M.B., 2018. Injection of bone marrow mesenchymal stem cells by intravenous or intraperitoneal routes is a viable alternative to spinal cord injury treatment in mice. Neural Regen Res. 13, 1046-1053. Ribotta, M.G., Provencher, J., Feraboli-Lohnherr, D., Rossignol, S., Privat, A., Orsal, D., 2000. Activation of locomotion in adult chronic spinal rats is achieved by transplantation of embryonic raphe cells reinnervating a precise lumbar level. J Neurosci. 20, 5144-52. Schira, J., Gasis, M., Estrada, V., Hendricks, M., Schmitz, C., Trapp, T., Kruse, F., Kogler, G., Wernet, P., Hartung, H.P., Muller, H.W., 2012. Significant clinical, neuropathological and behavioural recovery from acute spinal cord trauma by transplantation of a well-defined somatic stem cell from human umbilical cord blood. Brain. 135, 431-46. Singh, D., Harding, A.J., Albadawi, E., Boissonade, F.M., Haycock, J.W., Claeyssens, F., 2018. Additive manufactured biodegradable poly(glycerol sebacate methacrylate) nerve guidance conduits. Acta Biomater. 78, 48-63. Spejo, A.B., Chiarotto, G.B., Ferreira, A.D.F., Gomes, D.A., Ferreira, R.S., Jr., Barraviera,
B.,
Oliveira,
A.L.R.,
2018.
Neuroprotection
and
immunomodulation following intraspinal axotomy of motoneurons by treatment with adult mesenchymal stem cells. 15, 230. Stower, H., 2018. Cell therapy for spinal cord injury. Nat Med. 24, 1088. Takahashi, A., Nakajima, H., Uchida, K., Takeura, N., Honjoh, K., Watanabe, S.,
Kitade, M., Kokubo, Y., Johnson, W.E.B., Matsumine, A., 2018. Comparison of Mesenchymal Stromal Cells Isolated from Murine Adipose Tissue and Bone Marrow in the Treatment of Spinal Cord Injury. Cell Transplant. 27, 1126-1139. Tsenkina, Y., Ricard, J., Runko, E., Quiala-Acosta, M.M., Mier, J., Liebl, D.J., 2015. EphB3 receptors function as dependence receptors to mediate oligodendrocyte cell death following contusive spinal cord injury. Cell Death Dis. 6, e1922. Vaquero, J., Zurita, M., Rico, M.A., Bonilla, C., Aguayo, C., Fernandez, C., Tapiador, N., Sevilla, M., Morejon, C., Montilla, J., Martinez, F., Marin, E., Bustamante, S., Vazquez, D., Carballido, J., Rodriguez, A., Martinez, P., Garcia, C., Ovejero, M., Fernandez, M.V., 2017. Repeated subarachnoid administrations of autologous mesenchymal stromal cells supported in autologous plasma improve quality of life in patients suffering incomplete spinal cord injury. Cytotherapy. 19, 349-359. Wilems, T.S., Pardieck, J., Iyer, N., Sakiyama-Elbert, S.E., 2015. Combination therapy of stem cell derived neural progenitors and drug delivery of antiinhibitory molecules for spinal cord injury. Acta Biomater. 28, 23-32. Yan, J., Porch, M.W., Court-Vazquez, B., Bennett, M.V.L., Zukin, R.S., 2018. Activation of autophagy rescues synaptic and cognitive deficits in fragile X mice. Proc Natl Acad Sci U S A. Yang, R., Ouyang, Y., Li, W., Wang, P., Deng, H., Song, B., Hou, J., Chen, Z., Xie, Z., Liu, Z., Li, J., Cen, S., Wu, Y., Shen, H., 2016. Autophagy Plays a
Protective Role in Tumor Necrosis Factor-alpha-Induced Apoptosis of Bone Marrow-Derived Mesenchymal Stem Cells. Stem Cells Dev. 25, 788-97. Yao, R., Murtaza, M., Velasquez, J.T., Todorovic, M., Rayfield, A., Ekberg, J., Barton, M., St John, J., 2018. Olfactory Ensheathing Cells for Spinal Cord Injury: Sniffing Out the Issues. Cell Transplant. 27, 879-889. Zhang, S., Wang, X.J., Li, W.S., Xu, X.L., Hu, J.B., Kang, X.Q., Qi, J., Ying, X.Y., You,
J.,
Du,
Y.Z.,
2018.
Polycaprolactone/polysialic
acid
hybrid,
multifunctional nanofiber scaffolds for treatment of spinal cord injury. Acta Biomater. 77, 15-27.
Figure legends
Figure 1. Surface marker expression of bone marrow-derived mesenchymal stem cells (BMSCs) analyzed by flow cytometry.
Figure 2. (A) Adipogenic differentiation potentials of BMSCs were confirmed by oil red O staining. Scale bar = 100 µm. (B) Osteogenic differentiation potentials of BMSCs were confirmed by Alizarin Red staining. Scale bar = 200 µm. (C) Representative confocal microscopy images of cells after staining with antiMAP1LC3 antibody (green) for BMSCs after autophagy. (D) Representative confocal microscopy images of cells after staining with anti-MAP1LC3 antibody (green) for BMSCs after knock down expression of the autophagy gene Becn1 and then induced autophagy. The cell nuclei were counterstained with Hoechst 33342 (blue). Scale bar = 30 µm.
Figure 3. Surgical procedures of spinal cord injury (SCI) and ELISA analysis. (A) Morphology of MSCs under light microscope. Scale bar = 100 µm. (B) Photograph of spinal cord four weeks after surgery. (C) H&E staining at week 4 after surgery. Scale bar = 1 mm. (D). ELISA analyses of NGF, VEGF, BDNF and NT3 secretion from MSCs. Data are expressed as means ± SD; n = 3; *P < 0.05; **P < 0.01.
Figure 4. (A) Images of Hoechst labeled MSCs in each group. Scale bar, 30 µm. (B) Weekly post-SCI Basso, Beattie, and Bresnahan (BBB) open field locomotor scores. Data are expressed as means ± SD; n = 8; *P < 0.05; **P < 0.01.
Figure 5. Sections of the SCI at the lesion site stained with anti-NF antibody. (A) Representative images of the PBS, shBecn1-MSCs, MSCs, and autophagy-MSCs
treatment groups. The nuclei were stained using Hoechst 33342 (blue). Scale bar = 50 µm. (B) Quantification of the area of positive NF staining in each group. Data are expressed as means ± SD; n = 4; **P < 0.01.
Figure 6. Regeneration of serotonergic (5-HT) fibers at the SCI site. (A) Immunostained images of serotonergic (5-HT) fibers at the lesion site four weeks post-SCI. The nuclei were stained using Hoechst 33342 (blue). Scale bar = 50 µm. (B) Quantitative analysis of the area of 5-HT fibers in the different treatment groups. Data are expressed as means ± SD; n = 4; *P < 0.05; **P < 0.01.
Figure 7. Immunohistochemical analysis of the regenerated spinal cord four weeks after injury. (A) S-100 staining of sections showing Schwann cell regeneration. The nuclei were stained using Hoechst 33342 (blue). Scale bar = 50 µm. (B) Quantitative analysis of the area of Schwann cells in each group. Data are expressed as means ± SD; n = 4; **P < 0.01.
Figure 8. Immunohistochemical analysis of SCI sites four weeks after implantation. (A) Anti-von Willebrand factor (vWF) immunostained images of each treatment group. The nuclei were stained using Hoechst 33342 (blue). Scale bar = 50 µm. (B) Blood vessel density at the injury site. Statistical analysis performed using the ImagePro Plus software package. Data are expressed as means ± SD; n = 4; *P < 0.05; **P < 0.01.
Figure 9. Analysis of the degree of remyelination at the lesion site four weeks after surgery. (A) Transmission electron microscopy (TEM) images of regenerated spinal cord, showing differences in myelin sheath thickness among the treatment groups. Scale bar = 1 µm. (B) Myelin sheath thickness in each treatment group. Data are expressed as means ± SD; n = 4; *P < 0.05; **P < 0.01.
Transplantation with mesenchymal stem cells (MSCs) has shown beneficial effects in treating spinal cord injury (SCI). Autophagy is an evolutionarily conserved process of degradation
and recycling of cellular components that plays an important role in tissue homeostasis and cellular survival. Whether regulating autophagy in MSCs may affect their therapeutic potential in spinal cord injury repair has not yet been determined. In this study, we found that autophagy played important roles in MSCs during its application in SCI repairing. Regulation of autophagy in MSCs before in vivo transplantation could be a potential therapeutic intervention strategy for SCI.
S0006-8993(19)30367-1 https://doi.org/10.1016/j.brainres.2019.146321 146321 BRES 146321
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
13 May 2019 20 June 2019 2 July 2019
Please cite this article as: F. Ma, R. Li, H. Tang, T. Zhu, F. Xu, J. Zhu, Regulation of autophagy in mesenchymal stem cells modulates therapeutic effects on spinal cord injury, Brain Research (2019), doi: https://doi.org/10.1016/ j.brainres.2019.146321
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Regulation of autophagy in mesenchymal stem cells modulates therapeutic effects on spinal cord injury Fukai Maa,b,#, Ronggang Lia,c,#, Hailiang Tanga,#, Tongming Zhua, Feng Xua,d,*, and Jianhong Zhua,* aDepartment
of Neurosurgery, Fudan University Huashan Hospital and State Key
Laboratory of Medical Neurobiology, the Institutes of Brain Science, Shanghai Medical College, Fudan University, No.12 Urumqi Mid Road, Shanghai 200040, China. bNeurosurgery
Department, Shanghai Tenth People’s Hospital, Tongji University, No.
301 Middle Yanchang Road, Shanghai 200072, Shanghai, China. cDepartment
of Neurosurgery, Shanghai Public Health Clinical Center, Fudan
University, No. 2901 Caolanggong Road, Shanghai 201508, China. dDepartment
of Neurosurgery, Kashgar Prefecture Second People’s Hospital, No. 1
Jiankang Road, Kashgar 844000, China. *Correspondence: #These
[email protected] (F.Xu), [email protected] (J.Zhu)
authors contributed equally.
Abstract Transplantation with mesenchymal stem cells (MSCs) has shown beneficial effects in treating spinal cord injury. Autophagy is an evolutionarily conserved process of degradation and recycling of cellular components that plays an important role in tissue homeostasis and cellular survival. Whether regulating autophagy in MSCs may affect
their therapeutic potential in spinal cord injury repair has not yet been determined. In this study, autophagy was inhibited in MSCs with lentiviruses expressing short hairpin RNA (shRNA) to knock down Becn-1 expression, and autophagy was upregulated in MSCs under nutrient starvation. These MSCs were then labelled with Hoechst and applied to spinal cord-injured rats to evaluate their therapeutic effects. After transplanting MSCs into rats with spinal cord injuries, functional recovery, immunohistochemistry, and remyelination analyses were performed. After inducing autophagy, the MSCs exhibited an accumulation of LC3-positive autophagosomes in the cytoplasm. The expression levels of neurotrophic factors, including vascular endothelial growth factor and brain derived neurotrophic factor, were significantly higher in autophagic MSCs than normal MSCs. The in vivo study showed that more labelled MSCs migrated to the lesion site after induction of autophagy. Inducing autophagy in MSCs promoted functional recovery after spinal cord injury, whereas functional recovery was weak after inhibiting autophagy in MSCs. In contrast to the autophagy inhibition group, transplanting autophagic MSCs exhibited a greater positive impact on axon regeneration, growth of serotonergic fibers, blood vessel regeneration, and myelination, indicating a multifactorial contribution to spinal cord injury repair. These results suggest that autophagy plays important roles in MSCs during spinal cord injury repair. Regulation of autophagy in MSCs before in vivo transplantation may be a potential therapeutic interventional strategy for spinal cord injury.
Keywords Spinal cord injury, Autophagy, Mesenchymal stem cells, Regeneration
1. Introduction Spinal cord injury (SCI) is a devastating disease that is often caused by contusion, compression, or surgical intervention of the spinal cord (Cully, 2018; Takahashi et al., 2018). After injury, inflammation, ischemia, and apoptosis occur, contributing to spinal cord degeneration (Falnikar et al., 2016; Park et al., 2004). SCI often results in permanent loss of motor and sensory functions due to the inability to regenerate lost spinal cord tissue (Tsenkina et al., 2015; Wilems et al., 2015; Yao et al., 2018). Unlike peripheral nervous system regeneration, spontaneous regeneration after spinal cord injury is limited and incomplete (Comolli et al., 2009; Singh et al., 2018). SCI remains a unique clinical challenge requiring better treatment modalities (Anderson et al., 2018; Zhang et al., 2018). Various approaches have been investigated for restoring or improving regeneration in the hostile lesion environment after SCI (Stower, 2018). Improved therapies are still needed to enhance the long-term effectiveness of SCI treatment. The application of mesenchymal stem cells (MSCs) in regenerative medicine holds great promise for the treatment of various intractable central nervous system (CNS) diseases (Assinck et al., 2017; Khan et al., 2018). Previous studies demonstrated that MSCs are immune-privileged cells for in vivo transplantation due to their ability to regulate immune cell function and immune responses. MSCs exert
anti-inflammatory, neurotrophic, and angiogenic effects in SCI repair (Oliveri et al., 2014; Park, 2018). Many studies have shown that MSCs may restore the function of damaged spinal cord and promote functional recovery after SCI (Kang et al., 2012; Ma et al., 2018). Autophagy is an evolutionarily conserved cellular mechanism involving the formation of autophagosomes, which are responsible for degrading damaged and/or aged cytoplasm and cellular organelles (Yan et al., 2018). Autophagy is a crucial cellular process with important roles in maintaining homeostasis and remodeling during cellular development (Doherty and Baehrecke, 2018; Mizushima and Komatsu, 2011). Mounting evidence indicates that autophagic activation is required for selfrenewal, pluripotency, differentiation, and quiescence in stem cells, while dysfunctional autophagy may be related to various diseases (Hansen and Rubinsztein, 2018). However, the role of autophagy in the function of MSCs on SCI has yet to be explored. In this study, we first increased autophagy or inhibited autophagy in MSCs, and then transplanted the two types of MSCs into injured spinal cords to evaluate their effects on functional recovery.
2. Materials and methods 2.1 MSC isolation and culture All experimental procedures involving animals were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and approved by the Institutional
Animal Care and Use Committee. MSCs were isolated from Sprague-Dawley (SD) rats based on the protocols described in previous studies (Anokhina and Buravkova, 2007; Kim et al., 2009). After the rats were sacrificed, the femurs and tibias of the hind limbs were excised, and adherent tissues were removed. The bones were cut and the bone cavities were flushed with Dulbecco’s Modified Eagles Medium (DMEMF12; Hyclone) to obtain bone marrow. After passing through a 70-μm cell strainer, the marrow was centrifuged at 500 × g for 5 min. The pellet was resuspended in lowglucose DMEM supplemented with 15% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. A total of 106 cells were transferred to a T-25 cell culture flask and cultured at 37°C in 5% CO2. After 2 days, the nonadherent cells were removed, and the attached cells were cultured further. At the fourth or fifth passage, the cells were digested with 0.25% trypsin solution and collected for further experiments. 2.2 Flow cytometry analysis of cultured cells Digested cell aliquots (1 × 106 cells/mL) were placed on ice and stained with the following antibodies: anti-CD44 (BD Biosciences), anti-CD90.2 (Thy1.2; BD Biosciences), anti-stem cell antigen-1 (Sca-1; BD Biosciences), anti-CD34 (BD Biosciences), anti-CD45 (BD Biosciences), anti-CD117 (c-kit; BD Biosciences), antiCD11b (ITGAM; BD Biosciences), and anti-MHC II (eBioscience). Flow cytometry (FACSAria II, BD Biosciences) was used to analyze the data. 2.3 MSC differentiation assay Osteogenic differentiation and adipogenic differentiation were induced in MSCs
based on previously described methods (Pittenger et al., 1999). Briefly, the MSCs were cultured in differentiation medium for three weeks. Adipogenic differentiation was confirmed by Oil Red-O staining (Sigma-Aldrich, St. Louis, MO, USA). Osteogenic differentiation was confirmed with Alizarin Red staining (Sigma-Aldrich). Images were observed under an inverted phase-contrast microscope (Nikon, Tokyo, Japan). 2.4 Autophagic inhibition or induction in MSCs Oligonucleotides for Becn-1 short hairpin RNA (shRNA) (shBecn1) were synthesized by GenePharma. The shBecn1 sequence was as follows: 5′gatccGGAGAAAGGCAAGATTGAAGATTCAAGAGATCTTCAATCTTGCCTTT CTCCTTTTTTg-3′. Lentiviral vectors encoding the shRNA were then constructed to transfect MSCs to knock down expression of the essential autophagy gene Becn1 (Dang et al., 2014). MSCs were transfected with the lentiviral vectors according to the manufacturer’s instructions. Three days after MSC transfection, supernatants were replaced with culture medium as described above. Autophagy was induced in MSCs by serum starvation for 10 h before in vivo transplantation. 2.5 Immunocytochemistry Immunocytochemistry was performed to detect LC3, the cytoplasmic presence of which is used as an indicator of autophagy. The two types of MSCs were cultured in 48-well plates, post-fixed in 4% paraformaldehyde for 30 min and then permeabilized in 0.8% Triton X-100 for 5 min. Nonspecific binding was blocked with 10% normal goat serum for 30 min. The cells were incubated with primary mouse anti-
microtubule-associated protein 1 light chain 3 (MAP1LC3) antibody (Cell Signaling Technology), and then with 488-labeled secondary antibody (Invitrogen, Carlsbad, CA, USA). The cell nuclei were counterstained with Hoechst 33342. The staining was viewed under a confocal laser-scanning microscope (Nikon). 2.6 Enzyme-linked immunosorbent assay (ELISA) measurements The neurotrophins produced by MSCs were examined using ELISA kits. There MSCs were divided into three groups: MSCs in which autophagy was inhibited with shRNA (shBecn1-MSC group), normal MSCs (MSC group), and MSCs in which autophagy was induced (autophagy-MSC group). The cells were cultured in DMEM supplemented with 10% FBS for 18 h. The culture supernatant was collected, and cytokines were quantified using ELISA kits specific for nerve growth factor (R&D Systems), vascular endothelial growth factor (VEGF; R&D Systems), brain derived neurotrophic factor (BDNF; R&D Systems) and neurotrophin 3 (R&D Systems). 2.7 Spinal cord injury surgical procedures Adult female SD rats (200−220 g) were used in this study. Anesthesia was induced with an intraperitoneal injection of 4% chloral hydrate (400 mg/kg body weight). The hair on the back was removed, and the skin and muscle layers were cut open. A thoracic laminectomy at T8−T9 was performed with micro rongeurs to expose the spinal cord segment. Then, a left-sided spinal cord lateral hemisection was made with microscissors, which were plunged vertically into the spinal cord. After washing with phosphate buffered saline (PBS), gauze or gelatin foam was used to control bleeding in the surgical field. The muscles were sutured immediately using a
3-0 Vicryl suture (Ethicon) and the skin was then closed using a 5-0 chromic suture (Ethicon). During the operation, a heat lamp was used to maintain the body temperature at approximately 37°C. 2.8 MSC transplantation After surgical injury, the rats were randomly divided into four treatment groups (n = 8 per group): the PBS group, shBecn1-MSC group, MSC group, and autophagyMSC group. PBS was used as a control. The MSCs were labeled with Hoechst 33342 (5 µg/mL) (Sigma) before transplantation. For the groups treated with MSC transplantation, 1 × 106 cells in 200 µL of culture medium were administered intravenously (i.v.) via the tail vein. Rats were housed in a standard facility with free access to food and water. Four weeks after injury, the rats were sacrificed for histological analysis. 2.9 Functional evaluation Basso, Beattie, and Bresnahan (BBB) open field locomotor scoring was performed weekly by two blinded observers for 4 weeks to evaluate functional recovery, as described in previous studies(Basso et al., 1995). Rats were placed in an open field arena and allowed to move freely over a 4-min period. The BBB scale measures left hind limb movement involving paw placement, weight support, joint movements, and limb movement coordination. A score of 0 indicates no hindlimb movement, whereas the maximum score of 21 indicates the locomotion of a normal, healthy rat. 2.10
Immunohistochemistry
Four weeks following surgery and treatment, rats were perfused transcardially with 4% paraformaldehyde in phosphate buffer. A spinal cord segment containing the injury lesion was dissected and post-fixed in 4% paraformaldehyde overnight at 4°C. The segments were then transferred to 20% sucrose solution for 3 days, embedded in tissue-freezing medium and cut longitudinally in 20-μm sections on a cryostat. Every fifth slice was selected for immunohistochemical analysis. In total, 10 slices per sample were analyzed. Tissues were mounted onto gelatin-coated glass slides and stored at –80°C until use. The longitudinal sections were stained with hematoxylin and eosin to detect the structures. Hoechst 33342-labeled cells were observed under a microscope. Glass slides were fixed in acetone for 10 min, permeabilized with 0.8% Triton X-100 in 0.01 M PBS (pH 7.4) for 5 min and blocked with 10% normal goat serum for 30 min. Then, the slides were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used in this study: rabbit polyclonal antineurofilament (NF) heavy polypeptide antibody (Abcam, Cambridge, UK), 5-HT (serotonin) rabbit antibody (Immunostar), mouse monoclonal anti-S100 antibody (Abcam), and rabbit polyclonal anti-von Willebrand factor (vWF) antibody (Millipore). After washing with PBS, slides were incubated with secondary antibodies for 1 h at room temperature. Donkey anti-mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen) and Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Invitrogen) were used as the secondary antibodies. Cell nuclei were stained with Hoechst 33342. Negative
controls were performed using PBS without primary antibodies, and no positive labeling was observed (data not shown). The staining was examined using a laser scanning confocal microscope (Nikon). Five random fields in the middle of the lesion were selected within the dotted line on each slice at 200× magnification. Positive staining was quantified using the Image-Pro Plus software package (Media Cybernetics). 2.11 Examination of the myelin sheath The ultrastructure of the myelin sheath was observed 4 weeks after treatment. Specimens were post-fixed in 2.5% glutaraldehyde solution in PBS for two days at 4°C. After embedding in epoxy resin, they were cut into 70−90-nm ultrathin sections on a microtome. These sections were placed on a copper mesh and subjected to staining with 1% uranyl acetate and 1% lead citrate. After air-drying, the samples were examined under transmission electron microscopy (TEM; H-7650B, Tokyo, Japan). The thickness of the myelin sheaths was evaluated with the Image-Pro Plus software package. 2.12 Statistical analysis A two-way analysis of variance (ANOVA) was performed to compare the functional evaluation data using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Other data were analyzed by one-way ANOVA. The Student-Newman-Keuls posthoc test was used to calculate significance levels. Results are expressed as means ± standard deviation (SD). P < 0.05 was considered significant.
3. Results 3.1 MSC phenotypes and multipotent differentiation Consistent with typical bone marrow-derived MSCs (BMSCs), flow cytometry confirmed that the cells were positive for Sca-1, CD44, and CD90.2 (Thy1.2), and negative for CD34, CD117 (c-kit), CD45, CD11b, and MHC II (Fig. 1). After the BMSCs were cultured in differentiation medium for three weeks, obvious adipocytes and osteocytes were observed by staining with oil red O and Alizarin Red, respectively. For adipogenic differentiation, fat droplets were visualized by oil red O staining (Fig. 2A). Intracellular mineralized nodules, indicators of osteogenic differentiation, were stained by Alizarin Red (Fig. 2B). These differentiated cells represented the multipotent differentiation capacity of BMSCs. 3.2 Characteristics of MSCs after inducing or inhibiting autophagy Immunofluorescence detection of MAP1LC3 was performed to evaluate whether MSCs undergo autophagy. Cells displayed large numbers of MAP1LC3 dots in the cytoplasm after inducing autophagy (Fig. 2C). After transfecting MSCs with lentiviruses expressing shRNA for Becn1 knockdown, we further induced the cells by serum starvation to assess whether the inhibition was effective, and the results showed no obvious MAP1LC3 dots (Fig. 2D). These data indicate that MSCs underwent autophagy after induction, and that shBecn1 inhibited autophagy. 3.3 ELISA analysis The secretion levels of the four neurotrophins were decreased after inhibition of autophagy in MSCs (Fig. 3D). The secretion levels of VEGF and BDNF were
significantly higher in the autophagy-MSC group than in the normal MSC group. Although the secretion level of nerve growth factor were increased significantly in the autophagy-MSC and normal MSC groups compared with the normal MSC group, there was no significant difference between them (P > 0.05). The level of secreted neurotrophin 3 was not different among the three groups (P > 0.05). 3.4 Improvement of functional recovery MSCs (Fig. 3A) of various types, shBecn1-MSCs, normal MSCs, and autophagyMSCs, were used to treat SCI (Fig. 3B). At 4 weeks post-injury, hematoxylin and eosin staining revealed structures at the lesion site, and the dotted line indicated the boundary of the area analyzed by immunohistochemistry (Fig. 3C). Hoechst-labelled MSCs were present in the autophagy-MSC group, whereas very few labelled MSCs were present in the normal MSC group, and no labeled MSCs were observed in the shBecn1-MSC group (Fig. 4A). Recovery of motor function was assessed weekly by open field locomotor test. Left hindlimb locomotion improved gradually over the 4week observation period. Within the first two weeks, there were no differences among the treatment groups. Functional recovery in the PBS-treated group showed small degrees of improvement, while significant improvement in locomotion was observed over the course of the experiment in the autophagy-MSC group. At weeks 3 and 4, BBB scores were significantly higher in the autophagy-MSC group than in the other groups, but no difference was observed between the shBecn1-MSC and PBS groups (Fig. 4B). 3.5 Quantification of axonal fibers and 5-HT-positive fibers
At 4 weeks post-injury, an NF marker was used to evaluate axonal ingrowth at the injury site (Fig. 5A). As shown in Figure 5B, NF-positive fibers were promoted by treatment with autophagy-MSCs. The rats in the autophagy-MSC group exhibited the greatest axonal regeneration, while the shBecn1-MSC and PBS groups displayed only modest regeneration. There were significantly more NF-positive fibers in the groups treated with MSCs at week four after surgery than in the PBS group. The sprouting of serotonergic (5-HT) fibers from primary descending pathways of moto-sensory modulation in the mammalian spinal cord has been shown to aid recovery of locomotion (Courtine et al., 2008; Courtine et al., 2009). Regenerated 5HT fibers were more evident in the lesion site treated with autophagy-MSCs (Fig. 6A). The density of 5-HT-positive nerve fibers was significantly higher in the normal MSC group, and autophagy-MSC group than in the PBS group. Although 5-HT-positive nerve fibers in the normal MSC group were higher than in the shBecn1-MSC group, the difference was not significant (Fig. 6B). 3.6 Schwann cell regeneration analysis Schwann cells were evaluated by immunofluorescence staining for S100. Schwann cells within the lesioned area regenerated in all treatment groups, but were denser in the autophagy-MSC group than in the other treatment groups (Fig. 7A). A quantitative analysis also showed a greater number of S-100-positive cells in the center of the lesion in the MSC group than in the shBecn1-MSC and PBS groups, but this difference was not significant (Fig. 7B). 3.7 Autophagy-MSCs increased blood vessel formation
In terms of neovascularization, blood vessel formation (vWF staining) was observed four weeks after surgery (Fig. 8A). The blood vessel endothelial cell marker vWF signal in the lesion site in the autophagy-MSC group was higher than in the other treatment groups. Only a few blood vessel processes grew into the lesion site in the shBecn1-MSC and PBS groups. No differences in blood vessel density were found between the shBecn1-MSC and PBS groups (Fig. 8B). 3.8 Remyelination in the lesion zone The TEM images showed the high-resolution structure of myelinated axonal fibers (Fig. 9A). The thickness of the newly formed myelin sheath and remyelination of regenerated axons in the lesion site in the autophagy-MSC group was significantly higher than in the normal MSC and shBecn1-MSC groups. The PBS group showed limited myelination. The regenerated myelin sheath thickness at the SCI site was significantly greater in animals treated with MSCs and shBecn1-MSCs than in PBStreated animals (Fig. 9B). These data indicate that autophagy-MSCs not only increased the regeneration of axons within the lesion site, but also promoted myelination of the regenerated axons.
4. Discussion In this study, MSCs were used to treat SCI, due to their advantages of less immune rejection and fewer ethical concerns (Schira et al., 2012; Spejo et al., 2018). It has been reported that MSCs exert positive effects on axonal sprouting associated with accelerated functional recovery (Takahashi et al., 2018; Vaquero et al., 2017) It
remains unknown whether autophagy plays important roles in MSCs when MSCs are used to treat SCI. shRNA is an effective tool for prolonged knockdown of gene expression at the mRNA level for investigating gene function (Davidson and McCray, 2011). In the present study, lentiviral vectors were used to deliver shRNA to knock down Becn1 expression, which is essential for the autophagy process. The immunocytochemistry results showed significant elevation of MAP1LC3 in MSCs pretreated by induction of autophagy (Fig. 2C). In contrast, few MAP1LC3 immunofluorescence dots were present in MSCs after Becn1 knockdown (Fig. 2D). These results indicated that the inhibition or induction of autophagy in MSCs was successful. The results showed that the Hoechst-labelled MSCs migrated to the lesion site (Fig. 4A). This is consistent with previous reports that multiple chemokines at the injury site recruit transplanted MSCs after an injury (Li et al., 2012; Ramalho et al., 2018). However, we did not detect Hoechst-labelled MSCs in the shBecn1-MSC group, possibly because inhibiting autophagy limited the migration ability or cell survival in the inflammatory microenvironment at the lesion site (Fig. 4A). Autophagic MSCs secreted high levels of VEGF and BDNF, which are important growth factors for neural regeneration (Fig. 3D). Taken together, the homing MSCs exerted neuroprotective effects via the paracrine action of different neurotrophic factors after SCI. Before surgery, all rats exhibited normal locomotion (BBB score = 21). Immediately after surgery, all rats exhibited almost complete loss of left hindlimb
locomotion, indicating successful surgery. However, a significant difference was found between the autophagy-MSC group and the other treatment groups at the fourth week post-surgery, suggesting that inducing autophagy in MSCs further improved functional recovery, and inhibiting autophagy impaired MSC function. Sections of the injury site were stained with anti-NF antibody to evaluate axonal regeneration (Fig. 5A) and anti-5-HT antibody to evaluate newly sprouted serotonin fibers (Fig. 6A). Quantitative analysis revealed that inducing autophagy in MSCs promoted axonal and serotonin fiber regeneration. Serotonin plays an important role in locomotor behavior and in functional improvement after SCI (Ribotta et al., 2000). The survival and proliferation of Schwann cells following SCI were detected using S100 staining. The regeneration and migration of Schwann cells play key roles in successful axonal outgrowth and remyelination. After SCI, the loss of blood vessel integrity at the injury site partially results in cavity formation, cell death, and poor axonal regrowth (McCreedy and SakiyamaElbert, 2012). A previous study showed that autophagy enhances VEGF secretion from MSCs via ERK phosphorylation, thus promoting endothelial cell angiogenesis for cutaneous wound healing (An et al., 2018). In this study, we also found that the autophagic MSCs secreted a high level of VEGF (Fig. 3D). Thus, we stained for vWF to analyze angiogenesis in each group. The results showed enhanced vascularization in the autophagy-MSC group at the injured site after SCI, which is beneficial for nerve regeneration. In addition to axonal regeneration, improving the myelination of axons is an
important process in functional recovery. Previous studies have shown that remyelination of surviving or regenerated axons was beneficial for nerve function recovery of the spinal cord(Duncan et al., 2017). Our TEM data showed that treatment with autophagy-MSCs markedly improved myelination of the regenerated axons, as indicated by myelin sheath thickness. The regenerated axons, proliferation of Schwann cells, neovascularization, and remyelination of axons in the autophagy-MSC group all contributed to final functional recovery in SCI. These results indicate that inhibiting autophagy in MSCs limited the therapeutic effects of MSCs in SCI repair. Previous studies considered autophagy a detrimental factor in brain injury (Gao et al., 2018). Autophagy may have different effects on different cell types, but the specific mechanism needs further study. One study reported that elevated autophagy after irradiation leads to the accumulation of reactive oxygen species (ROS) in MSCs resulted in DNA damage and loss of stemness. Inducing autophagy by starvation reduced ROS accumulationassociated DNA damage and maintained MSC stemness, whereas inhibiting autophagy augmented ROS accumulation and DNA damage, resulting in a loss of stemness in MSCs (Hou et al., 2013). It has also been reported that autophagy may prevent radiation deteriorative processes in MSCs. MSCs exposed to low-dose irradiation showed impaired autophagy, leading to cellular senescence (Alessio et al., 2015). Autophagy also plays a fundamental role in MSC differentiation. Ablation of an essential component of mammalian autophagy led to multiple autophagic defects in osteoblasts and defective osteoblast terminal differentiation in primary bone
marrow, suggesting an important role of autophagy in support of osteoblast differentiation (Liu et al., 2013). Additionally, autophagy maintained cellular homeostasis and had a protective effect against environmental stresses. Enhancing autophagy
in
MSCs
may
elevate
cellular
survival
in
an
inflammatory
microenvironment. Inhibiting autophagy made MSCs more susceptible to cell death (Yang et al., 2016). In this study, the autophagy-enhanced therapeutic effect of MSCs may involve multiple aspects.
5. Conclusion We successfully induced or inhibited autophagy in MSCs and then applied these types of MSCs in a rat T8−T9 SCI model. The in vitro study showed that the secretion of neurotrophic and growth factors from autophagic MSCs was increased. We demonstrated that enhancing autophagy in MSCs resulted in a positive impact on neuronal generation, Schwann cell regeneration, and promotion of angiogenesis. The locomotion recovery results were consistent with the immunohistochemical results. On the other hand, inhibiting autophagy downregulated the utility of MSCs for treating SCI. Our study suggests that autophagy is important for MSC function, and that regulating autophagy may provide a novel strategy for improving the regenerative capacity of MSCs in SCI repair.
Data Availability The data used to support the findings of this study are included within the article.
Acknowledgments This work was supported by grants (2018YFA0107900, 31771491, 81471242, 81601069) from the National Nature Science Foundation and Ministry of Science and Technology of China, Shanghai Sailing Program (19YF1404600) and Shanghai Municipal Government (2019CXJQ01). Many thanks for Dr Congfeng Xu and Dr Jiefang Huang in chinese academy of sciences for providing technical support regarding autophagy inhibiting.
Author Disclosure Statement No competing financial interests exist.
Reference
Alessio, N., Del Gaudio, S., Capasso, S., Di Bernardo, G., Cappabianca, S., Cipollaro, M., Peluso, G., Galderisi, U., 2015. Low dose radiation induced senescence of human mesenchymal stromal cells and impaired the autophagy process. Oncotarget. 6, 8155-66. An, Y., Liu, W.J., Xue, P., 2018. Autophagy promotes MSC-mediated vascularization in cutaneous wound healing via regulation of VEGF secretion. 9, 58. Anderson, M.A., O'Shea, T.M., Burda, J.E., Ao, Y., Barlatey, S.L., Bernstein, A.M., Kim, J.H., James, N.D., Rogers, A., Kato, B., Wollenberg, A.L., Kawaguchi,
R., Coppola, G., Wang, C., Deming, T.J., He, Z., Courtine, G., Sofroniew, M.V., 2018. Required growth facilitators propel axon regeneration across complete spinal cord injury. Nature. 561, 396-400. Anokhina, E.B., Buravkova, L.B., 2007. [Heterogeneity of stromal precursor cells isolated from rat bone marrow]. Tsitologiia. 49, 40-7. Assinck, P., Duncan, G.J., Hilton, B.J., Plemel, J.R., 2017. Cell transplantation therapy for spinal cord injury. 20, 637-647. Basso, D.M., Beattie, M.S., Bresnahan, J.C., 1995. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 12, 1-21. Comolli, N., Neuhuber, B., Fischer, I., Lowman, A., 2009. In vitro analysis of PNIPAAm-PEG, a novel, injectable scaffold for spinal cord repair. Acta Biomater. 5, 1046-55. Courtine, G., Song, B., Roy, R.R., Zhong, H., Herrmann, J.E., Ao, Y., Qi, J., Edgerton, V.R., Sofroniew, M.V., 2008. Recovery of supraspinal control of stepping via indirect propriospinal relay connections after spinal cord injury. Nat Med. 14, 69-74. Courtine, G., Gerasimenko, Y., van den Brand, R., Yew, A., Musienko, P., Zhong, H., Song, B., Ao, Y., Ichiyama, R.M., Lavrov, I., Roy, R.R., Sofroniew, M.V., Edgerton, V.R., 2009. Transformation of nonfunctional spinal circuits into functional states after the loss of brain input. Nat Neurosci. 12, 1333-42. Cully, M., 2018. Spinal cord injury: Ion transporter agonist improves recovery. Nat Rev Drug Discov. 17, 621.
Dang, S., Xu, H., Xu, C., Cai, W., Li, Q., Cheng, Y., Jin, M., Wang, R.X., Peng, Y., Zhang, Y., Wu, C., He, X., Wan, B., Zhang, Y., 2014. Autophagy regulates the therapeutic potential of mesenchymal stem cells in experimental autoimmune encephalomyelitis. Autophagy. 10, 1301-15. Davidson, B.L., McCray, P.B., Jr., 2011. Current prospects for RNA interferencebased therapies. Nat Rev Genet. 12, 329-40. Doherty, J., Baehrecke, E.H., 2018. Life, death and autophagy. Nat Cell Biol. Duncan, I.D., Marik, R.L., Broman, A.T., Heidari, M., 2017. Thin myelin sheaths as the hallmark of remyelination persist over time and preserve axon function. Proc Natl Acad Sci U S A. 114, E9685-e9691. Falnikar, A., Hala, T.J., Poulsen, D.J., Lepore, A.C., 2016. GLT1 overexpression reverses established neuropathic pain-related behavior and attenuates chronic dorsal horn neuron activation following cervical spinal cord injury. Glia. 64, 396-406. Gao, Y., Zhang, M.Y., Wang, T., Fan, Y.Y., Yu, L.S., Ye, G.H., Wang, Z.F., Gao, C., Wang, H.C., Luo, C.L., Tao, L.Y., 2018. IL-33/ST2L Signaling Provides Neuroprotection Through Inhibiting Autophagy, Endoplasmic Reticulum Stress, and Apoptosis in a Mouse Model of Traumatic Brain Injury. Front Cell Neurosci. 12, 95. Hansen, M., Rubinsztein, D.C., 2018. Autophagy as a promoter of longevity: insights from model organisms. 19, 579-593. Hou, J., Han, Z.P., Jing, Y.Y., Yang, X., Zhang, S.S., Sun, K., Hao, C., Meng, Y., Yu,
F.H., Liu, X.Q., Shi, Y.F., Wu, M.C., Zhang, L., Wei, L.X., 2013. Autophagy prevents irradiation injury and maintains stemness through decreasing ROS generation in mesenchymal stem cells. Cell Death Dis. 4, e844. Kang, K.N., Kim, D.Y., Yoon, S.M., Lee, J.Y., Lee, B.N., Kwon, J.S., Seo, H.W., Lee, I.W., Shin, H.C., Kim, Y.M., Kim, H.S., Kim, J.H., Min, B.H., Lee, H.B., Kim, M.S., 2012. Tissue engineered regeneration of completely transected spinal cord using human mesenchymal stem cells. Biomaterials. 33, 4828-35. Khan, I.U., Yoon, Y., Kim, A., Jo, K.R., Choi, K.U., Jung, T., Kim, N., Son, Y., Kim, W.H., Kweon, O.K., 2018. Improved Healing after the Co-Transplantation of HO-1 and BDNF Overexpressed Mesenchymal Stem Cells in the Subacute Spinal Cord Injury of Dogs. Cell Transplant. 27, 1140-1153. Kim, K., Dean, D., Mikos, A.G., Fisher, J.P., 2009. Effect of initial cell seeding density on early osteogenic signal expression of rat bone marrow stromal cells cultured on cross-linked poly(propylene fumarate) disks. Biomacromolecules. 10, 1810-7. Li, L., Yang, M., Wang, C., Zhao, Q., Liu, J., Zhan, C., Liu, Z., Li, X., Wang, W., Yang, X., 2012. Effects of cytokines and chemokines on migration of mesenchymal stem cells following spinal cord injury. Neural Regen Res. 7, 1106-12. Liu, F., Fang, F., Yuan, H., Yang, D., Chen, Y., Williams, L., Goldstein, S.A., Krebsbach, P.H., Guan, J.L., 2013. Suppression of autophagy by FIP200 deletion leads to osteopenia in mice through the inhibition of osteoblast
terminal differentiation. J Bone Miner Res. 28, 2414-30. Ma, Y.H., Zeng, X., Qiu, X.C., Wei, Q.S., Che, M.T., Ding, Y., Liu, Z., Wu, G.H., Sun, J.H., Pang, M., Rong, L.M., Liu, B., Aljuboori, Z., Han, I., Ling, E.A., Zeng, Y.S., 2018. Perineurium-like sheath derived from long-term surviving mesenchymal stem cells confers nerve protection to the injured spinal cord. Biomaterials. 160, 37-55. McCreedy, D.A., Sakiyama-Elbert, S.E., 2012. Combination therapies in the CNS: engineering the environment. Neurosci Lett. 519, 115-21. Mizushima, N., Komatsu, M., 2011. Autophagy: renovation of cells and tissues. Cell. 147, 728-41. Oliveri, R.S., Bello, S., Biering-Sorensen, F., 2014. Mesenchymal stem cells improve locomotor recovery in traumatic spinal cord injury: systematic review with meta-analyses of rat models. Neurobiol Dis. 62, 338-53. Park, E., Velumian, A.A., Fehlings, M.G., 2004. The role of excitotoxicity in secondary mechanisms of spinal cord injury: a review with an emphasis on the implications for white matter degeneration. J Neurotrauma. 21, 754-74. Park, K., 2018. Functional recovery in spinal cord injury using mesenchymal stem cells. J Control Release. 278, 159. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R., 1999. Multilineage potential of adult human mesenchymal stem cells. Science. 284, 143-7.
Ramalho, B.D.S., Almeida, F.M., Sales, C.M., de Lima, S., Martinez, A.M.B., 2018. Injection of bone marrow mesenchymal stem cells by intravenous or intraperitoneal routes is a viable alternative to spinal cord injury treatment in mice. Neural Regen Res. 13, 1046-1053. Ribotta, M.G., Provencher, J., Feraboli-Lohnherr, D., Rossignol, S., Privat, A., Orsal, D., 2000. Activation of locomotion in adult chronic spinal rats is achieved by transplantation of embryonic raphe cells reinnervating a precise lumbar level. J Neurosci. 20, 5144-52. Schira, J., Gasis, M., Estrada, V., Hendricks, M., Schmitz, C., Trapp, T., Kruse, F., Kogler, G., Wernet, P., Hartung, H.P., Muller, H.W., 2012. Significant clinical, neuropathological and behavioural recovery from acute spinal cord trauma by transplantation of a well-defined somatic stem cell from human umbilical cord blood. Brain. 135, 431-46. Singh, D., Harding, A.J., Albadawi, E., Boissonade, F.M., Haycock, J.W., Claeyssens, F., 2018. Additive manufactured biodegradable poly(glycerol sebacate methacrylate) nerve guidance conduits. Acta Biomater. 78, 48-63. Spejo, A.B., Chiarotto, G.B., Ferreira, A.D.F., Gomes, D.A., Ferreira, R.S., Jr., Barraviera,
B.,
Oliveira,
A.L.R.,
2018.
Neuroprotection
and
immunomodulation following intraspinal axotomy of motoneurons by treatment with adult mesenchymal stem cells. 15, 230. Stower, H., 2018. Cell therapy for spinal cord injury. Nat Med. 24, 1088. Takahashi, A., Nakajima, H., Uchida, K., Takeura, N., Honjoh, K., Watanabe, S.,
Kitade, M., Kokubo, Y., Johnson, W.E.B., Matsumine, A., 2018. Comparison of Mesenchymal Stromal Cells Isolated from Murine Adipose Tissue and Bone Marrow in the Treatment of Spinal Cord Injury. Cell Transplant. 27, 1126-1139. Tsenkina, Y., Ricard, J., Runko, E., Quiala-Acosta, M.M., Mier, J., Liebl, D.J., 2015. EphB3 receptors function as dependence receptors to mediate oligodendrocyte cell death following contusive spinal cord injury. Cell Death Dis. 6, e1922. Vaquero, J., Zurita, M., Rico, M.A., Bonilla, C., Aguayo, C., Fernandez, C., Tapiador, N., Sevilla, M., Morejon, C., Montilla, J., Martinez, F., Marin, E., Bustamante, S., Vazquez, D., Carballido, J., Rodriguez, A., Martinez, P., Garcia, C., Ovejero, M., Fernandez, M.V., 2017. Repeated subarachnoid administrations of autologous mesenchymal stromal cells supported in autologous plasma improve quality of life in patients suffering incomplete spinal cord injury. Cytotherapy. 19, 349-359. Wilems, T.S., Pardieck, J., Iyer, N., Sakiyama-Elbert, S.E., 2015. Combination therapy of stem cell derived neural progenitors and drug delivery of antiinhibitory molecules for spinal cord injury. Acta Biomater. 28, 23-32. Yan, J., Porch, M.W., Court-Vazquez, B., Bennett, M.V.L., Zukin, R.S., 2018. Activation of autophagy rescues synaptic and cognitive deficits in fragile X mice. Proc Natl Acad Sci U S A. Yang, R., Ouyang, Y., Li, W., Wang, P., Deng, H., Song, B., Hou, J., Chen, Z., Xie, Z., Liu, Z., Li, J., Cen, S., Wu, Y., Shen, H., 2016. Autophagy Plays a
Protective Role in Tumor Necrosis Factor-alpha-Induced Apoptosis of Bone Marrow-Derived Mesenchymal Stem Cells. Stem Cells Dev. 25, 788-97. Yao, R., Murtaza, M., Velasquez, J.T., Todorovic, M., Rayfield, A., Ekberg, J., Barton, M., St John, J., 2018. Olfactory Ensheathing Cells for Spinal Cord Injury: Sniffing Out the Issues. Cell Transplant. 27, 879-889. Zhang, S., Wang, X.J., Li, W.S., Xu, X.L., Hu, J.B., Kang, X.Q., Qi, J., Ying, X.Y., You,
J.,
Du,
Y.Z.,
2018.
Polycaprolactone/polysialic
acid
hybrid,
multifunctional nanofiber scaffolds for treatment of spinal cord injury. Acta Biomater. 77, 15-27.
Figure legends
Figure 1. Surface marker expression of bone marrow-derived mesenchymal stem cells (BMSCs) analyzed by flow cytometry.
Figure 2. (A) Adipogenic differentiation potentials of BMSCs were confirmed by oil red O staining. Scale bar = 100 µm. (B) Osteogenic differentiation potentials of BMSCs were confirmed by Alizarin Red staining. Scale bar = 200 µm. (C) Representative confocal microscopy images of cells after staining with antiMAP1LC3 antibody (green) for BMSCs after autophagy. (D) Representative confocal microscopy images of cells after staining with anti-MAP1LC3 antibody (green) for BMSCs after knock down expression of the autophagy gene Becn1 and then induced autophagy. The cell nuclei were counterstained with Hoechst 33342 (blue). Scale bar = 30 µm.
Figure 3. Surgical procedures of spinal cord injury (SCI) and ELISA analysis. (A) Morphology of MSCs under light microscope. Scale bar = 100 µm. (B) Photograph of spinal cord four weeks after surgery. (C) H&E staining at week 4 after surgery. Scale bar = 1 mm. (D). ELISA analyses of NGF, VEGF, BDNF and NT3 secretion from MSCs. Data are expressed as means ± SD; n = 3; *P < 0.05; **P < 0.01.
Figure 4. (A) Images of Hoechst labeled MSCs in each group. Scale bar, 30 µm. (B) Weekly post-SCI Basso, Beattie, and Bresnahan (BBB) open field locomotor scores. Data are expressed as means ± SD; n = 8; *P < 0.05; **P < 0.01.
Figure 5. Sections of the SCI at the lesion site stained with anti-NF antibody. (A) Representative images of the PBS, shBecn1-MSCs, MSCs, and autophagy-MSCs
treatment groups. The nuclei were stained using Hoechst 33342 (blue). Scale bar = 50 µm. (B) Quantification of the area of positive NF staining in each group. Data are expressed as means ± SD; n = 4; **P < 0.01.
Figure 6. Regeneration of serotonergic (5-HT) fibers at the SCI site. (A) Immunostained images of serotonergic (5-HT) fibers at the lesion site four weeks post-SCI. The nuclei were stained using Hoechst 33342 (blue). Scale bar = 50 µm. (B) Quantitative analysis of the area of 5-HT fibers in the different treatment groups. Data are expressed as means ± SD; n = 4; *P < 0.05; **P < 0.01.
Figure 7. Immunohistochemical analysis of the regenerated spinal cord four weeks after injury. (A) S-100 staining of sections showing Schwann cell regeneration. The nuclei were stained using Hoechst 33342 (blue). Scale bar = 50 µm. (B) Quantitative analysis of the area of Schwann cells in each group. Data are expressed as means ± SD; n = 4; **P < 0.01.
Figure 8. Immunohistochemical analysis of SCI sites four weeks after implantation. (A) Anti-von Willebrand factor (vWF) immunostained images of each treatment group. The nuclei were stained using Hoechst 33342 (blue). Scale bar = 50 µm. (B) Blood vessel density at the injury site. Statistical analysis performed using the ImagePro Plus software package. Data are expressed as means ± SD; n = 4; *P < 0.05; **P < 0.01.
Figure 9. Analysis of the degree of remyelination at the lesion site four weeks after surgery. (A) Transmission electron microscopy (TEM) images of regenerated spinal cord, showing differences in myelin sheath thickness among the treatment groups. Scale bar = 1 µm. (B) Myelin sheath thickness in each treatment group. Data are expressed as means ± SD; n = 4; *P < 0.05; **P < 0.01.
Transplantation with mesenchymal stem cells (MSCs) has shown beneficial effects in treating spinal cord injury (SCI). Autophagy is an evolutionarily conserved process of degradation
and recycling of cellular components that plays an important role in tissue homeostasis and cellular survival. Whether regulating autophagy in MSCs may affect their therapeutic potential in spinal cord injury repair has not yet been determined. In this study, we found that autophagy played important roles in MSCs during its application in SCI repairing. Regulation of autophagy in MSCs before in vivo transplantation could be a potential therapeutic intervention strategy for SCI.