Simulated Microgravity Culture Enhances the Neuroprotective Effects of Human Cranial Bone-Derived Mesenchymal Stem Cells in Traumatic Brain Injury

Abstract

Fundamental cures of central nervous system (CNS) diseases are rarely achieved due to the low regenerative ability of the CNS. Recently, cell-based therapy using mesenchymal stem cells (MSCs) has been explored as an effective treatment for CNS diseases. Among the various tissue-derived MSCs, we have isolated human cranial bone-derived MSCs (cMSCs) in our laboratory. In addition, we have focused on simulated microgravity (MG) as a valuable culture environment of MSCs. However, detailed mechanisms underlying functional recovery from transplantation of MSCs cultured under MG conditions remain unclear. In this study, we investigated the therapeutic mechanisms of transplantation of cMSCs cultured under MG conditions in traumatic brain injury (TBI) model mice. Human cMSCs were cultured under 1G and MG conditions, and cMSCs cultured under MG conditions expressed significantly higher messenger RNA (mRNA) levels of hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-β). In TBI model mice, the transplantation of cMSCs cultured under MG conditions (group MG) showed greater motor functional improvement compared with only phosphate-buffered saline administration (group PBS). Moreover, the protein expression levels of tumor necrosis factor alpha (TNF-α) and the Bcl-2-associated X protein (Bax)/b cell leukemia/lymphoma 2 protein (Bcl-2) ratio were significantly lower at brain injury sites in mice of group MG than those of group PBS. In addition, an in vitro study showed that the conditioned medium of cMSCs cultured under MG conditions significantly suppressed the cell death of NG108-15 cells exposed to oxidative or inflammatory stress through anti-inflammatory and antiapoptosis effects. These findings demonstrate that culturing cMSCs under simulated MG increases the neuroprotective effects, suggesting that simulated MG cultures may be a useful method for cell-based therapy strategies for CNS diseases.

Introduction

Central nervous system (CNS) diseases, including stroke, traumatic brain injury (TBI), and spinal cord injury (SCI), rarely achieve a radical cure because the CNS has limited regenerative capacity. Recently, cell-based therapy has attracted increasing attention as a novel strategy for CNS diseases. In particular, mesenchymal stem cells (MSCs) are considered valuable candidates for cell-based therapy because they can be isolated from various tissues and have self-renewal and multilineage differentiation potentials [1,2]. Moreover, MSCs are able to secrete diverse humoral factors [3 –5]. Indeed, animal experiments have shown that motor function in CNS disease models is significantly improved by MSC transplantation [6,7]. In addition, clinical trials of cell-based therapy using MSCs have been reported [8].

Human MSCs can be isolated from various tissues such as bone marrow, adipose tissue, dental pulp, Wharton jelly, and synovial membranes [9 –11]. Previous studies have suggested that the unique properties of specific MSCs differ depending on the derived tissue [10]. Thus, it is necessary to choose individually suitable MSCs for each patient's symptoms. Recently, we focused on MSCs derived from human cranial bone-derived MSCs (cMSCs), which originate from the neural crest. Shinagawa et al. showed that cMSCs have a higher neurogenic potential than MSCs derived from iliac bone marrow [12]. In addition, Mead et al. reported that MSCs derived from neural crest-originated dental pulp have a higher neuroprotective effect than those derived from iliac bone marrow or adipose tissue [13]. From these studies, cMSCs are thought to be useful candidates for cell-based therapy.

The cell culture environment is one of the important factors in MSC transplantation [14 –16]. We have previously reported that MSCs cultured under simulated microgravity (MG) conditions have higher migration ability and promote greater functional recovery compared with MSCs cultured under normal gravity conditions in an SCI model [17]. However, the mechanisms of functional recovery from transplantation of MSCs cultured under simulated MG remain unclear. Therefore, the present study aimed to investigate whether differences in the gravity environment affect the neuroprotective effects of cMSCs in vivo and in vitro.

Materials and Methods

Isolation and culture of cMSCs

Human cranial bone marrow samples were collected from frontotemporal cranial bone waste following neurosurgical procedures. Tissue samples were seeded onto tissue culture dishes (Sumitomo Bakelite Co., Tokyo, Japan). The growth medium for cMSCs consisted of Dulbecco's modified Eagle's medium (DMEM)-low glucose (Sigma-Aldrich Co., St Louis, MO) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA), penicillin (100 U/mL), and streptomycin (100 μg/mL: both from Sigma-Aldrich Co.). After observing cell adhesion in the dishes, adherent cells were collected and used as cMSCs. Cells were maintained at 37°C in 5% CO₂, and the medium was changed every 3 days. Human cranial bone samples were obtained upon written informed consent by the patients. This study was approved by the Hiroshima University ethical committee.

Cell culture under simulated MG conditions

cMSCs were seeded (3.5 × 10³ cells/cm²) onto culture flasks (Corning, Inc., Corning, NY). After 24 h of culture, cells were divided into the 1G or MG conditions. MG conditions were produced using Gravite^® (Space Bio-Laboratories Co. Ltd., Hiroshima, Japan). This device can establish 10⁻³ G conditions similar to the International Space Station over a time average by controlled rotation of the two axes, with consequent minimization of the cumulative gravitational vector at the center of the device.

Multilineage cell differentiation

cMSCs, at passage 3 or 4, were used for differentiation into osteoblasts, adipocytes, or neurons. To induce osteogenic differentiation, cells were cultured in Mesenchymal Stem Cell Osteogenic Differentiation Medium (Promocell, Heidelberg, Germany) for 21 days. The medium was changed every 3 or 4 days. Cells were finally stained with Alizarin red S solution (Sigma-Aldrich Co.) for 30 min to confirm calcium deposition. To induce adipogenic differentiation, cells were cultured in Mesenchymal Stem Cell Adipogenic Differentiation Medium (Promocell) for 14 days. The medium was changed every 3 or 4 days. Cells were finally stained with Oil red O solution (Wako Pure Chemical Industries, Osaka, Japan) for 15 min to confirm neutral triglycerides and lipid droplets. To induce neural differentiation, cells were cultured under modified neural differentiation conditions, including neural conditioning medium and neural differentiation medium, according to previously reported methods [15,16]. Neural conditioning medium consisted of DMEM: Nutrient Mixture F-12 (Invitrogen Co., Carlsbad, CA) with 1% FBS, basic fibroblast growth factor (bFGF; 100 ng/mL; PeproTech, Rocky Hill NJ), penicillin (100 U/mL), and streptomycin (100 μg/mL). After incubation in this neural conditioning medium for 3 days, cells were cultured in neural differentiation medium comprising a neural conditioning medium with forskolin (10 μM; Sigma-Aldrich Co.) added for 7 days. The differentiation medium was changed every 3 or 4 days. After neural differentiation, cMSCs were incubated with neurofilament medium chain (NF-M) mouse mAb (1:200; Cell Signaling Technology, Inc.) as the primary antibody overnight at 4°C, followed by Alexa Fluor 488-conjugated anti-mouse IgG antibody (1:500; Molecular Probes Europe BV Co., Leiden, Netherlands) as the fluorescent secondary antibody, for 60 min at room temperature. 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI; 1:1,000; Kirkegaard & Perry Laboratories, Gaithersburg, MD) was used to stain nuclei. Stained cMSCs were examined with a multifunctional microscope (BZ-9000; KEYENCE Co., Osaka, Japan). Differentiation potential after culturing under the 1G condition or MG condition for 5 days was also evaluated.

Flow cytometry analysis

cMSCs at passage 3 were harvested with TrypLE^™ Select (Thermo Fisher Scientific), centrifuged at 1,500 g for 5 min, and resuspended in phosphate-buffered saline (PBS; n = 3). Aliquots containing 1 × 10⁵ cells were incubated with fluorochrome-conjugated [either phycoerythrin (PE) or fluorescein isothiocyanate (FITC)] antibodies against human CD34, CD44, CD45, CD73, CD90 (Biolegend Co., San Diego, CA), and CD105 (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). PE mouse IgG1 and FITC mouse IgG1 (Biolegend Co.) were used as isotype controls. Data acquisition and analysis were performed using FACSVerse (BD Biosciences, San Jose, CA).

Reverse transcription and real-time polymerase chain reaction

After 5 days of culture under either 1G or MG conditions, cMSCs were collected in PBS (n = 5). Total RNA was extracted using NucleoSpin^® RNA (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized using ReverTra Ace-α- (Toyobo Co., Ltd., Osaka, Japan). Real-time polymerase chain reaction (PCR) was performed with the 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions. Real-time PCR was performed using the Fast Start Universal Probe Master (Roche, Basel, Switzerland) and oligonucleotide primer sets corresponding to the cDNA sequences of human bFGF, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primer sequences used in this study are listed in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/scd).

Preparation of TBI model mice and cell transplantation

The TBI model was developed by the cryogenic procedure using adult, male C57BL/6 mice (6–8 weeks old, n = 39). The mice were anesthetized with isoflurane (Mylan Seiyaku Co., Osaka, Japan) before the surgical procedure. After a skin incision on the top of the head, a metal probe (diameter, 3 mm; length, 100 mm) that had been chilled in liquid nitrogen was applied to the left motor cortex area (midpoint between the coronal structure and bregma and 3 mm lateral to the left from the sagittal suture) for 30 s, followed by a resting period of 30 s; this procedure was repeated four times. Mice were divided into the following three groups according to the treatment received: only PBS administration after brain injury (group PBS, n = 13); transplantation of cMSCs cultured under 1G conditions after brain injury (group 1G, n = 13); and transplantation of cMSCs cultured under MG conditions after brain injury (group MG, n = 13). Mice of group 1G and group MG were injected with cMSCs (3 × 10⁵ cells/100 μL PBS) intravenously 24 h after the surgical procedure. All experimental protocols in the present study were approved by the Animal Testing Committee Guidelines at Hiroshima University. In addition, animal care and handling procedures were in accordance with National Institutes of Health guidelines.

Motor functional analyses in TBI model mice

To evaluate motor function, the rotarod test and the beam-walking test were used. The rotarod test can evaluate motor function and coordination, while the beam-walking test can evaluate forelimb and hind limb locomotor activity [18]. In the rotarod test, mice were placed on the rotarod (KN-75; Natsume Seisakusyo, Tokyo, Japan) at a speed of 20 rpm, and the time until they fall (for a maximum of 60 s) was measured. In the beam-walking test, mice were placed on a narrow wooden beam (width, 6 mm; length, 120 mm). Then, the number of foot faults for the contralateral hind limb per 50 steps was counted. Motor functional analyses were performed during the dark cycle pretransplantation and days 7, 14, 21, and 28 after transplantation (n = 5 in each group).

Messenger RNA expression analysis of brain injury sites

Mice were sacrificed 24 h after cMSC transplantation (n = 4 in each group). Brains were obtained and soaked in RNA Later (Sigma-Aldrich Co.). Total RNA was extracted from the injured sites (left motor cortex area) using Isogen (Nippon Gene, Tokyo, Japan). RNA extraction and reverse transcription were performed as described in the above-referenced method. Real-time PCR was performed using oligonucleotide primer sets corresponding to the cDNA sequences of mouse b cell leukemia/lymphoma 2 protein (Bcl-2), Bcl-2-associated X protein (Bax), and tumor necrosis factor alpha (Tnf-α). Beta-actin (Actb) was used as an internal control. The primer sequences used in this study are listed in Supplementary Table S2.

Western blotting analysis

Brain injury site samples were collected 24 h after cMSC transplantation and total proteins were extracted using radioimmunoprecipitation buffer (Nacalai Tesque, Kyoto, Japan; n = 4 in each group). The protein concentration was measured by the BioRad protein assay (Bio-Rad Laboratories, Hercules, CA). Proteins (20 μg per lane) were separated using electrophoresis on a 15% sodium dodecyl sulfate–polyacrylamide gel and transferred to a nitrocellulose membrane (HybondTM-ECL; GE Healthcare, Little Chalfont, UK). The membrane was blocked with a blocking buffer (20 mM Tris-HCl pH 7.4/137 mM NaCl/0.1%Tween-20 containing 1%, 2%, or 5% bovine serum albumin) for 1 h at room temperature. Membranes were incubated overnight at 4°C with primary antibodies against Bax (sc-7480, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), Bcl-2 (sc-492, 1:200; Santa Cruz Biotechnology), TNF-α (ab6671, 1:500; Abcam, Cambridge, UK), and GAPDH (G9495, 1:40,000; Sigma-Aldrich Co.). After washing with Tris-buffered saline with Tween 20, membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:2,000 for Bax; Cell Signaling Technology, Inc.) and anti-rabbit IgG (1:2,000 for Bcl-2, 1:10,000 for TNF-α and GAPDH; Cell Signaling Technology, Inc.) as the secondary antibody. The immunoreaction was visualized using either SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) or Trident femto-ECL (Gene Tex, San Antonio, TX) and images were acquired with the Versa Doc imaging system (Bio-Rad Laboratories). Target protein expression was quantified by measuring the band density with ImageJ software (NIH, Bethesda, MD). Data were normalized using GAPDH.

Preparation of cMSC-conditioned medium

cMSCs were seeded onto culture flasks and cultured under 1G or MG conditions in the growth medium. At the point when they reached 80% confluency, the medium was changed to fresh growth medium without FBS, and cells were cultured under both conditions. After 24 h of culture, culture supernatants were collected from both conditions as conditioned medium (CM). After 0.2 μm filtration, 1G-CM and MG-CM were stored at −80°C until use.

Inflammatory or oxidative stress exposure of NG108-15 cells and cell death assays

To evaluate the neuroprotective effects of 1G-CM or MG-CM, NG108-15 cells were exposed to inflammatory or oxidative stress. Lipopolysaccharide (LPS; Wako Pure Chemical Industries, Osaka, Japan) was used to mimic inflammatory stress in cells. H₂O₂ (Santoku Chemical Industries, Tokyo, Japan) was used to mimic oxidative stress in cells. NG108-15 (ECACC, Porton Down, UK) neural cells were seeded onto culture dishes (Corning, Inc.) and cultured in DMEM-high glucose (DMEM-H; Sigma-Aldrich Co.) and 10% FBS (Thermo Fisher Scientific), penicillin (100 U/mL), streptomycin (100 μg/mL; both from Sigma-Aldrich Co.), and HAT supplement (Thermo Fisher Scientific). Cells were cultured at 37°C in 5% CO₂. After 48 h of culture, the medium was changed to fresh cMSC growth medium (absence of cMSC culture and without FBS; Ctrl), 1G-CM, or MG-CM (with 500 μM H₂O₂ or 200 ng/mL LPS) [19]. After 24 h of exposure to stress, cells were collected, centrifuged, and suspended in PBS (n = 8). Cell survival rates were determined with a counting chamber using Trypan blue staining. The remaining cells were collected for messenger RNA (mRNA) expression analysis.

mRNA expression analysis of stress-exposed NG108-15 cells

Total RNA was extracted from NG108-15 cell samples and reverse transcription was performed as described in the above-referenced method (n = 6). Real-time PCR was performed using oligonucleotide primer sets corresponding to the cDNA sequences of Bcl-2, Bax, Tnf-α, and tumor necrosis factor receptor superfamily, member 1A (Tnfrsf1a). Actb was used as the internal control. The primer sequences used in this study are listed in Supplementary Table S3.

Statistical analyses

Statistical analyses were performed using the JSTAT software (Sato). Data were evaluated using a paired t-test for mRNA expression of cMSC analysis. For comparison of the three groups, if Bartlett's test showed significant equality of variance, one-way analysis of variance (ANOVA) with the Bonferroni test was applied. When Bartlett's test did not show a significant equality of variance, the Kruskal–Wallis test with modified Tukey's test was applied. In the motor functional analysis, two-way ANOVA with the Bonferroni test was used. P value <0.05 was considered significant.

Results

Differentiation potential and surface marker expression in cMSCs

The differentiation potential of isolated cMSCs into adipocytes, osteoblasts, and neurons was investigated. cMSCs were negative for specific staining [Oil red O staining, Alizarin red S staining, and immunostaining (NF-M)] before differentiation, but positive cells were observed after differentiation (Fig. 1A–F).

FIG. 1.

Specific staining of isolated cMSCs in multilineage cell differentiation. Alizarin red S staining before and after osteogenic differentiation of cMSCs (A, B). Oil red O staining before and after adipogenic differentiation of cMSCs (C, D). Immunostaining of NF-M before and after neural differentiation of cMSCs (E, F). All scale bars indicate 100 μm. cMSCs, cranial bone-derived mesenchymal stem cells; NF-M, neurofilament medium chain. Color images available online at www.liebertpub.com/scd

Among the cell surface markers, isolated cMSCs were strongly positive for CD44, CD73, CD90, and CD105, but almost completely negative for CD34 and CD45 (Table 1).

Table 1.

Surface Marker Expression of Cranial Bone-Derived Mesenchymal Stem Cells

	Percent positive
MSC markers
CD44	99.47 ± 0.38
CD73	100.00 ± 0.00
CD90	100.00 ± 0.00
CD105	99.93 ± 0.07
Hematopoietic markers
CD34	0.10 ± 0.03
CD45	0.20 ± 0.08

Data represent the mean ± SEM of independent experiments. n = 3.

MSC, mesenchymal stem cell; SEM, standard error of the mean.

There were no differences in the differentiation potential of cells after culturing under 1G condition or MG condition for 5 days (Fig. 2A–F).

FIG. 2.

Specific staining of cMSCs cultured under 1G or MG conditions in multilineage cell differentiation. Alizarin red S staining after osteogenic differentiation of cMSCs that were cultured under 1G or MG conditions (A, B). Oil red O staining after adipogenic differentiation of cMSCs that were cultured under 1G or MG conditions (C, D). Immunostaining of NF-M after neural differentiation of cMSCs that were cultured under 1G or MG conditions (E, F). All scale bars indicate 100 μm. MG, microgravity. Color images available online at www.liebertpub.com/scd

Effects of MG culture conditions on the mRNA expression associated with neurotrophic and anti-inflammatory factors in cMSCs

cMSCs were cultured under 1G or MG conditions and analyzed for mRNA expression in both conditions. Expression levels of HGF and TGF-β in the MG conditions were significantly higher than those in the 1G conditions (Fig. 3A, B). On the other hand, expression levels of BDNF, bFGF, GDNF, and VEGF did not show significant differences between the two conditions (Fig. 3C–F).

FIG. 3.

mRNA expression of cMSCs cultured under MG conditions. mRNA expression levels of HGF (A), TGF-β (B), BDNF (C), bFGF (D), GDNF (E), and VEGF (F). Data represent the mean ± SEM of independent experiments. n = 5, *p < 0.05. BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; GDNF, glial cell-derived neurotrophic factor; HGF, hepatocyte growth factor; mRNA, messenger RNA; SEM, standard error of the mean; TGF-β, transforming growth factor beta; VEGF, vascular endothelial growth factor.

Motor functional improvements in TBI model mice

We evaluated motor function using the rotarod test and beam-walking test to compare the functional recovery of mice of group PBS, group 1G, and group MG. Mice of group MG showed more significant improvement than those of group PBS 7 days after the transplantation and also showed more significant improvement than those of group 1G on day 14 (Fig. 4A). In the beam-walking test, mice of group MG had significantly greater improvement than those of group PBS 7 days after transplantation (Fig. 4B). Mice of group MG showed the most functional recovery among the three groups on day 28 after transplantation.

FIG. 4.

Effects of MG-cultured cMSC transplantation on motor functional improvements in TBI model mice. Results of the rotarod test (A) and the beam-walking test (B). Data represent the mean ± SEM of independent experiments. n = 5, *p < 0.05 (group MG vs. group PBS), ^§ p < 0.05 (group MG vs. group 1G), ^† p < 0.05 (group 1G vs. group PBS). PBS, phosphate-buffered saline; TBI, traumatic brain injury.

Effects of MG-cultured cMSC transplantation on the mRNA expression of brain injury sites

To estimate the effects of MG-cultured cMSC transplantation at the brain injury sites, mRNA expression levels of inflammatory and apoptotic factors were analyzed. Tnf-α expression levels were significantly lower in mice of group MG than those of group PBS (Fig. 5A). No significant differences were observed for Bax expression, Bcl-2 expression, or the Bax/Bcl-2 ratio (Fig. 5B–D).

FIG. 5.

mRNA expression levels in mouse brain injury sites. mRNA expression levels of Tnf-α (A), Bax (B), Bc1-2 (C), and Bax/Bcl-2 (D). Data represent the mean ± SEM of independent experiments. n = 4, *p < 0.05. Bax, Bcl-2-associated X protein; Bcl-2, b cell leukemia/lymphoma 2 protein; Tnf-α, tumor necrosis factor alpha.

Effects of MG-cultured cMSC transplantation on the protein expression of brain injury sites

Inflammation and apoptosis-related protein expression levels in the brain injury sites were analyzed. TNF-α and Bax expression levels were significantly lower in mice of group MG than those of group PBS (Fig. 6A–C). Bcl-2 expression levels were significantly higher in mice of group MG and group 1G than those of group PBS (Fig. 6A, D). The Bax/Bcl-2 ratio was significantly lower in mice of group MG and group 1G than those of group PBS (Fig. 6A, E).

FIG. 6.

Protein expression levels in mouse brain injury sites. Protein expression levels of TNF-α (A, B), Bax (A, C), Bcl-2 (A, D), and Bax/Bcl-2 (A, E). Data represent the mean ± SEM of independent experiments. n = 4, *p < 0.05; **p < 0.01.

Survival rates and mRNA expression of stress-exposed NG108-15 cells

We estimated paracrine effects of MG-cultured cMSCs against inflammatory or oxidative stress-induced cell death. As a result, the survival rates of stress-exposed NG108-15 cells were significantly higher in the MG-CM than in the 1G-CM or Ctrl (Fig. 7A, B).

FIG. 7.

Effects of cMSC-conditioned medium on the survival rate of stress-exposed NG108-15 cells. Survival rates of NG108-15 cells exposed to inflammatory stress (A) and oxidative stress (B). Data represent the mean ± SEM of independents experiments. n = 8, *p < 0.05; **p < 0.01.

mRNA expression in stress-exposed NG108-15 cells was also analyzed. In inflammatory stress-exposed experiments, Tnf-α expression levels of inflammatory stress-exposed NG108-15 were significantly lower in cells cultured in MG-CM than in Ctrl (Fig. 8A). On the other hand, there were no significant differences in Tnfrsf1a or beclin 1 (Becn1) expression levels or the Bax/Bcl-2 ratio among the three groups (Fig. 8B–D). In oxidative stress-exposed experiments, the Bax/Bcl-2 ratio of oxidative stress-exposed NG108-15 cells was significantly lower in cells cultured in MG-CM than in Ctrl (Fig. 8E). There were no significant differences in Becn1 expression levels among the three groups (Fig. 8F).

FIG. 8.

mRNA expression levels of stress-exposed NG108-15 cells. mRNA expression of Tnf-α (A), Tnfrsf1a (B), Bax/Bcl-2 (C), and Becn1 (D) in NG108-15 cells exposed to inflammatory stress. mRNA expression of Bax/Bcl-2 (E) and Becn1 (F) in NG108-15 cells exposed to oxidative stress. Data represent the mean ± SEM of independent experiments. n = 6, *p < 0.05; **p < 0.01. Becn1, beclin 1; Tnfrsf1a, tumor necrosis factor receptor superfamily, member 1A.

Discussion

In the present study, we examined whether cMSCs cultured under simulated MG promoted functional recovery in a TBI model after cell transplantation and whether they have an inhibitory effect on acute inflammation or apoptosis after injury in vivo and in vitro.

We investigated differences of the gravity environment on mRNA expression levels of cMSCs. Our results showed that culturing under simulated MG enhanced HGF and TGF-β mRNA expression in cMSCs compared with normal gravity culture. HGF is an important factor of cell growth, migration, survival, angiogenesis, wound healing, and tissue regeneration [20 –23]. Furthermore, it has been reported that HGF has a role in axonal growth, dendrite elongation [24,25], and neuroprotection from various stresses such as glutamate toxicity or hypoxia–reoxygenation [26,27]. TGF-β is an inducing factor of cell transformation [28], and it has been reported that TGF-β is closely related to cellular proliferation, differentiation, and migration [29 –31]. These processes are involved in the immune system through the promotion of wound healing and the expression of anti-inflammatory cytokines [32 –34]. The expression levels of HGF and TGF-β are affected by physical stimulus. For instance, previous studies have suggested that HGF expression is promoted through physical stimulus induced by MAP Kinase signaling pathway activation [35 –38]. In addition, it has also been suggested that hypertension induces the gene expression of TGF-β [39]. Therefore, differences of the gravity environment, a type of physical stimulus, may affect the mRNA expression of HGF and TGF-β in cMSCs. Although detailed mechanisms are still not clear, our results suggest that simulated MG promotes neuroprotection and anti-inflammation-related gene expression.

Many previous studies have reported that various secondary injuries in the acute phase of CNS diseases, such as inflammation and oxidative stress, lead to further cell death and functional disability [40 –42]. Secondary injuries in CNS diseases have been reported to reach a peak at 1 to 2 days after the injury [43,44]. For these reasons, cell transplantation was performed 1 day after injury in this study. Motor function recovery was significantly higher in mice belonging to group MG than those of group PBS 7 days after transplantation. In addition, TNF-α expression levels and Bax/Bcl-2 ratio in brain injury sites of group MG were significantly lower than those of group PBS. Previous studies have reported that inflammatory reactions, one of the secondary injuries in CNS disorders, are caused by secretion of inflammatory cytokines such as TNF-α and interleukin through activation of astrocytes, microglia, and macrophages that have passed through the collapsed blood–brain barrier after brain injury [45 –48]. Other studies have demonstrated that HGF inhibits inflammation through suppression of activation of TNF-α transcription factor NF-κB [49,50]. Indeed, administration of TGF-β to inflammation areas suppresses the expression of inflammatory cytokines, including TNF-α in microglia, astrocytes, and macrophages [51,52]. Moreover, it has been reported that TNF-α induces apoptosis-related factor expression through a p38MAPK cascade [53]. From these studies, our results suggest that transplantation of cMSCs cultured under simulated MG promotes functional recovery in the TBI model from the early stage through inhibition of the inflammatory response and apoptosis at the injury sites, which may be mediated by HGF and TGF-β.

In the present study, neuroprotective effects of the CM of cMSCs cultured under simulated MG were analyzed. To mimic the environment of secondary injury, we used LPS for inflammation and H₂O₂ for oxidative stress [54,55]. CM was replaced in NG108-15 cells exposed to inflammatory or oxidative stress. The survival rates of NG108-15 cells exposed to inflammatory stress and oxidative stress were significantly higher in group MG-CM than in group 1G-CM or group Ctrl. For mRNA expression in NG108-15 cells, TNF-α expression levels on inflammatory stress in group MG-CM were significantly lower than in group Ctrl. It is known that LPS stimulation induces the gene expression of TNF-α by an intracellular signaling pathway through the Toll-like receptor [56]. Another study demonstrated that TGF-β suppresses the expression of TNF-α in inflammation of the spinal cord [52]. Several previous studies have reported that HGF suppresses intracellular signal transduction mediated by the Toll-like receptor [49,57]. Significant suppression of TNF-α expression in group MG-CM suggested that the suppression of the inflammatory response in NG108-15 cells by the involvement of HGF and TGF-β might contribute to improvement in cell viability. Moreover, the Bax/Bcl-2 ratio on oxidative stress in group MG-CM was significantly lower than in group Ctrl. It is reported that H₂O₂-induced oxidative stress promotes the expression of apoptosis-related factors in nerve cells [57]. Previous research has shown that HGF plays a role in neuroprotection against injured nerve cells by inhibiting apoptosis-related factors through increasing both phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation [26]. Our study indicated that cMSCs cultured under simulated MG were protected against oxidative stress induced in NG108-15 cells.

In conclusion, these results demonstrate novel evidence that transplantation of cMSCs cultured under simulated MG facilitated functional recovery in TBI model mice through inhibition of inflammation and apoptosis. Although further study regarding secretion is needed, we consider that secretion related to neuroprotection and anti-inflammation of cMSCs might contribute to the present results. Collectively, our novel results suggest that culturing MSCs under simulated MG conditions is a valuable strategy for cell-based therapy.

Footnotes

Acknowledgments

The authors thank Dr. Masahiro Kino-oka and Dr. Mee-Hae Kim from the Department of Biotechnology, Graduate School of Engineering, Osaka University, for their support. They also thank Tomoyuki Kurose from the Division of Bio-Environmental Adaptation Sciences, Graduate School of Biomedical & Health Sciences, Hiroshima University, for technical support. This work was carried out, in part, at the Analysis Center of Life Sciences, Natural Science Center for Basic Research and Development, Hiroshima University.

Author Disclosure Statement

L.Y. is a director of Space Bio-Laboratories Co., Ltd., (SBL) and Y.K. is a president of SBL. They are shareholders. The interest conflicts of this research have been approved by the Conflict of Interest Management Committee. By regularly reporting research progress to the Conflicts of Interest Management Committee, we will maintain fairness regarding the interests of this research. All other authors declare no conflicts exist.

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